MHC 1b-mediated alpha-synuclein-specific tolerance induction as novel therapeutic method 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 invention also relates to methods of producing such recombinant polypeptides, pharmaceutical compositions comprising such recombinant polypeptides, and their use for the treatment of Parkinson's disease.

Description

Field of the Invention

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

[0002] background

[0003] Parkinson's disease (PD) is the most common neurodegenerative movement disorder, affecting more than 7 million people worldwide, and there is currently no cure. Patients suffer from severe motor and non-motor symptoms such as tremor, bradykinesia, rigidity, postural instability, and depression. The pathological disease is characterized by the degeneration of dopaminergic neurons in the substantia nigra and the formation of protein aggregates containing α-synuclein (called Lewy bodies). Although the physiological role of α-synuclein (aSyn) points to the function of synaptic transmitter release and immune response amplification (Alam et al, Cell Reports 38:110090.doi:10.1016 / j.celrep.2021.110090), the pathogenic role of insoluble α-synuclein remains a mystery. Studies have found that duplication and mutation of the aSyn encoding gene SNCA can cause hereditary PD, which closely links aSyn to the pathogenesis of PD.

[0004] Anatomical features of the brain in human PD patients include activation of microglia and infiltration of T cells 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). The ratio of IFN-γ-producing to IL-4-producing T cells in the peripheral blood of PD patients is increased, indicating a systemic shift to a proinflammatory environment (Baba Y et al., 2005). Recently, aSyn-specific T cell responses were detected in peripheral blood cells of PD patients and were found to precede motor symptoms, indicating that the immune system is actively and specifically involved in disease progression. Two aSyn-derived peptides have been shown to trigger the release of MHC-restricted cytokines from peripheral blood monocytes (Sulzer D et al., 2017).

[0005] Currently, PD treatment relies on the use of L-dopamine or its derivatives to compensate for the loss of substantia nigra neurons. This intervention can initially improve disease symptoms but cannot stop the progression of the disease.

[0006] A population-based case-control study (including approximately 48,000 PD patients) analyzed the risk of developing PD after taking immunosuppressants. Patients taking azathioprine had a significantly reduced risk of developing PD (Racette BA et al., 2018). A recent cohort study further showed that patients receiving anti-TNF-α treatment for inflammatory bowel disease had a reduced risk of developing PD (Peter I et al., 2018). Small-scale open-label clinical trials have explored the effects of immunomodulators such as β2-adrenergic receptor agonists or anti-IL-17 therapies, and have achieved partially encouraging results (Magistrelli Land Comi C, 2020; Storelli E et al., 2019).

[0007] The side effects of potent immunosuppressants are a major barrier to trials in patients with early-stage PD, given the often slow-progressing nature of this chronic disease. However, for patients with advanced disease, irreversible neurodegeneration limits the benefits that can be gained by preventing further progression. In contrast, highly targeted immunomodulatory drugs could enable early treatment, thereby maximizing the therapeutic effect.

[0008] To this end, the aSyn-specific antibody cinpanemab (BIIB054) was tested in a clinical trial (https: / / clinicaltrials.gov / ct2 / show / NCT03318523). It was hoped that sequestration of free aSyn by cinpanemab would prevent further aSyn aggregation and thus disease progression. Unfortunately, this study failed to meet its primary or secondary endpoints. Therefore, there remains an urgent need for targeted and effective disease-modifying therapies for PD.

