MHC 1b-mediated α-synuclein-specific tolerance as a novel therapeutic approach for Parkinson's disease

Abstract

The present 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. The present invention more specifically relates to recombinant polypeptides comprising a peptide antigen and one or more domains of a non-classical MHC class Ib molecule. The present invention also relates to methods for producing such recombinant polypeptides, pharmaceutical compositions comprising them, and their use for treating Parkinson's disease.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present 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. The present invention more specifically relates to recombinant polypeptides comprising a peptide antigen and one or more domains of a non-classical MHC class Ib molecule. The present invention also relates to methods for producing such recombinant polypeptides, pharmaceutical compositions comprising them, and their use for treating Parkinson's disease. [Background technology]

[0002] Parkinson's disease (PD) is the most common neurodegenerative movement disorder affecting over 7 million people worldwide and for which there is still no cure. Patients suffer from severe motor and non-motor symptoms such as tremor, bradykinesia, rigidity, postural instability, and depression. The pathological hallmark of the disease is the degeneration of dopaminergic neurons in the substantia nigra and the formation of α-synuclein-containing protein aggregates called Lewy bodies. Although the physiological role of α-synuclein (aSyn) points to functions in synaptic transmitter release and amplifying immune responses (Alam et al., Cell Reports 38:110090. doi:10.1016 / j.celrep.2021.110090), the pathological role of insoluble α-synuclein remains enigmatic. The observation that multiple mutations and mutations in the SNCA gene that encodes aSyn can cause hereditary forms of PD have firmly linked aSyn to the development of PD.

[0003] Autopsies of human 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). Proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interferon (IFN)-γ and interleukins IL-1β and IL-6 in the nigrostriatal system further confirm that PD is accompanied by significant neuroinflammation (Mogi M et al., 1994; Mogi M et al., 1994). An increased ratio of IFN-γ to IL-4 producing T cells in the peripheral blood of PD patients indicates a systematic shift towards a pro-inflammatory environment (Baba Y et al., 2005). Recently, aSyn-specific T cell responses have been detected in the peripheral blood of PD patients and were found to precede motor symptoms, indicating an active and specific involvement of the immune system in disease progression. Two aSyn-derived peptides were shown to induce MHC-restricted cytokine release from peripheral blood mononuclear cells (Sulzer D et al., 2017).

[0004] Currently, treatment of PD relies primarily on counteracting SN neuronal loss by administering L-dopamine or derivative substances, an intervention that initially ameliorates disease symptoms but does not prevent disease progression.

[0005] A population-based case-control study including approximately 48,000 PD patients analyzed the risk of developing PD according to the intake of immunosuppressants. Patients medicated with azathioprine had a significantly lower risk of PD (Racette BA et al., 2018). A recent cohort study further showed that patients receiving anti-TNF-α therapy for inflammatory bowel disease had a lower risk of developing PD (Peter I et al., 2018). The effect of immunomodulatory agents such as β2-adrenergic receptor agonists or anti-IL-17 therapy was explored in small open-label clinical trials with some promising results (Magistrelli L and Comi C, 2020; Storelli E et al., 2019).

[0006] Given the often slowly progressive nature of this chronic disease, the side effects of potent immunosuppressants are a major obstacle to their testing in early-stage PD patients. However, in later-stage patients, irreversible neurodegeneration limits the benefits that can be achieved by blocking further progression. Highly targeted immunomodulatory drugs, in contrast, would allow early treatment, thereby maximizing therapeutic benefit.

[0007] To this end, a clinical trial (https: / / clinicaltrials.gov / ct2 / show / NCT03318523) was conducted with the aSyn-specific antibody simpanemab (BIIB054). It was hoped that sequestration of free aSyn by simpanemab would prevent further aSyn aggregation and thus prevent disease progression. Unfortunately, this study failed to meet its primary or secondary endpoints. Thus, there remains an urgent need for targeted and effective disease-modifying therapeutic agents for PD.

[0008] The pathogenetic role of T cells in PD has been mainly analyzed in toxin-based models such as the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or 6-hydroxydopamine (6-OHDA) models. However, given the lack of aSyn aggregation and Lewy bodies, these models poorly reflect the molecular aspects of the human disease. This prompted Dr. Ip to generate a new mouse model for PD that more faithfully recapitulates the essential features of the human disease. Human α-synuclein into the substantia nigra. A53TStereotaxic-guided unilateral injection of AAV1 / 2 vectors encoding haSyn induces behavioral deficits, loss of dopaminergic nigral neurons and striatal fibers and reduced striatal dopamine, as well as the formation of Lewy bodies (Ip CW et al., 2017). The high face validity of this model is achieved by injecting α-synuclein mutants known to be associated with disease in humans, so the etiological relevance of this model is also supported by its disease-related construct validity. Furthermore, insoluble aSyn aggregates exhibiting S129 phosphorylation and Lewy body-like neurites and Lewy bodies are detectable in this animal model (Ip CW et al., 2017). The haSyn PD model thus provides an important tool to advance preclinical and translational research toward novel and better therapies for patients with PD. In a collaborative study, we confirmed the broad pro-inflammatory profile of brain immune cells in this haSyn PD model and demonstrated the upregulation of CD4+ into the brains of haSyn PD mice. + and CD8 + The researchers showed that activated T cell subsets were infiltrating the brain. These T cells induced neurodegeneration that resembled PD. - / - ) reduced dopaminergic neurodegeneration, whereas T cell reconstitution exacerbated neuronal loss in vivo. T cells isolated from the brains of haSyn PD mice were cytotoxic to neuronal cells expressing haSyn in vitro (Karikari et al., Brain Behav Immun. 2022 Mar;101:194-210).

[0009] Immunosuppressive MHC class Ib molecules, such as HLA-G, are important for tolerance induction during pregnancy. They exert immunosuppressive effects on various immune cells through immunosuppressive receptors, such as ILT2, ILT4 and Kir2DL4. WO 2018 / 215340 concerns the combination of MHC class Ib molecules with peptides for targeted therapeutic immunomodulation, but is silent on the treatment of Parkinson's disease.

[0010] Thus, there is an urgent need for effective targeted drugs for the treatment of Parkinson's disease. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] WO 2018 / 215340 [Non-patent literature]

[0012] [Non-Patent Document 1] Alam et al., Cell Reports 38:110090. doi:10.1016 / j.celrep.2021.110090 [Non-Patent Document 2] Brochard V et al., 2009 [Non-Patent Document 3] McGeer PL et al., 1988 [Non-Patent Document 4] Mogi M et al., 1994 [Non-Patent Document 5] Baba Y et al., 2005 [Non-Patent Document 6] Sulzer D et al., 2017 [Non-Patent Document 7] Racette BA et al., 2018 [Non-Patent Document 8] Peter I et al., 2018 [Non-Patent Document 9] Magistrelli L and Comi C, 2020 [Non-Patent Document 10] Storelli E et al., 2019 [Non-Patent Document 11] Ip CW et al., 2017 [Non-Patent Document 12] Karikari et al., Brain Behav Immun. 2022 Mar;101:194-210 Summary of the Invention [Problem to be solved by the invention]

[0013] Description of the invention The inventors have found that human MHC class Ib molecules, such as HLA-G, have the ability to induce antigen-specific tolerance to peptide antigens presented. Thus, MHC class Ib molecules can be advantageously used according to the invention to suppress immune responses in an antigen-specific manner, despite having a structure and sequence similar to the classical human MHC class Ia molecules that induce antigen peptide-specific immune responses. Furthermore, the inventors have found that for the suppression of immune responses according to the invention, molecules other than naturally occurring MHC class Ib molecules can be used, in particular polypeptides that comprise at least one domain of an MHC class Ib molecule, preferably at least the [α]3 domain of an MHC class Ib molecule only: the [α]1 and [α]2 domains of variable class Ia molecules can be combined with the [α]3 domain of human MHC class Ib molecules to suppress immune responses to peptides presented by these antigens.

[0014] Antigen-loaded HLA-G molecules can be unstable. Therefore, the inventors designed soluble recombinant polypeptides containing peptide antigens, MHC class Ib molecules 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 MHC class Ib molecules such as HLA-G were exchanged with the respective domains of other MHC molecules to increase the flexibility and versatility of these recombinant polypeptides (see, e.g., FIG. 2). These alternative recombinant polypeptides can be designed taking into account the antigen-binding domains of other human HLA molecules. It has already been found that a construct containing the α1 and α2 domains of mouse H2-Kb can present the ovalbumin-derived peptide SIINFEKL to OT-1 T cells, which express a transgenic T cell receptor that specifically recognizes this antigen (WO 2018 / 215340).

[0015] The literature teaches that Parkinson's disease is a neurodegenerative disease caused by pathogenic accumulation of α-synuclein. The more recent concept that a key pathogenic event results from an immune response to α-synuclein has yet to be translated towards therapeutic development.

[0016] Surprisingly, the inventors have found that a therapeutic effect in the treatment of Parkinson's disease can be achieved by suppressing the immune response to alpha-synuclein using a surrogate of the recombinant polypeptide of the present invention in a mouse model that closely mimics human Parkinson's disease. Thus, according to the present invention, Parkinson's disease can be treated by the recombinant polypeptide of the present invention.

[0017] Furthermore, according to the present invention, the recombinant polypeptides of the present invention hardly cause systemic immunosuppression, so they are expected to be very advantageous in the immunotherapeutic treatment of Parkinson's disease. Importantly, early stage patients suffering from Parkinson's disease should not be exposed to the strong side effects of systemic immunosuppression, because this is likely to result in opportunistic potentially fatal infections. However, in later stage patients, neurodegeneration is often irreversible. However, the highly specific tolerance induction achieved by presenting α-synuclein antigen to the recombinant polypeptides of the present invention will already be tolerant at early stages of the disease, such as prodromal Parkinson's disease, and will pave the way for new therapeutic agents. [Means for solving the problem]

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

[0019] 1. A recombinant polypeptide capable of presenting a peptide antigen, the recombinant polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen presented by the recombinant polypeptide, the peptide antigen being a peptide of human α-synuclein; ii) optionally, a linker sequence; iii) optionally a sequence of a human polypeptide domain that includes a sequence of human β2 microglobulin, or an amino acid sequence that is at least 90% identical to the amino acid sequence of human β2 microglobulin represented by SEQ ID NO:5; iv) optionally, a linker sequence; v) optionally, the [α]1 domain of an MHC molecule; vi) optionally, the [α]2 domain of an MHC molecule; vii) an [α]3 domain of an MHC class Ib molecule, or a derivative of an [α]3 domain of an MHC class Ib molecule, which derivative is capable of binding to ILT2 or ILT4; viii) optionally, a protease cleavage site; ix) optionally, a spacer sequence; and x) Possibly, an affinity tag.

[0020] 2. The recombinant polypeptide according to item 1, wherein the peptide antigen according to i) is 7 to 11 amino acids in length, preferably 8 to 10 amino acids in length.

[0021] 3. The recombinant polypeptide according to item 1 or 2, wherein the peptide antigen according to i) is composed of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46.

[0022] 4. The recombinant polypeptide of item 3, wherein the peptide antigen is composed of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44.

[0023] 5. The recombinant polypeptide according to item 3, wherein the peptide antigen is composed of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, and SEQ ID NO:33.

[0024] 6. The recombinant polypeptide according to item 3, wherein the peptide antigen consists of the amino acid sequence of SEQ ID NO: 20.

[0025] 7. The recombinant polypeptide according to item 3, wherein the peptide antigen is composed of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, and SEQ ID NO:33.

[0026] 8. A recombinant polypeptide described in any one of the preceding paragraphs, wherein the [α]1 domain described in (v) and the [α]2 domain described in (vi) are derived from a human MHC class Ia molecule or a human MHC class Ib molecule.

[0027] 9. The recombinant polypeptide according to item 8, wherein the [α]1 domain described in (v) and the [α]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.

[0028] 10. The recombinant polypeptide according to item 8, wherein the [α]1 domain described in (v) and the [α]2 domain described in (vi) are derived from a human MHC class Ia molecule.

[0029] 11. The recombinant polypeptide according to item 10, wherein the [α]1 domain described in (v) and the [α]2 domain described in (vi) are derived from a human HLA-A2 molecule.

[0030] 12. The recombinant polypeptide according to item 10, wherein the [α]1 domain described in (v) and the [α]2 domain described in (vi) are derived from a human HLA-A1 molecule.

[0031] 13. The recombinant polypeptide according to item 10, wherein the [α]1 domain described in (v) and the [α]2 domain described in (vi) are derived from a human HLA-A11 molecule.

[0032] 14. A recombinant polypeptide according to any one of the preceding paragraphs, wherein the [α]3 domain of the MHC class Ib molecule described in (vii) is the [α]3 domain of human HLA-E, human HLA-F or human HLA-G.

[0033] 15. A recombinant polypeptide according to any one of the preceding paragraphs, wherein the [α]3 domain of the MHC class Ib molecule described in (vii) is the [α]3 domain of human HLA-G.

[0034] 16. A recombinant polypeptide described in any one of the preceding paragraphs, wherein the [α]3 domain or derivative described in (vii) is identical to or has at least 80% amino acid sequence identity, preferably at least 90% amino acid sequence identity, to an [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 2.

[0035] 17. The recombinant polypeptide according to item 16, wherein the [α]3 domain or derivative according to (vii) is identical to or has at least 92% amino acid sequence identity with an [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 2.

[0036] 18. The recombinant polypeptide according to item 16, wherein the [α]3 domain or derivative according to (vii) is identical to or has at least 94% amino acid sequence identity with an [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 2.

[0037] 19. The recombinant polypeptide according to item 16, wherein the [α]3 domain or derivative according to (vii) is identical to or has at least 96% amino acid sequence identity with an [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 2.

[0038] 20. The recombinant polypeptide according to item 16, wherein the [α]3 domain or derivative according to (vii) is identical to or has at least 98% amino acid sequence identity with an [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 2.

[0039] 21. The recombinant polypeptide according to item 16, wherein the [α]3 domain or derivative according to (vii) is identical to or has at least 99% amino acid sequence identity with an [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 2.

[0040] 22. The recombinant polypeptide according to item 16, wherein the [α]3 domain described in (vii) is identical to the [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 2.

[0041] 23. A recombinant polypeptide according to any one of the preceding paragraphs, wherein the linker sequence described in (ii) and / or the linker sequence described in (iv) comprises the amino acid sequence (GGGGS)n, where n is an integer equal to or greater than 1.

[0042] 24. The recombinant polypeptide according to item 23, wherein the linker sequence according to (ii) comprises the amino acid sequence (GGGGS)n, where n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, preferably selected from the group consisting of 2, 3, 4 and 5.

[0043] 25. The recombinant polypeptide according to item 23 or 24, wherein the linker sequence according to (iv) comprises the amino acid sequence (GGGGS)n, where n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, preferably selected from the group consisting of 2, 3, 4 and 5.

[0044] 26. A recombinant polypeptide according to any one of the preceding paragraphs, 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.

[0045] 27. A recombinant polypeptide according to any one of the preceding claims, wherein the polypeptide is a dimer or multimer.

[0046] 28. A recombinant polypeptide according to any one of the preceding claims, wherein the polypeptide comprises or consists of all of components i) to vii).

[0047] 29. A recombinant polypeptide according to any one of the preceding claims, wherein the polypeptide does not comprise components viii) to x).

[0048] 30. A recombinant polypeptide according to any one of items 1 to 28, wherein the polypeptide comprises or consists of all of components i) to x).

[0049] 31. The recombinant polypeptide of any one of the preceding claims, further comprising an N-terminal secretory signal peptide sequence.

[0050] 32. The recombinant polypeptide according to any one of items 1 to 30, wherein the recombinant polypeptide is composed of an amino acid sequence consisting of the following ((a) and (b)) in the order from the N-terminus to the C-terminus: (a) a peptide antigen selected from the group consisting of the amino acid sequences of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46, and (b) The amino acid sequence of SEQ ID NO:16.

[0051] 33. The recombinant polypeptide of any one of the preceding claims, wherein the recombinant polypeptide is soluble.

[0052] 34. A nucleic acid encoding one or more polypeptides according to any one of the preceding paragraphs.

[0053] 35. The nucleic acid according to item 34, wherein the nucleic acid is a vector.

[0054] 36. A pharmaceutical composition or kit comprising at least one nucleic acid according to item 34 or 35.

[0055] 37. A pharmaceutical composition or kit comprising at least one recombinant polypeptide according to any one of items 1 to 33.

[0056] 38. The pharmaceutical composition or kit according to item 37, wherein the pharmaceutical composition or kit comprises at least two different recombinant polypeptides according to any one of items 1 to 33, each of the different polypeptides comprising a different peptide antigen as defined in any one of items 3 to 7.

[0057] 39. A pharmaceutical composition or kit according to any one of items 36 to 38 for use in treating Parkinson's disease in a human patient.

[0058] 40. The pharmaceutical composition or kit for use according to item 39, wherein the Parkinson's disease is prodromal Parkinson's disease.

[0059] 41. A pharmaceutical composition or kit for use according to item 39 or 40, wherein the treatment is treatment of Parkinson's disease by immunotherapy.

[0060] 42. A pharmaceutical composition or kit for use according to any one of items 39 to 41, wherein the treatment is by inducing immune tolerance to the autoantigenic human α-synuclein.

[0061] 43. A recombinant host cell comprising the nucleic acid or vector according to item 34 or 35 and expressing a recombinant polypeptide according to any one of items 1 to 33.

[0062] 44. A method for obtaining a pharmaceutical composition comprising a polypeptide according to any one of items 1 to 33, the method comprising the steps of: (a) culturing a recombinant host cell according to item 43 under conditions allowing expression of the recombinant polypeptide from the nucleic acid molecule; (b) recovering the recombinant polypeptide; (c) purifying the recombinant polypeptide; and (d) formulating the recombinant polypeptide into a pharmaceutical composition. [Brief description of the drawings]