[0009] The pathogenic role of T cells in PD has been mainly analyzed in toxin-based models, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or 6-hydroxydopamine (6-OHDA) models. However, these models do not reflect the molecular aspects of the human disease well due to the lack of aSyn aggregation and Lewy body formation. This prompted Dr. Ip to develop a new PD mouse model that faithfully reproduces the basic features of the human disease. Under stereotactic guidance, the gene encoding human α-synuclein was inserted into the mouse model. A53TUnilateral injection of an AAV1 / 2 vector of (“haSyn”) into the substantia nigra induced behavioral deficits, loss of dopaminergic nigral neurons and striatal fibers, and reduction of striatal dopamine, as well as Lewy body formation (Ip CW et al., 2017). Since the high face validity of this model was achieved by injection of α-synuclein mutants known to be associated with human disease, the pathogenic relevance of this model is also supported by the validity of its disease-relevant constructs. In addition, insoluble aSyn aggregates and Lewy-like neurites and bodies showing S129 phosphorylation can be detected in this animal model (Ip CW et al., 2017). Therefore, the haSyn PD model provides an important tool for advancing preclinical and translational work to develop new and better therapies for PD patients. In a collaborative project, the inventors confirmed the broad pro-inflammatory characteristics of brain immune cells in this haSyn PD model and demonstrated that CD4 + and CD8 + Activated T cell subsets infiltrate the brains of haSyn PD mice. These T cells induce neurodegeneration with PD-like symptoms. - / - ) reduced dopaminergic neurodegeneration, while T cell reconstitution exacerbated neuronal loss in vivo. T cells isolated from the brains of haSyn PD mice were cytotoxic to haSyn-expressing neuronal cells in vitro (Karikari et al., Brain Behav Immun. 2022 Mar; 101: 194-210.).

[0010] Immunosuppressive MHC class Ib molecules (such as HLA-G) are essential for tolerance induction during pregnancy. They exert immunosuppressive effects on various immune cells through immunosuppressive receptors (such as ILT2, ILT4 and Kir2DL4). WO2018 / 215340 relates to a combination of MHC class Ib molecules and peptides for targeted therapeutic immunomodulation, but does not mention the treatment of Parkinson's disease.

[0011] Therefore, there is an urgent need for targeted drugs to effectively treat Parkinson's disease. Summary of the invention

[0012] The inventors have found that human MHC class Ib molecules, such as HLA-G, have the ability to induce antigen-specific tolerance to presented peptide antigens. Therefore, although the structure and sequence of MHC class Ib molecules are similar to the classical human MHC class Ia molecules that induce antigen peptide-specific immune responses, according to the present invention, MHC class Ib molecules can be advantageously used to suppress immune responses in an antigen-specific manner. In addition, the inventors have found that molecules other than naturally occurring MHC class Ib molecules, particularly polypeptides that only comprise at least one domain of MHC class Ib molecules, preferably at least α3 domains of MHC class Ib molecules, can be used to suppress immune responses as described in the present invention: α1 and α2 domains of various Ia class molecules can be combined with α3 domains of human MHC class Ib molecules to suppress immune responses to peptides presented by these antigens. HLA-G molecules loaded with antigens may be unstable. Therefore, the inventors designed a soluble recombinant polypeptide comprising a peptide antigen, an MHC class Ib molecule, such as HLA-G, and β2-microglobulin (b2m), and covalently linked these three components (e.g., via a covalent linker). Alternatively, the antigen-binding α1 and α2 domains of the MHC class Ib molecule (e.g., HLA-G) were replaced with corresponding domains of other MHC molecules to enhance the flexibility and versatility of these recombinant polypeptides (e.g., see Figure 2 ). These alternative recombinant polypeptides can be designed together with the antigen binding domains of other human HLA molecules. b The construct of the α1 and α2 domains can present the ovalbumin-derived peptide SIINFEKL to OT-1 T cells. (OT-1 T cells express a transgenic T cell receptor that specifically recognizes the antigen) (WO2018 / 215340).

[0013] Literature suggests that Parkinson's disease is a neurodegenerative disorder caused by pathogenic accumulation of α-synuclein. Recent concepts suggest that key pathogenic events are caused by immune responses against α-synuclein, but this concept has not yet been translated into therapeutic development.

[0014] Surprisingly, the inventors found that the effect of treating Parkinson's disease can be achieved by suppressing the immune response to α-synuclein using a surrogate molecule of the recombinant polypeptide of the present invention in a mouse model that faithfully simulates human Parkinson's disease. Therefore, according to the present invention, Parkinson's disease can be treated by the recombinant polypeptide of the present invention.