[0063] [Figure 1]Schematic diagram of peptide-loaded soluble MHC Ib molecules suitable for achieving therapeutically effective antigen-specific immune modulation. The presented peptide antigens are represented as dotted spheres, the HLA-Gα1-3 domains are in light grey and the β2 microglobulin domains are in dark grey. The optional linkers connecting the antigenic peptides to the β2 microglobulin molecules are shown in grey stick form and the optional disulfide traps are depicted as black spheres. The diagram was generated using Pymol and is based on structures published in Clements et al., Proc Natl Acad Sci US A. 2005 Mar 1;102(9):3360-5 and Hansen et al., Trends Immunol. 2010 Oct;31(10):363-9. [Diagram 2]Example of a vector construct encoding a single-chain MHC Ib molecule suitable for therapeutic peptide-specific immunomodulation HLA-G1 and HLA-G5 each consist of three [α] domains (shown in black in the figure), a non-covalently bound β2 microglobulin subunit (shown in dark grey in the figure), and an antigenic peptide (short black arrow) presented on HLA-G. HLA-G1 further contains a transmembrane domain and a short intracellular chain (not shown in the figure). As shown in the figure, the [α]3 domain can bind to the receptors ILT2 (see Shiroishi et al., Proc Natl Acad Sci US A. 2003 July 22;100(15):8856-8861) and ILT4 (see Shiroishi et al., Proc Natl Acad Sci US A. 2006 October 31;103(44):16412-7) on immune cells. Physiologically, these sequences form the MHC class 1 complex by non-covalent association. To facilitate purification of the complex MHC Ib molecule, one or more protein tags (such as Spot, myc and / or His(6x) tags) may be introduced for subsequent optional removal by cleavage with an optional factor Xa cleavage site. Furthermore, the antigen peptide, β2 microglobulin and MHC Ib[α] chain can be linked to increase stability. Vector maps are generated using Snapgene Viewer Software. [Diagram 3]Prediction and validation of antigenic aSyn peptides. Results from IFN-γ ELISpot analysis after stimulation of mononuclear cells containing CD4+, CD8+ T cells from the spleens of haSyn PD (grey) and control mice (black) 10 weeks after EV or A53T vector injection with predicted peptides. Five aSyn peptides elicit 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 / ), the haSyn protein sequence was aligned with mouse α-synuclein and screened for potential neo-antigens likely to be presented on MHC I (H-2Db and H-2Kb) and MHC II (I-Ab) molecules. The three highest scoring peptides for each MHC molecule (8-15 amino acids long) were synthesized in aqua basic quality by Thermo Scientific. Each peptide was used to stimulate cells derived from lymph nodes or spleens, followed by ELISpot assays. Splenocytes from two initially non-responsive haSyn mice and two controls were cultured for 10 days in the presence of all 10 peptides (1 μg / ml each) in RPMI containing 10% FCS and 20 ng / ml IL-2. Multiscreen PVDF plates (MSIPS4510, Millipore) were activated with 35% ethanol, washed with sterile PBS, and coated overnight at 4°C with 2 μg / ml of filtered anti-mouse IFN-γ antibody (AN18, Mabtech). Isolated single cell suspensions derived from cervical lymph nodes and spleens were seeded in IMDM containing 7.5% FCS with or without 5 μg / ml of synthetic peptides. IFN-γ spots were then detected using 2 μg / ml biotinylated anti-mouse IFN-γ antibody (R4-6A2, Mabtech) followed by horseradish peroxidase-conjugated streptavidin (Cell Signaling) in PBS-T supplemented with 0.05% BSA.Assays were developed with filtered "ready-to-use" TMB substrate (Mabtech) and analyzed using an Immunospot S6 Core Analyzer. [Figure 4-1] aSyn-loaded AIM Bio surrogate molecules can prevent neuronal cell death and improve endurance in PD models. Weekly treatment for nine cycles with a peptide, here an aSyn peptide, a polypeptide containing an MHC class 1 antigen-presenting domain and an HLA-Gα3 domain (AIM Bio), resulted in significant, dose-dependent neuroprotection of dopaminergic neurons in haSyn PD mice compared to vehicle-treated haSyn PD mice with significantly higher numbers of tyrosine hydroxylase (TH)+ neurons in the substantia nigra of the treatment group. Immunohistological staining was performed using 40 μm PFA-fixed cryosections processed 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). Development was performed using (DAB)-HCl-peroxidase (Vector Labs). An estimate of the number of dopaminergic neurons in the substantia nigra was performed by unbiased stereological analysis using the Stereo Investigator software package (v11.07, MicroBrightField Biosciences). The endurance of treated and untreated mice was tested by comparing pre-op and post-op latencies in the Rotarod test as described in (Ip CW et al., 2017). A: Images (scale bar = 100 micrometers); B: quantification; C: latencies compared to pre-op assessed by the Rotarod test. [Figure 4-2]aSyn-loaded AIM Bio surrogate molecules can prevent neuronal cell death and improve endurance in PD models. Weekly treatment for nine cycles with a peptide, here an aSyn peptide, a polypeptide containing an MHC class 1 antigen-presenting domain and an HLA-Gα3 domain (AIM Bio), resulted in significant, dose-dependent neuroprotection of dopaminergic neurons in haSyn PD mice compared to vehicle-treated haSyn PD mice with significantly higher numbers of tyrosine hydroxylase (TH)+ neurons in the substantia nigra of the treatment group. Immunohistological staining was performed using 40 μm PFA-fixed cryosections processed 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). Development was performed using (DAB)-HCl-peroxidase (Vector Labs). An estimate of the number of dopaminergic neurons in the substantia nigra was performed by unbiased stereological analysis using the Stereo Investigator software package (v11.07, MicroBrightField Biosciences). The endurance of treated and untreated mice was tested by comparing pre-op and post-op latencies in the Rotarod test as described in (Ip CW et al., 2017). A: Images (scale bar = 100 micrometers); B: quantification; C: latencies compared to pre-op assessed by the Rotarod test. [Figure 4-3]aSyn-loaded AIM Bio surrogate molecules can prevent neuronal cell death and improve endurance in PD models. Weekly treatment for nine cycles with a peptide, here an aSyn peptide, a polypeptide containing an MHC class 1 antigen-presenting domain and an HLA-Gα3 domain (AIM Bio), resulted in significant, dose-dependent neuroprotection of dopaminergic neurons in haSyn PD mice compared to vehicle-treated haSyn PD mice with significantly higher numbers of tyrosine hydroxylase (TH)+ neurons in the substantia nigra of the treatment group. Immunohistological staining was performed using 40 μm PFA-fixed cryosections processed 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). Development was performed using (DAB)-HCl-peroxidase (Vector Labs). An estimate of the number of dopaminergic neurons in the substantia nigra was performed by unbiased stereological analysis using the Stereo Investigator software package (v11.07, MicroBrightField Biosciences). The endurance of treated and untreated mice was tested by comparing pre-op and post-op latencies in the Rotarod test as described in (Ip CW et al., 2017). A: Images (scale bar = 100 micrometers); B: quantification; C: latencies compared to pre-op assessed by the Rotarod test. [Figure 5-1]AIM Bio surrogate molecules loaded with aSyn prevent infiltration of CD11b+ myeloid cells (microglia) but induce infiltration of CD8+ T cells into the striatum in a PD model. Immunohistochemical staining of mouse tissues for CD11b+ myeloid cells and CD8+ T cells was performed using 10 μm fresh coronal cryosections of the striatum. After 4% PFA fixation or acetone fixation, respectively, sections were incubated with rat anti-mouse CD11b (1:100, Serotec) or rat anti-CD8 MCA609G 1:500 antibodies, respectively, followed by biotinylated rabbit anti-rat secondary antibodies (Vector Labs). Development was performed using (DAB)-HCl-peroxidase (Vector Labs). Microglia were quantified at 200x magnification in the SN and striatal regions as delineated by serial sections stained for TH on a BH2 light microscope (Olympus). A: Images showing CD11b+ microglial cells in the striatum (scale bars = 50 micrometers for the whole image and 20 micrometers for the magnified area in the lower left corner, respectively); B: Quantification; C: Images showing CD8+ T cells in the striatum (scale bars = 100 micrometers for the whole image and 50 micrometers for the magnified area in the lower left corner, respectively); D: Quantification. [Figure 5-2]AIM Bio surrogate molecules loaded with aSyn prevent infiltration of CD11b+ myeloid cells (microglia) but induce infiltration of CD8+ T cells into the striatum in a PD model. Immunohistochemical staining of mouse tissues for CD11b+ myeloid cells and CD8+ T cells was performed using 10 μm fresh coronal cryosections of the striatum. After 4% PFA fixation or acetone fixation, respectively, sections were incubated with rat anti-mouse CD11b (1:100, Serotec) or rat anti-CD8 MCA609G 1:500 antibodies, respectively, followed by biotinylated rabbit anti-rat secondary antibodies (Vector Labs). Development was performed using (DAB)-HCl-peroxidase (Vector Labs). Microglia were quantified at 200x magnification in the SN and striatal regions as delineated by serial sections stained for TH on a BH2 light microscope (Olympus). A: Images showing CD11b+ microglial cells in the striatum (scale bars = 50 micrometers for the whole image and 20 micrometers for the magnified area in the lower left corner, respectively); B: Quantification; C: Images showing CD8+ T cells in the striatum (scale bars = 100 micrometers for the whole image and 50 micrometers for the magnified area in the lower left corner, respectively); D: Quantification. [Figure 5-3]AIM Bio surrogate molecules loaded with aSyn prevent infiltration of CD11b+ myeloid cells (microglia) but induce infiltration of CD8+ T cells into the striatum in a PD model. Immunohistochemical staining of mouse tissues for CD11b+ myeloid cells and CD8+ T cells was performed using 10 μm fresh coronal cryosections of the striatum. After 4% PFA fixation or acetone fixation, respectively, sections were incubated with rat anti-mouse CD11b (1:100, Serotec) or rat anti-CD8 MCA609G 1:500 antibodies, respectively, followed by biotinylated rabbit anti-rat secondary antibodies (Vector Labs). Development was performed using (DAB)-HCl-peroxidase (Vector Labs). Microglia were quantified at 200x magnification in the SN and striatal regions as delineated by serial sections stained for TH on a BH2 light microscope (Olympus). A: Images showing CD11b+ microglial cells in the striatum (scale bars = 50 micrometers for the whole image and 20 micrometers for the magnified area in the lower left corner, respectively); B: Quantification; C: Images showing CD8+ T cells in the striatum (scale bars = 100 micrometers for the whole image and 50 micrometers for the magnified area in the lower left corner, respectively); D: Quantification. [Figure 5-4]AIM Bio surrogate molecules loaded with aSyn prevent infiltration of CD11b+ myeloid cells (microglia) but induce infiltration of CD8+ T cells into the striatum in a PD model. Immunohistochemical staining of mouse tissues for CD11b+ myeloid cells and CD8+ T cells was performed using 10 μm fresh coronal cryosections of the striatum. After 4% PFA fixation or acetone fixation, respectively, sections were incubated with rat anti-mouse CD11b (1:100, Serotec) or rat anti-CD8 MCA609G 1:500 antibodies, respectively, followed by biotinylated rabbit anti-rat secondary antibodies (Vector Labs). Development was performed using (DAB)-HCl-peroxidase (Vector Labs). Microglia were quantified at 200x magnification in the SN and striatal regions as delineated by serial