[0015] In addition, according to the present invention, the recombinant polypeptides of the present invention are expected to play a huge advantage in the immunotherapy of Parkinson's disease because they hardly cause any systemic immunosuppression. Importantly, patients with early stage Parkinson's disease should not be affected by the strong side effects of systemic immunosuppression, as this may lead to opportunistic infections and potentially fatal infections. However, in advanced patients, neurodegenerative lesions are generally irreversible. However, the highly specific tolerance induction achieved by presenting the alpha-synuclein antigen on the recombinant polypeptides of the present invention is already tolerable in the early stages of the disease (e.g., prodromal Parkinson's disease), which will open up new therapeutic approaches.

[0016] Therefore, the present invention relates to the following preferred embodiments:

[0017] 1. A recombinant polypeptide capable of presenting a peptide antigen, the recombinant polypeptide comprising, in order from N-terminus to C-terminus:

[0018] i) a peptide antigen presented by the recombinant polypeptide, wherein the peptide antigen is a peptide of human α-synuclein;

[0019] ii) optionally, a linker sequence;

[0020] iii) optionally, a human polypeptide domain sequence, the human polypeptide domain sequence comprising a human β2 microglobulin sequence, or an amino acid sequence that is at least 90% identical to the human β2 microglobulin amino acid sequence shown in SEQ ID No: 5;

[0021] iv) an optional linker sequence;

[0022] v) optionally, an α1 domain of an MHC molecule;

[0023] vi) optionally, an α2 domain of an MHC molecule;

[0024] vii) an α3 domain of an MHC class Ib molecule or a derivative of an α3 domain of an MHC class Ib molecule, said derivative

[0025] Ability to bind to ILT2 or ILT4;

[0026] viii) optionally, a protease cleavage site;

[0027] ix) optionally, a spacer sequence; and

[0028] x) Optionally, an affinity tag.

[0029] 2. The recombinant polypeptide according to item 1, wherein the peptide antigen according to i) has a length of 7 to 11 amino acids, preferably a length of 8 to 10 amino acids.

[0030] 3. The recombinant polypeptide according to item 1 or 2, wherein the peptide antigen according to i) consists of an amino acid sequence selected from the group consisting 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.

[0031] 4. The recombinant polypeptide according to item 3, wherein the peptide antigen 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: 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.

[0032] 5. The recombinant polypeptide according to item 3, wherein the peptide antigen consists of an amino acid sequence selected from the following group, 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.

[0033] 6. The recombinant polypeptide according to item 3, wherein the peptide antigen consists of the amino acid sequence of SEQ ID No: 20.

[0034] 7. The recombinant polypeptide according to item 3, wherein the peptide antigen consists of an amino acid sequence selected from the following group, 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.

[0035] 8. The recombinant polypeptide according to any one of the above items, wherein the α1 domain according to (v) and the α2 domain according to (vi) are derived from human MHC class Ia molecules or from human MHC class Ib molecules.

[0036] 9. The recombinant polypeptide according to item 8, wherein the α1 domain according to (v) and the α2 domain according to (vi) are from a human MHC class Ib molecule, wherein the human MHC class Ib molecule is a human HLA-G molecule.

[0037] 10. The recombinant polypeptide according to item 8, wherein the α1 domain according to (v) and the α2 domain according to (vi) are from human MHC class Ia molecules.

[0038] 11. The recombinant polypeptide according to item 10, wherein the α1 domain according to (v) and the α2 domain according to (vi) are from human HLA-A2 molecules.

[0039] 12. The recombinant polypeptide according to item 10, wherein the α1 domain according to (v) and the α2 domain according to (vi) are from human HLA-A1 molecules.

[0040] 13. The recombinant polypeptide according to item 10, wherein the α1 domain according to (v) and the α2 domain according to (vi) are from human HLA-A11 molecules.

[0041] 14. The recombinant polypeptide according to any one of the above items, wherein the α3 domain of the MHC class Ib molecule according to (vii) is the α3 domain of human HLA-E, human HLA-F or human HLA-G.

[0042] 15. The recombinant polypeptide according to any one of the above items, wherein the α3 domain of the MHC class Ib molecule according to (vii) is the α3 domain of human HLA-G.