sections stained for TH on a BH2 light microscope (Olympus). A: Images showing CD11b+ microglial cells in the striatum (scale bars = 50 micrometers for the whole image and 20 micrometers for the magnified area in the lower left corner, respectively); B: Quantification; C: Images showing CD8+ T cells in the striatum (scale bars = 100 micrometers for the whole image and 50 micrometers for the magnified area in the lower left corner, respectively); D: Quantification. [Figure 6] aSyn-loaded AIM Bio surrogate molecules reduce aSyn accumulation in the substantia nigra in PD models. Scale bar = 100 micrometers. Neurons were fixed in 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. No. 111-165-003) secondary antibodies, with DAPI counterstaining. Less intense aSyn staining was observed in mice treated with aSyn-loaded AIM Bio surrogate molecules. [Figure 7-1]aSyn-loaded AIM Bio protects tyrosine hydroxylase+ fibers in striatal and substantia nigra neurons. For methods, see FIG. 4. A significant reduction in tyrosine hydroxylase+ fibers was observed, especially in the superior part of the striatum on the side (here, the right side) where A53T aSyn AAV (A53T) was injected. Injection with aSyn-loaded AIM Bio significantly reduced the unilateral loss of tyrosine hydroxylase+ fibers in a dose-dependent manner. A: Image; Scale bar = 1000 micrometers. B: Quantification. C: Repeat experiment of FIG. 4 using alternative Nissl staining to show neurons. [Figure 7-2] aSyn-loaded AIM Bio protects tyrosine hydroxylase+ fibers in striatal and substantia nigra neurons. For methods, see FIG. 4. A significant reduction in tyrosine hydroxylase+ fibers was observed, especially in the superior part of the striatum on the side (here, the right side) where A53T aSyn AAV (A53T) was injected. Injection with aSyn-loaded AIM Bio significantly reduced the unilateral loss of tyrosine hydroxylase+ fibers in a dose-dependent manner. A: Image; Scale bar = 1000 micrometers. B: Quantification. C: Repeat experiment of FIG. 4 using alternative Nissl staining to show neurons. [Figure 7-3] aSyn-loaded AIM Bio protects tyrosine hydroxylase+ fibers in striatal and substantia nigra neurons. For methods, see FIG. 4. A significant reduction in tyrosine hydroxylase+ fibers was observed, especially in the superior part of the striatum on the side (here, the right side) where A53T aSyn AAV (A53T) was injected. Injection with aSyn-loaded AIM Bio significantly reduced the unilateral loss of tyrosine hydroxylase+ fibers in a dose-dependent manner. A: Image; Scale bar = 1000 micrometers. B: Quantification. C: Repeat experiment of FIG. 4 using alternative Nissl staining to show neurons. [Figure 8]aSyn-loaded AIM Bio induces CD8+CD122+ regulatory T cells. Significant induction of CD8+CD122+ regulatory T cells in both spleen and cervical lymph nodes was observed in response to aSyn-loaded AIM Bio treatment. Frozen sections were fixed with acetone for 10 min at -20°C, washed, and blocked with 5% BSA; 5% NGS, 0.2% Triton-X100 in PBS for 1 h. CD122 was stained overnight with anti-CD122i rabbit polyclonal antibody (MyBiosource, 1:100) and anti-CD8i clone YTS191.1 rat (Biorad, 1:200) in 1% BSA; 1% NGS, 0.2% Triton-X100. Secondary antibody staining used anti-rabbit Cy3 (Dianova, 1:300) and anti-rat AF488 (Invitrogen, 1:300) for 1 h. [Figure 9-1] Stability of purified single-chain MHC Ib molecules. After purification, single-chain MHC Ib molecules were analyzed for stability after one and three freeze-thaw cycles, after storage at room temperature for 5 days, and after heating at 50° C. for 30 min. For this, A) Coomassie gel staining of a 12% polyacrylamide gel with 2 μg AIM Bio and B) aHLA-G Western blot using 2A12aHLA-G antibody (1:1000) blot with 1 μg protein was performed under non-reducing conditions. Both monomers and dimers are detectable. [Figure 9-2] Stability of purified single-chain MHC Ib molecules. After purification, single-chain MHC Ib molecules were analyzed for stability after one and three freeze-thaw cycles, after storage at room temperature for 5 days, and after heating at 50° C. for 30 min. For this, A) Coomassie gel staining of a 12% polyacrylamide gel with 2 μg AIM Bio and B) aHLA-G Western blot using 2A12aHLA-G antibody (1:1000) blot with 1 μg protein was performed under non-reducing conditions. Both monomers and dimers are detectable. [Figure 10]Single-chain MHC Ib molecules are heat stable. For the Thermal Shift Assay (TSA), 3 μg of each single-chain MHC Ib molecule or Motavizumab as a control molecule was diluted in PBS and 5x SYPRO Orange dye (stock solution 5000x, final concentration: 5x) to a volume of 25 μl. A melting curve program was set up in the StepOnePlus Instrument using StepOnePlus Software 2.3. The starting temperature was 25°C for 1 min, then the temperature was increased by 1°C every minute, to a final temperature of 95°C for 2 min, and autofluorescence was measured in arbitrary units. Data was exported and graphed in Prism V7.04. A Boltzmann sigmoid function was used to determine the melting temperature (Tm). [Figure 11] Single-chain MHC Ib molecules induce Tregs in a dose-dependent manner. OT-I mice were injected intraperitoneally with the indicated amounts of single-chain H2_Kbα1+2 and HLA-Gα3 domain constructs (with human β2 microglobulin and the indicated peptides) or vehicle (PBS). Ova is the cognate peptide of the OT-I TCR in these mice, and Gp34 is a control peptide derived from an irrelevant virus. After 14 days, mice were sacrificed and splenocytes were tested for IL-10-secreting cells in a recall mouse IL-10 ELISpot (200,000 cells per well, MabTech mouse IL-10 ELISpot kit, 5 μg / ml of the indicated peptide or PBS alone was added, 48 hours). A clear induction of IL-10-secreting cells in response to Ova peptide was observed when 50 μg and 500 μg of mouse-matched Ova_KbG were injected. [Figure 12]Single-chain MHC Ib molecules inhibit T cell lysis in a dose-dependent manner. OT1 / BL6 mice were sacrificed, splenocytes were harvested, and washed once in RPMI 5% FCS. Red blood cells were removed with 2 ml of 1x sterile red blood cell lysis buffer for 3 min. Cells were cultured at high density (107 cells / ml) for 72 h in RPMI 10% FCS medium containing 20 ng / ml GMCSF, 20 ng / ml IL-2, and 10 ng / ml IL-4, and increasing doses of Ova_KbG. Cells were then scraped off the plate and CD8+ cells were purified with magnetic beads. Sterile 96-well white plates were used. Luciferase-expressing Panc02 target cells were supplemented with 20 μg / ml Ova peptide (SIINFEKL) and shaken at 500 rpm for 60 min at 37 °C. CD8+ effector T cells as well as luciferin were added at a ratio of 50:1. Luminescence was measured after 0, 24 and 48 hours. [Figure 13]Single-chain MHC Ib molecules induce the expression of IL-10 in EAE-ODC Ova mice. Serum cytokines in EAE-ODC Ova mice were measured using a Th1 / Th2 10plex Flowcytomix Kit (eBioscience) according to the manufacturer's instructions. This kit was used to simultaneously detect mouse granulocyte-macrophage colony-stimulating factor (GMCSF), interleukin 1α (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 α (TNFα) in one sample. Beads coated with 10 different specific capture antibodies were mixed. Then, 25 μL of mixed capture beads, 25 μL of unknown serum sample or standard dilution, and 25 μL of phycoerythrin (PE) detection reagent were added consecutively to each well of a 96-V bottom well plate and incubated for 2 h at room temperature in the dark. Samples were washed with 1 mL of wash buffer for 5 min and centrifuged. After removing the supernatant, the bead pellet was resuspended in 200 μL buffer. Samples were measured on an Attune™ NxT flow cytometer and analyzed with Attune Cytometric Software (Thermo Fisher Scientific). [Figure 14] 1 is a graphical representation showing the development process of preferred polypeptide constructs of the present invention. [Figure 15]Temperature shift assay. For temperature shift assay (TSA), 3 μg of each single-chain MHCIb molecule was diluted with PBS and 5x SYPRO Orange dye (stock 5000x, final concentration: 5x) to a volume of 25 μl. The melting curve program was set up on the StepOnePlus Instrument using StepOnePlus Software 2.3. The starting temperature was 25°C for 1 min, then the temperature was increased at 1°C per min to a final temperature of 95°C over 2 min, while measuring autofluorescence throughout the process. The data was exported and graphs were drawn in Prism V7.04. The melting temperature (Tm) was determined by Boltzmann's sigmoid function. A high melting temperature indicates good protein stability for therapeutic use. [Figure 16-1] Stability of the polypeptide constructs of the invention (A, B). sc HLA-A2 or A11α2 HLA-Gα3 constructs containing the indicated peptides were purified by spot cap purification according to the manufacturer's protocol followed by ON dialysis. Constructs that underwent three freeze-thaw cycles were then heated to 50°C for 30 min or kept at room temperature for 6 days. Degradation was then analyzed on a Coomassie genl (12% gel, 4% stacking gel, 1 μg purified protein, non-reducing conditions, 15 μl sample + 5 μl of 4x Laemmli). Roti Mark TRICOLOR XTRA (Roth, 2244) was used for Coomassie staining for 5-6 h. Destaining was performed with 12.5 ml methanol + 37.5 ml H2O dest for 5 min and another round of destaining was performed with H2O dest. [Figure 16-2]Stability of the polypeptide constructs of the invention (A, B). sc HLA-A2 or A11α2 HLA-Gα3 constructs containing the indicated peptides were purified by spot cap purification according to the manufacturer's protocol followed by ON dialysis. Constructs that underwent three freeze-thaw cycles were then heated to 50°C for 30 min or kept at room temperature for 6 days. Degradation was then analyzed on a Coomassie genl (12% gel, 4% stacking gel, 1 μg purified protein, non-reducing conditions, 15 μl sample + 5 μl of 4x Laemmli). Roti Mark TRICOLOR XTRA (Roth, 2244) was used for Coomassie staining for 5-6 h. Destaining was performed with 12.5 ml methanol + 37.5 ml H2O dest for 5 min and another round of destaining was performed with H2O dest. [Figure 17-1] ELISpot results from PBMCs of healthy donors. Induction of regulatory IL-10 secreting cells by the indicated constructs of the invention over a 14 day period in PBMCs from healthy donors is shown. [Figure 17-2] ELISpot results from PBMCs of healthy donors. Induction of regulatory IL-10 secreting cells by the indicated constructs of the invention over a 14 day period in PBMCs from healthy donors is shown. [Figure 18-1] ELISpot results of IL-10 from Parkinson's disease patients (A) and healthy / age-matched individuals (B). Induction of regulatory IL-10 secreting cells by the indicated constructs of the invention and age-matched healthy donors (C1-C3) or normal healthy donors (B10, B12, B13) over a 14 day period is shown. [Figure 18-2] ELISpot results of IL-10 from Parkinson's disease patients (A) and healthy / age-matched individuals (B). Induction of regulatory IL-10 secreting cells by the indicated constructs of the invention and age-matched healthy donors (C1-C3) or normal healthy donors (B10, B12, B13) over a 14 day period is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0064] Detailed Description of the Invention Definitions and General Techniques Unless otherwise defined below, the terms used in the present invention shall be understood according to the general meaning known to those skilled in the art. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. Publications mentioned herein may be cited by specifying the full literature reference in the text, or by citing the first author and publication date of the document (e.g., "Brochard V et al., 2009"), followed by citing the corresponding full literature in the "References" section of this specification.