[0043] 16. A recombinant polypeptide according to any of the above items, wherein the α3 domain or derivative according to (vii) is identical to the α3 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, preferably at least 90% amino acid sequence identity, with the α3 domain having the amino acid sequence of SEQ ID No: 9 or SEQ ID No: 2.

[0044] 17. A recombinant polypeptide according to item 16, wherein the α3 domain or derivative according to (vii) is identical to the α3 domain having the amino acid sequence of SEQ ID No: 9 or SEQ ID No: 2, or has at least 92% amino acid sequence identity with the α3 domain having the amino acid sequence of SEQ ID No: 9 or SEQ ID No: 2.

[0045] 18. A recombinant polypeptide according to item 16, wherein the α3 domain or derivative according to (vii) is identical to the α3 domain having the amino acid sequence of SEQ ID No: 9 or SEQ ID No: 2, or has at least 94% amino acid sequence identity with the α3 domain having the amino acid sequence of SEQ ID No: 9 or SEQ ID No: 2.

[0046] 19. A recombinant polypeptide according to item 16, wherein the α3 domain or derivative according to (vii) is identical to the α3 domain having the amino acid sequence of SEQ ID No: 9 or SEQ ID No: 2, or has at least 96% amino acid sequence identity with the α3 domain having the amino acid sequence of SEQ ID No: 9 or SEQ ID No: 2.

[0047] 20. A recombinant polypeptide according to item 16, wherein the α3 domain or derivative according to (vii) is identical to the α3 domain having the amino acid sequence of SEQ ID No: 9 or SEQ ID No: 2, or has at least 98% amino acid sequence identity with the α3 domain having the amino acid sequence of SEQ ID No: 9 or SEQ ID No: 2.

[0048] 21. A recombinant polypeptide according to item 16, wherein the α3 domain or derivative according to (vii) is identical to the α3 domain having the amino acid sequence of SEQ ID No: 9 or SEQ ID No: 2, or has at least 99% amino acid sequence identity with the α3 domain having the amino acid sequence of SEQ ID No: 9 or SEQ ID No: 2.

[0049] 22. The recombinant polypeptide according to item 16, wherein the α3 domain according to (vii) is identical to the α3 domain having the amino acid sequence of SEQ ID No: 9 or SEQ ID No: 2.

[0050] 23. A recombinant polypeptide according to any of the above items, wherein the linker sequence according to (ii) and / or the linker sequence according to (iv) comprises the amino acid sequence (GGGGS)n, wherein n is an integer equal to or greater than 1.

[0051] 24. A recombinant polypeptide according to item 23, wherein the linker sequence according to (ii) comprises the amino acid sequence (GGGGS)n, wherein n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, and is preferably selected from the group consisting of 2, 3, 4 and 5.

[0052] 25. A recombinant polypeptide according to item 23 or 24, wherein the linker sequence according to (iv) comprises the amino acid sequence (GGGGS)n, wherein n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, and is preferably selected from the group consisting of 2, 3, 4 and 5.

[0053] 26. A recombinant polypeptide according to any of the above items, wherein the sequence of the human polypeptide domain according to (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.

[0054] 27. The recombinant polypeptide according to any one of the above items, wherein the polypeptide is a dimer or a multimer.

[0055] 28. The recombinant polypeptide according to any one of the above items, wherein the polypeptide comprises or consists of all components i) to vii).

[0056] 29. The recombinant polypeptide according to any one of the above items, wherein the polypeptide does not comprise components viii) to x).

[0057] 30. The recombinant polypeptide according to any one of items 1 to 28, wherein the polypeptide comprises or consists of all components i) to x).

[0058] 31. The recombinant polypeptide according to any one of the above items, further comprising an N-terminal secretion signal peptide sequence.

[0059] 32. The recombinant polypeptide according to any one of items 1 to 30, wherein the recombinant polypeptide consists of the following amino acid sequence, which consists of the following ((a) and (b)) in order from N-terminus to C-terminus:

[0060] (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;

[0061] (b) The amino acid sequence of SEQ ID No:16.

[0062] 33. The recombinant polypeptide according to any of the above items, wherein the recombinant polypeptide is soluble.

[0063] 34. A nucleic acid encoding one or more polypeptides according to any one of the above.