[0065] All proteins of the present invention, including recombinant polypeptides of the present invention, can be obtained by methods known in the art. Such methods include the method of producing recombinant polypeptides. Recombinant polypeptides of the present invention can be expressed in recombinant host cells according to the present invention. Recombinant host cells of the present invention are preferably mammalian cells, such as CHO cells and HEK cells.

[0066] It will be understood that the recombinant polypeptides of the present invention are intended to optionally include a secretory signal peptide sequence. Similarly, the recombinant polypeptides of the present invention are intended to optionally include an affinity tag, e.g., to facilitate purification, and also to optionally include a protease cleavage site between the tag and the polypeptide, e.g., to facilitate removal of the tag by protease cleavage.

[0067] It is also understood that any reference to amino acid sequences referred to herein is intended to encompass not only the unmodified amino acid sequences but also typical post-translational modifications of these amino acid sequences (e.g., glycosylation or deamidation of amino acids, clipping of specific amino acids, or other post-translational modifications) that occur in cellular expression systems known in the art, including mammalian cells such as CHO cells and HEK cells.

[0068] Likewise, it will be understood that the recombinant polypeptides of the present invention are intended to optionally include the respective propeptides.

[0069] It will also be understood that the recombinant polypeptide of the present invention may be in soluble or 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 standard conditions indicated above. As used herein, soluble means that at least 95% of the recombinant polypeptide is determined to be soluble under these standard conditions.

[0070] 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.

[0071] According to the present invention, the MHC molecule is preferably a human MHC molecule.

[0072] The recombinant polypeptide of the present invention is preferably an isolated recombinant polypeptide.

[0073] It will be understood how recombinant polypeptides capable of binding and presenting the peptide antigens of the present invention can be prepared. For example, peptide antigen binding domains such as [α]1 and [α]2 domains are well known and modifications of these domains can be made. The ability of the polypeptides of the present invention and peptide antigens to bind to MHC molecules can be determined by techniques known in the art, including but not limited to exploratory methods such as mass spectrometry after MHC peptide elution and in silico bioinformatics prediction, and confirmatory methods such as MHC peptide multimer binding and stimulation assays.

[0074] According to the present invention, the recombinant polypeptides, pharmaceutical compositions and kits of the invention are preferably suitable for use in human patients.

[0075] According to the present invention, the recombinant polypeptides, pharmaceutical compositions and kits of the present invention are preferably suitable for use in the treatment of Parkinson's disease in human patients.

[0076] According to the invention, the recombinant polypeptides, pharmaceutical compositions and kits of the invention are preferably suitable for inducing immune tolerance to human α-synuclein, eg in a human patient.

[0077] In accordance with the present invention, it is understood that the recombinant polypeptides, pharmaceutical compositions and kits of the present invention are stable.

[0078] It will be understood that any length of these peptide antigens referred to herein (e.g., "7-11 amino acids long") in relation to the peptide antigens used in accordance with the present invention is intended to refer to the length of the peptide antigen itself. Thus, the length of the peptide antigen referred to herein does not include length contributed by additional amino acids that are not part of the peptide antigen, such as additional amino acids from potential linker sequences, etc.

[0079] In accordance with the present invention, each occurrence of the term "comprising" may optionally be replaced with the term "consisting of."

[0080] Methods and Techniques Generally, unless otherwise defined herein, the methods used in the present invention (e.g., cloning methods or antibody-related methods) are performed according to procedures known in the art, e.g., as described in Sambrook et al. ("Molecular Cloning: A Laboratory Manual.", 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1989), Ausubel et al. ("Current Protocols in Molecular Biology." Greene Publishing Associates and Wiley Interscience; New York 1992), and Harlow and Lane ("Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1988), all of which are incorporated herein by reference.

[0081] Protein-protein binding, such as the binding of antibodies to their respective target proteins, can be assessed by methods known in the art. Protein-protein binding is preferably assessed by surface plasmon resonance spectroscopy.

[0082] For example, the binding of MHC class Ib molecules or recombinant polypeptides of the present invention to their receptors, such as ILT2 and ILT4, is preferably assessed by surface plasmon resonance spectroscopy. More preferably, the binding of MHC class Ib molecules or recombinant polypeptides of the present invention to their receptors is assessed by surface plasmon resonance at 25° C. Suitable conditions for such surface plasmon resonance measurements are described in Shiroishi et al., Proc Natl Acad Sci US A. 2003 July 22;100(15):8856-8861.

[0083] Sequence alignment of the sequences of 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). Suitable parameters for sequence alignment of short peptides by the BLAST algorithm, suitable for the peptide antigens of the present invention, are known in the art. Most software tools using the BLAST algorithm automatically adjust the parameters for sequence alignment of short input sequences. In one embodiment, the following parameters are used: Max target sequences 10; Word size 3; BLOSUM 62 matrix; gap costs; existence 11, extension 1; conditional compositional score matrix adjustment. Thus, when used in the context of sequences, terms such as "identity" or "identical" preferably refer to the identity value obtained using the BLAST algorithm.

[0084] Preparation of the Pharmaceutical Composition of the Invention The pharmaceutical compositions of the present invention are prepared in accordance with known standards for the preparation of pharmaceutical compositions.

[0085] For example, pharmaceutical compositions are prepared so that they can be appropriately stored and administered. Thus, the pharmaceutical compositions of the present invention may contain pharma- ceutical acceptable components, such as carriers, excipients, and / or stabilizers.

[0086] Such pharma- ceutically acceptable ingredients are non-toxic in the amounts used when the pharmaceutical composition is administered to a human patient. The pharma- ceutically acceptable ingredients added to a pharmaceutical composition may depend on the chemical nature of the active ingredients present in the composition, the particular intended use of the pharmaceutical composition, and the route of administration.

[0087] Generally, pharma- ceutically acceptable ingredients used in connection with the present invention are used according to the knowledge available in the art, e.g., information from Remington's Pharmaceutical Sciences, Ed. AR Gennaro, 20th edition, 2000, Williams & Wilkins, PA, USA. Pharmaceutical compositions containing the nucleic acids (e.g., RNA) of the present invention can also be formulated according to the knowledge available in the art, e.g., using liposomal formulations that target dendritic cells.

[0088] Peptide antigens of the present invention Peptide antigens that can be used according to the present invention, including those defined above, are not particularly limited except by their ability to be presented on MHC molecules. The "peptide antigen presented by said recombinant polypeptide" referred to in the context of the present invention is understood to be a peptide antigen that is presented by said recombinant polypeptide to human T cells in a form that binds to a T cell receptor on the human T cells, if human T cells are present.

[0089] Peptides that can be presented on MHC molecules can be generated as known in the art (see, e.g., 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).

[0090] Peptide antigens are widely known in the art.Generally, the peptide antigen of the present invention can bind to MHC class I protein.Those skilled in the art will understand that for each MHC class Ib molecule or polypeptide that can present the peptide of the present invention, preferably, peptide antigens that can bind to said MHC class Ib molecule or recombinant polypeptide are used.These peptide antigens can be selected based on methods known in the art.

[0091] Binding of a peptide antigen to an MHC class Ib molecule, or to a polypeptide capable of binding a peptide antigen according to the invention, can be assessed by methods known in the art, for example the following method: 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.

[0092] Such methods include experimental methods and methods that predict peptide-antigen binding.

[0093] Anchor residues that serve to anchor peptide antigens onto MHC class I molecules and ensure binding of peptide antigens to MHC class I molecules are known in the art.

[0094] In a preferred embodiment consistent with all embodiments of the invention, the peptide antigens used in accordance with the invention contain either anchor residues or preferred amino acid residues at positions predicted for MHC class I molecules.

[0095] Such prediction can preferably be performed as described in any 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.

[0096] In the present invention, the peptide antigen is derived from human alpha-synuclein.

[0097] It is understood that the non-anchor amino acid residues of the peptide antigens of the present invention may or may not contain conservative substitutions, preferably no more than two conservative substitutions, more preferably one conservative substitution, with respect to the corresponding amino acid sequence of the peptide antigen derived from human alpha-synuclein.

[0098] The peptide antigens of the invention are preferably composed of naturally occurring amino acids. However, non-naturally occurring amino acids can also be used, as can modified amino acids. For example, in one embodiment, the peptide antigens of the invention include peptidomimetics of the indicated peptide antigen amino acid sequence of human α-synuclein.

[0099] Methods for the synthesis of peptidic antigens, including those of the present invention, are well known in the art.

[0100] array Preferred amino acid sequences referred to in this application can be independently selected from the following sequences: The sequences are represented in N-terminal to C-terminal order; in addition, the sequences are represented in the one-letter amino acid code.

[0101] Examples of sequences which are part of the recombinant polypeptides of the invention: Leader peptide (which is deleted from the recombinant polypeptide due to processing during cellular expression): e.g., MSRSVALAVLALLSLSGLEA (SEQ ID NO: 1) Peptide antigen: any MHC class I peptide corresponding to the MHC class I [α] 1 and 2 domains, e.g., GAPQEGIL (SEQ ID NO: 20) First linker: for example, GGGGSGGGSGGGGS (SEQ ID NO: 3) or GCGASGGGGSGGGGS (SEQ ID NO: 4) β2 microglobulin, for example: IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 5, human beta 2 microglobulin) A second linker, for example: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 6) [α]1 and 2 domains derived from either human HLA-G, or any other MHC class I [α]1 and 2 domains suitable for presenting a selected antigenic peptide, Y84 may be C in the DT variant For example, the [α]1 and 2 domains from human HLA-G: GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRA (SEQ ID NO: 7) Or: human HLA-A2[α] 1 and 2 domains: e.g. GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGCYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRT (SEQ ID NO: 8) Human HLA-G[α]3 domain (or any MHC Ib[α]3 domain, such as HLA-F, which also interacts with the ILT2 and ILT4 receptors), for example: DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDL (SEQ ID NO: 9; sequence of HLA-G[alpha]3).

[0102] Of note, the following underlined amino acids in this sequence are relevant for ILT2 or ILT4 receptor interaction: TIFF2025514872000001.tif14158

[0103] Alternatively, a truncated human HLA-G[α]3 domain may be used, lacking the optional C-terminal amino acid sequence from intron 4 (SKEGDGGIMSVRESRSLSEDL; SEQ ID NO: 47), i.e.: DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO: 2), Factor Xa restriction site: IEGRTGTKLGP (SEQ ID NO: 10) SpotTag: PDRVRAVSHWSSC (SEQ ID NO: 11) Myc tag: EQKLISEEDL (SEQ ID NO: 12) His tag: HHHHHH* (SEQ ID NO: 13) Spacer sequence: for example NSAVD (SEQ ID NO: 14) or GS.

[0104] Further (alternative) examples of peptide antigens which can be part of the recombinant polypeptide of the invention are:

[0105] Most preferred: Preferably, GAPQEGIL (SEQ ID NO: 20) is used in a recombinant polypeptide containing human HLA-G[α]1 and 2 domains. Preferably, KTKEGVLYV (SEQ ID NO: 28) is used in a recombinant polypeptide containing human HLA-A2[α]1 and 2 domains. Preferably, AVVTGVTAV (SEQ ID NO: 33) is used in a recombinant polypeptide containing human HLA-A2[α]1 and 2 domains. Preferably, GVVHGVTTV (SEQ ID NO: 31) is used in a recombinant polypeptide containing human HLA-A2 [α] 1 and 2 domains. Preferably, MDVFMKGLSK (SEQ ID NO: 22) is used in a recombinant polypeptide containing the human HLA-A11 [α] 1 and 2 domains. Preferably, GVVAAAEKTK (SEQ ID NO: 25) is used in a recombinant polypeptide containing the human HLA-A11 [α] 1 and 2 domains.

[0106] preferable: Preferably, MPVDPDNEAY (SEQ ID NO: 21) is used in a recombinant polypeptide containing human HLA-A1 [α] 1 and 2 domains. Preferably, PVDPDNEAY (SEQ ID NO: 44) is used in a recombinant polypeptide containing the human HLA-A1 [α] 1 and 2 domains. Preferably, SIAAATGFV (SEQ ID NO: 37) is used in a recombinant polypeptide containing human HLA-A2[α]1 and 2 domains. Preferably, VVTGVTAVA (SEQ ID NO: 34) is used in a recombinant polypeptide containing human HLA-A2[α]1 and 2 domains. Preferably, VVAAAEKTK (SEQ ID NO: 26) is used in a recombinant polypeptide containing the human HLA-A11 [α] 1 and 2 domains. Preferably, VFMKGLSKAK (SEQ ID NO: 24) is used in a recombinant polypeptide containing human HLA-A11 [α] 1 and 2 domains. Preferably, AAATGFVKKD (SEQ ID NO: 42) is used in a recombinant polypeptide containing the human HLA-A11 [α] 1 and 2 domains. Preferably, AATGFVKK (SEQ ID NO: 43) is used in a recombinant polypeptide containing the human HLA-A11 [α] 1 and 2 domains.

[0107] Examples of recombinant polypeptides of the present invention (without leader peptide): GAPQEGILGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM GGGGSGGGGSGGGGSGGGGSGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGCYNQSEDGSHTIQIMYGCDV GPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC* (SEQ ID NO: 15; note that the asterisk represents the position of the stop codon). It should be noted here that the sequence of the peptide antigen of the full-length protein (here: GAPQEGIL) can be replaced with any peptide antigen sequence of the present invention, i.e., any peptide antigen presented by the recombinant polypeptide, which is a peptide of human α-synuclein. That is, the recombinant polypeptide of the present invention can be composed of a sequence consisting of a peptide antigen that is a peptide of human α-synuclein (e.g., any one of the peptide antigens of SEQ ID NOs: 20 to 46), followed by the following sequence: GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGSG GGGSGGGGSGGGGSGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGCYNQSEDGSHTIQIMYGCDVGPDG RFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC* (SEQ ID NO: 16; note that the asterisk represents the position of the stop codon).

[0108] The receptors ILT2 (also known as LILRB1) and ILT4 (also known as LILRB2) are known in the art. Preferred sequences of these receptors of the invention are as follows: ILT2: (SEQ ID NO:17)

[0109] ILT4: (SEQ ID NO:18)

[0110] The sequence of human alpha-synuclein is known in the art. A preferred human alpha-synuclein is as follows: MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA (SEQ ID NO: 19)

[0111] Therapeutic Applications of the Invention: Parkinson's disease (PD), including prodromal Parkinson's disease (prodromal PD), and its classifications are known in the art.

[0112] Prodromal PD refers to 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 indicate a higher than average risk of developing motor symptoms in the future and a diagnosis of PD. Many prodromal symptoms are non-motor and greatly affect the quality of life of both patients with prodromal PD and those whose disease stage has progressed to motor PD. Thus, early detection and treatment of these prodromal symptoms is essential for high quality care. The best-characterized symptoms of prodromal PD include anosmia, constipation, mood disorders, and REM sleep behavior disorder (RBD).

[0113] The present invention is further illustrated by the following non-limiting examples: EXAMPLES

[0114] Reference example: Screening for target antigens in mice To screen for suitable target antigens, mice injected with haSyn or control virus were sacrificed. Immune cells were isolated from the brain, peripheral lymph nodes and spleen. These cells were then incubated with in silico predicted (e.g., by NetMHC) A53T-aSyn peptides. By screening aSyn-specific immune responses in C57BL / 6 mice, we identified MHC I and MHC II restricted haSyn peptides that induced IFN-γ release from T cells of diseased mice (Figure 3). T cell reactivity against haSyn peptides was also shown for T cells derived from cervical lymph nodes and brain.

[0115] Example 1 Prevention of neuronal cell death in in vivo models of PD Given the important role of T cells in neurodegenerative processes (as demonstrated by depletion / reconstitution experiments), the inventors reasoned that this haSyn PD mouse model, which closely resembles the human disease, is ideally suited to evaluate the efficacy of immune modulation in the context of aSyn-based pathology such as PD and to evaluate the suitability of recombinant polypeptides of the invention for the treatment of PD.

[0116] The inventors further determined that induction of immune tolerance to aSyn induces tissue-wide protection through local antigen-specific activation of Tregs. After identifying the A53T-aSyn-derived aSyn 68-78 peptide as immunogenic in mice, the inventors developed a recombinant polypeptide of the invention that corresponds to this peptide antigen, which is immunogenic in mice, as well as mouse H2-D. b A recombinant polypeptide carrying the α1 and α2 domains was developed (hereafter referred to as the “mouse AIM Bio surrogate molecule” or “AIM Bio”). aSyn In pilot studies, haSyn PD mice were treated with 0.5 μg / g AIM Bio aSyn or 2 μg / g AIM Bio aSyn or an off-target irrelevant 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, traditionally designated Gp33, binds to H2-K b The epitope presented above was later found to contain only amino acids 34-41 (the epitope beginning at amino acid 33, in contrast, is not associated with H2-K d Therefore, we follow the most recent recommendations and use H2-K bThe epitope is referred to as Gp34. Furthermore, there is ambiguous use of the nomenclature of Gp33 and Gp34 in the literature. The sequence of the peptide 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 with Gp34_KbG had lost approximately 45% of nigral neurons compared to animals injected with a non-pathogenic empty vector control (EV). Surprisingly, this neuronal loss was consistent with both low (0.5 μg / g) and high (2 μg / g) doses of AIM Bio aSyn In addition, the inventors demonstrated that aSyn-loaded AIM Bio surrogate molecule inhibited the uptake of CD11b into the striatum in a PD model. + We found that aSyn-loaded AIM Bio surrogate molecules prevented myeloid cell infiltration (Figure 5), reduced aSyn accumulation in the substantia nigra in PD models (Figure 6), and aSyn-loaded AIM Bio protected tyrosine hydroxylase+ fibers in the striatum (Figure 7).

[0117] Example 2 aSyn-specific MHC class I-based therapeutics for the treatment of PD in human patients Our findings indicate that single-chain proteins containing aSyn peptide antigen and HLA-Gα3 domain can largely prevent neuronal cell death in animal models of Parkinson's disease, i.e., they can be used for the treatment of PD. Based on these findings, we have generated aSyn peptide and MHC class I fusion molecules for therapeutic use in human PD patients.

[0118] Target sequence (human α-synuclein): MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTK EQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSE EGYQDYEPEA (SEQ ID NO: 19)

[0119] Related peptides: GAPQEGIL (SEQ ID NO:20) MPVDPDNEAY (SEQ ID NO:21) MDVFMKGLSK (SEQ ID NO:22) DVFMKGLSK (SEQ ID NO:23) VFMKGLSKAK (SEQ ID NO:24) GVVAAAEKTK (SEQ ID NO:25) VVAAAEKTK (SEQ ID NO:26) GVAEAAGKTK (SEQ ID NO:27) KTKEGVLYV (SEQ ID NO:28) GVLYVGSKTK (SEQ ID NO:29) VLYVGSKTK (SEQ ID NO:30) GVVHGVTTV (SEQ ID NO:31) (based on the A53T mutant) GVTTVAEKTK (SEQ ID NO:32) (based on the A53T mutant) AVVTGVTAV (SEQ ID NO:33) VVTGVTAVA (SEQ ID NO:34) VTGVTAVAQK (SEQ ID NO:35) GSIAAATGF ​​(SEQ ID NO:36) SIAAATGFV (SEQ ID NO:37) SIAAATGFVK (SEQ ID NO:38) IAAATGFVK (SEQ ID NO:39) GSIAAATGFVK (SEQ ID NO:40) AAATGFVKK (SEQ ID NO:41) AAATGFVKKD (SEQ ID NO:42) AATGFVKK (SEQ ID NO:43) PVDPDNEAY (SEQ ID NO:44) MPSEEGYQDY (SEQ ID NO:45) PSEEGYQDY (SEQ ID NO:46)

[0120] Most preferred: SIAAATGFV (SEQ ID NO: 37) + HLA-A2 presentation domain KTKEGVLYV (SEQ ID NO:28) + HLA-A2 presentation domain AVVTGVTAV (SEQ ID NO:33) + HLA-A2 presentation domain GVVHGVTTV (SEQ ID NO:31) + HLA-A2 presentation domain VVTGVTAVA (SEQ ID NO: 34) + HLA-A2 presentation domain GSIAAATGFVK (SEQ ID NO: 40) + HLA-A11 presentation domain AAATGFVKK (SEQ ID NO: 41) + HLA-A11 presentation domain VLYVGSKTK (SEQ ID NO:30) + HLA-A11 presentation domain

[0121] preferable: MPVDPDNEAY (SEQ ID NO:21) + HLA-A1 presentation domain PVDPDNEAY (SEQ ID NO: 44) + HLA-A1 presentation domain VVAAAEKTK (SEQ ID NO: 26) + HLA-A11 presentation domain VFMKGLSKAK (SEQ ID NO:24) + HLA-A11 presentation domain AAATGFVKKD (SEQ ID NO: 42) + HLA-A11 presentation domain AATGFVKK (SEQ ID NO: 43) + HLA-A11 presentation domain MDVFMKGLSK (SEQ ID NO: 22) + HLA-A11 presentation domain GVVAAAEKTK (SEQ ID NO: 25) + HLA-A11 presentation domain GAPQEGIL (SEQ ID NO: 20) + HLA-G presentation domain

[0122] The most preferred and preferred peptides indicated above are preferred because the inventors have found that these epitopes are highly likely to induce a tolerogenic effect in Parkinson's patients based on online prediction algorithms (Syfpeithi, NetMHC), literature review of immunodominant regions, 3D protein folding predictions (αFold2) and protein expression data as well as the experimental results reported herein.

[0123] Example 3 Methods used in the present invention Methods for Producing Recombinant Polypeptides of the Invention Expi-293F cells (Thermo Fisher), grown in Expi-293™ Expression Medium (Thermo Fisher): for DNA complex formation with Expifectamine, Opti-MEM (Thermo Fisher) was used, and 1 μg DNA was transfected at 2.5 × 10 6 Transfect cells / ml, add enhancer according to the protocol after 18-20 hours, and collect supernatant after 4-6 days (37°C, 8% CO 2 , humidified incubator), 19mm 2 Orbital shaker 125 rpm.

[0124] Purification of Spot-Tag proteins: Equilibration of Spot-Cap resin: Transfer desired amount of slurry to appropriate tube, pellet beads by centrifugation (4°C, 4 min, 2500g), remove and discard supernatant, add 10 bed volumes of PBS (chilled) to beads, mix by inversion, pellet beads by centrifugation (4°C, 4 min, 2500g), remove and discard supernatant, repeat twice.

[0125] Add the required volume of beads to the supernatant, incubate overnight at 4° C. on a rotator, wash the beads by repeated centrifugation (4° C., 4 min, 2500 g), and remove the supernatant.

[0126] Prepare 500 μM Spot peptide solution in PBS, remove supernatant, incubate with ⅓ volume of Spot peptide solution for 5-10 min.

[0127] The beads are pelleted by centrifugation. Proteins are concentrated and Spot peptides are removed using Amicon Ultra-4 centrifugal filters (15 kDa cutoff) over a 15 kDa Amicon cutoff column.

[0128] Amicon Ultra-4 centrifugal filters (15 kDa cutoff) are rinsed with PBS followed by 0.1 N NaOH (centrifugation at 4000 g, 4° C.) to remove traces of glycerol.

[0129] ELISPOT: 1) Cell culture A) PBMC isolation (under laminar flow hood) To isolate peripheral blood mononuclear cells (PBMCs), density gradient centrifugation was performed using leukocytes from the leukapheresis chamber and density gradient medium (e.g. Ficoll, or ROTI Sep 1077). Cells were centrifuged at 1200xg for 20 min with the brake off, after which the interphase ring was collected and washed with 1x PBS (5 min, 300xg). PBMCs were stored frozen until further use.

[0130] B) PBMC pulsing (under laminar flow hood) PBMCs were thawed one day (d-1) prior to PBMC pulsing and placed in wells of a 6-well plate in 5 ml of X-VIVO 15 medium containing 5% human AB serum at 37° C. overnight.

[0131] The next day (d0), cells were counted and cultured at 3 × 10 in X-VIVO 15 complete medium (5% hAB serum and cytokine cocktail: 20 ng / ml hIL-2, 20 ng / ml hGM-CSF, 10 ng / ml hIL-4 and 10 ng / ml hTGF-b1). 6The cells were resuspended at a cell density of 1000 cells / ml.

[0132] For the experiments, 3×10 cells were plated into each well of a 12-well plate in a final volume of 1000 μl of X-VIVO complete medium (containing a cytokine cocktail and 5 μg / ml of AIM Biomolecule (recombinant polypeptide of the invention) or respective control). 6 cells were seeded.

[0133] On day 3, 1 ml of complete medium (containing cytokines) was added, and on day 6, a second pulse treatment was performed (after removing the medium) with 5 μg / ml of a recombinant polypeptide of the invention or its surrogate (collectively referred to as "AIM Bio" molecules). On days 7, 10, and 12, 1 ml of complete medium (containing cytokines) was added.

[0134] What you'll need: X-VIVO 15 medium + 5% human AB serum X-VIVO 15 complete medium: X-VIVO 15 medium + 2% human AB serum (containing cytokine cocktail): 10ng / ml TGF-b1, 10ng / ml IL-4, 20ng / ml IL-2, 20ng / ml GM-CSF 6-well plates 12 well plate.

[0135] 2) ELISPOT Laminar flow hood On day 13, ELISPOT plates were coated with anti-hIL10 (clone 9D-7, 1:500 dilution in PBS, sterile filtered) and aIL10 (10G8-biotin), and on day 14, 200,000 cells were plated per well in duplicate on ELISPOT plates, including a negative control (cells + PBS) and a positive control (e.g., LPS).

[0136] PFDF membranes were activated with 50 μl / well EtOH (35% v / v) for 1 min and then washed 5 times with 200 μl sterile distilled water. Plates were coated overnight at 4 °C with 100 μl / well antibody solution. The next day, unbound coating antibody was removed and after 5 washing steps with 200 μl PBS, 200 μl blocking buffer (X-VIVO 15 5% hAB serum) was added and plates were incubated at room temperature for 30 min to 2 h.

[0137] The respective antigen peptides in DMSO, or DMSO as control, were prepared and a final amount of 5 μg peptide / ml was added to a final volume of 100 μl / well. 150,000 cells were seeded per well in X-VIVO 15 medium containing 5% human AB serum. The blocking buffer (X-VIVO 15 medium + 5% human AB serum) was carefully removed and medium containing PBS as a negative control and stimuli (5 μg / ml total per well) were added to other wells and incubated at 37°C overnight.

[0138] Outside the laminar flow hood Secondary antibodies were prepared: 1 μg / ml aIL-10 biotinylated antibody (1:1000 dilution) in 0.5% BSA / 1x PBS and horseradish peroxidase-conjugated streptavidin (1:750 in 0.5% BSA / PBS), tetramethylbenzidine solution was filtered using a 0.45 μm filter and stored at 4° C. until use.

[0139] The cell supernatant was removed and the cells were washed five times with 100 μl PBS, and the final excess buffer was removed with a paper towel.

[0140] 25 μl of diluted HRP-streptavidin (1:750) was added per well and incubated for 1 hour at room temperature in the dark, followed by 5 washing steps with sterile 1×PBS.

[0141] 100 μl of filtered TMB substrate was added per well and allowed to sit for 15-25 min until blue spots developed. The reaction was stopped by thoroughly washing the wells with tap water.

[0142] The plastic underdrain of the plate was removed and the bottom and sides of the plate were washed with tap water and dried.

[0143] Plates were read using an ImmunoSpot S6 Ultra-V Analyzer (Cellular Technology Limited), analyzed in Excel, and graphs / statistics were performed in Graphad Prism.

[0144] What you'll need: Capture antibodies: anti-hIL10 (clone: ​​9D-7, Mabtech #3430-3-250; 1:500 dilution), anti-hIL10 biotinylated (Mabtech #3430-6-250) 1x PBS (sterile) 35% EtOH (v / v) Blocking buffer: X-vivo 5% hAB serum (sterile) [blocking is performed in the same medium as cell culture] Dilution buffer: 0.5% BAS in PBS Washing buffer: 1x PBS Culture medium: For T cells, X-VIVO 15 medium (Lonza) Filter syringe: Millex GV ELISPOT PVDF plate (#MSIP4510, Millipore) TMB substrate.

[0145] Example 4: Further proof of principle regarding the stability and efficacy of the recombinant polypeptides of the invention. Furthermore, the inventors set out to obtain and test recombinant polypeptides having the general structure of the recombinant polypeptides of the present invention but containing a variety of different peptide antigens to further provide proof of principle that the recombinant polypeptides of the present invention and their surrogates are stable and effective. As shown in Figures 9 and 10, respectively, the tested recombinant polypeptides are stable during freeze-thaw and storage, and are heat stable. Furthermore, these recombinant polypeptides induce Tregs in a dose-dependent manner (Figure 11) and inhibit T cell lysis in a dose-dependent manner (Figure 12). The effect of the recombinant polypeptides on serum cytokine profile in EAE-ODC Ova mice is shown in Figure 13. There is an induction of IL-10, and possibly IL-4, both of which are known immunosuppressive cytokines that downregulate immune responses in an inflammatory environment. This requires the HLA-Gα3 domain and the cognate peptide. IL-2 appears to be induced in response to presentation of the cognate peptide to cells, independent of the α3 domain. IL-2 is required for T cell activation and survival.

[0146] Further evidence of the stability of the recombinant polypeptides of the present invention is also provided in FIGS.

[0147] Example 5 ELISpot results from PBMCs of healthy donors (Figure 17) and IL-10 ELISpot results from Parkinson's patients (Figure 18A) and healthy / age-matched individuals (Figure 18B). These experiments show that a significant induction of immunosuppressive cells in response to the constructs of the invention can be observed already after 14 days in both PBMCs of healthy donors and patients.

[0148] (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.

[0149] Industrial Applicability The pharmaceutical compositions, polypeptides, nucleic acids, cells and products used in the present invention can be industrially applicable, for example, they can be used in the manufacture of medicines or as medicines.

Claims

1. A recombinant polypeptide capable of presenting a peptide antigen, the recombinant polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen presented by the recombinant polypeptide, wherein the peptide antigen is a peptide of human alpha-synuclein; ii) optionally, a linker sequence; iii) optionally a sequence of a human polypeptide domain comprising the sequence of human β2 microglobulin, or an amino acid sequence that is at least 90% identical to the amino acid sequence of human β2 microglobulin represented by SEQ ID NO: 5; iv) optionally, a linker sequence; v) optionally the [α]1 domain of an MHC molecule; vi) optionally, the [α]2 domain of an MHC molecule; vii) an [α]3 domain of an MHC class Ib molecule, or a derivative of an [α]3 domain of an MHC class Ib molecule, which derivative is capable of binding to ILT2 or ILT4; viii) optionally, a protease cleavage site; ix) optionally, a spacer sequence; and x) Possibly affinity tags The recombinant polypeptide comprising:

2. The recombinant polypeptide of claim 1, wherein the peptide antigen according to i) is 7 to 11 amino acids in length, preferably 8 to 10 amino acids in length.

3. 2. The recombinant polypeptide of claim 1, 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 NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:

46.

4. The recombinant polypeptide of claim 1, wherein the [α]1 domain described in (v) and the [α]2 domain described in (vi) are derived from a human MHC class Ia molecule or a human MHC class Ib molecule.

5. The recombinant polypeptide of claim 4, wherein the [α]1 domain described in (v) and the [α]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.

6. The recombinant polypeptide of claim 5, wherein the [α]1 domain described in (v) and the [α]2 domain described in (vi) are derived from a human MHC class Ia molecule, and the [α]1 domain described in (v) and the [α]2 domain described in (vi) are derived from a human HLA-A2 molecule, a human HLA-A1 molecule, or a human HLA-A11 molecule.

7. A recombinant polypeptide according to claim 1, wherein the [α]3 domain of the MHC class Ib molecule described in (vii) is the [α]3 domain of human HLA-E, human HLA-F or human HLA-G, preferably the [α]3 domain of human HLA-G.

8. The recombinant polypeptide of claim 1, wherein the [α]3 domain or derivative described in (vii) is identical to an [α]3 domain having the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:2, 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 identity, or at least 99% amino acid sequence identity, or is identical to an [α]3 domain having the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:

2.

9. 2. The recombinant polypeptide of claim 1, wherein the linker sequence described in (ii) and / or the linker sequence described in (iv) comprises the amino acid sequence (GGGGS)n, where n is an integer greater than or equal to 1, and n is preferably an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, preferably selected from the group consisting of 2, 3, 4 and 5.

10. 2. The recombinant polypeptide of claim 1, 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.

11. The recombinant polypeptide of claim 1, wherein the polypeptide is a dimer or a multimer.

12. 2. The recombinant polypeptide of claim 1, wherein the polypeptide comprises or consists of: all of components i) to vii); no components viii) to x); or comprises or consists of all of components i) to x).

13. The recombinant polypeptide of claim 1, further comprising an N-terminal secretory signal peptide sequence.

14. The recombinant polypeptide comprises, in order from N-terminus to C-terminus, the following ((a) and (b)): (a) a peptide antigen selected from the group consisting of the amino acid sequences of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46; and (b) the amino acid sequence of SEQ ID NO: 16 2. The recombinant polypeptide of claim 1, comprising an amino acid sequence consisting of:

15. The recombinant polypeptide of claim 1 , wherein the recombinant polypeptide is soluble.

16. 2. A nucleic acid encoding one or more polypeptides according to claim 1, wherein the nucleic acid is preferably a vector.

17. A pharmaceutical composition or kit comprising at least one nucleic acid according to claim 16.

18. A pharmaceutical composition or kit comprising at least one recombinant polypeptide according to claim 1.

19. 19. The pharmaceutical composition or kit of claim 18, wherein the pharmaceutical composition or kit comprises at least two different recombinant polypeptides according to claim 1, each of the different polypeptides comprising a different peptide antigen as defined in claim 3.

20. 18. A pharmaceutical composition or kit according to claim 17 for use in treating Parkinson's disease in a human patient.

21. 21. The pharmaceutical composition or kit for use according to claim 20, wherein the Parkinson's disease is prodromal Parkinson's disease.

22. 21. The pharmaceutical composition or kit for use according to claim 20, wherein the treatment is treatment of Parkinson's disease by immunotherapy, preferably by inducing immune tolerance to the autoantigenic human alpha-synuclein.

23. A recombinant host cell comprising the nucleic acid or vector of claim 16 and expressing the recombinant polypeptide of claim 1.

24. A method for obtaining a pharmaceutical composition comprising a polypeptide described in claim 1, the method comprising the steps of: (a) culturing a recombinant host cell that expresses the recombinant polypeptide described in claim 1, the recombinant host cell comprising a nucleic acid encoding one or more polypeptides described in claim 1, under conditions that allow expression of the recombinant polypeptide from the nucleic acid molecule; (b) recovering the recombinant polypeptide; (c) purifying the recombinant polypeptide; and (d) formulating the recombinant polypeptide into a pharmaceutical composition.

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