[0064] 35. The nucleic acid according to item 34, wherein the nucleic acid is a vector.

[0065] 36. A pharmaceutical composition or kit comprising at least one nucleic acid according to item 34 or 35.

[0066] 37. A pharmaceutical composition or kit comprising at least one recombinant polypeptide according to any one of items 1-33.

[0067] 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 1-33, wherein each different polypeptide comprises a different peptide antigen as defined in any one of items 3 to 7.

[0068] 39. The pharmaceutical composition or kit according to any one of items 36-38, for use in treating Parkinson's disease in a human patient.

[0069] 40. The pharmaceutical composition or kit for use according to item 39, wherein the Parkinson's disease is prodromal Parkinson's disease.

[0070] 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.

[0071] 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 against the self-antigen human alpha-synuclein.

[0072] 43. A recombinant host cell comprising the nucleic acid or vector of item 34 or 35, and expressing the recombinant polypeptide of any one of items 1 to 33.

[0073] 44. A method for obtaining a pharmaceutical composition comprising a polypeptide according to any one of items 1 to 33, the method comprising the following steps: (a) culturing the recombinant host cell of item 43 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 : Description of peptide-loaded soluble MHC Ib molecules suitable for achieving therapeutic antigen-specific immunomodulation.

[0075] The presented peptide antigen is represented by a dotted sphere, the HLA-Gα1-3 domain is represented by a light gray, and the β2 microglobulin domain is represented by a dark gray. The optional linker connecting the antigenic peptide and the β2 microglobulin molecule is represented by a gray rod, and the optional disulfide bond capture is represented by a black sphere. This figure was generated using Pymol and modified from the 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.

[0076] Figure 2 : Examples of vector-based constructs encoding single-chain MHC Ib molecules suitable for therapeutic peptide-specific immunomodulation.

[0077] HLA-G1 and HLA-G5 each consist of three α domains (shown in black here), a non-covalently bound β2-microglobulin subunit (shown in dark gray here) and an antigenic peptide presented on HLA-G (short black arrow). HLA-G1 also contains a transmembrane domain and a short intracellular chain (not shown here). As shown, the α3 domain is able to 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 Oct 31; 103 (44): 16412-7) on immune cells. Physiologically, these sequences form non-covalently linked MHC class 1 complexes. To simplify the purification of the composite MHC Ib molecule, one or more protein tags (e.g., SpotTag, myc tag, and / or His (6x) tag) can be introduced. These tags are introduced in such a way that they can be selectively removed by subsequent cleavage using an optional Factor Xa cleavage site. In addition, the antigenic peptide, β2-microglobulin, and MHC Ib α chain can be linked in sequence to improve stability. The vector map was generated using Snapgene Viewer software.

[0078] Figure 3 : Prediction and confirmation of antigenic aSyn-peptides.

[0079] CD4+ T cells from spleens of haSyn PD (grey) and control mice (black) were stimulated with predicted peptides 10 weeks after EV or A53T vector injection. + 、CD8 + The results of IFN-γ ELISpot analysis were performed after mononuclear detachment of T cells. Five aSyn peptides can induce antigen-specific IFN-γ immune responses. The haSyn protein sequence was aligned with mouse α-synuclein using NCBI Protein Blast, www.syfpeithi.de (Ver.1.0) and netMHC4.0 (http: / / www.cbs.dtu.dk / services / NetMHC / ), and the possible expression of aSyn peptides in MHC I (H-2D b and H-2K b ) and MHC II(IA b) molecules. The three peptides (8-15 amino acids in length) with the highest scores for each MHC molecule were synthesized by Thermo Scientific in water-based quality. Each peptide was used to stimulate lymph node or spleen cells, followed by ELISpot assays. Splenocytes from two initially unresponsive haSyn mice and two control mice were cultured for 10 days in RPMI containing 10% FCS and 20ng / ml IL-2 containing all 10 peptides (1μg / ml each). Multiscreen PVDF plates (MSIPS4510, Millipore) were activated with 35% ethanol, washed with sterile PBS, and coated overnight at 4°C with 2μg / ml filtered anti-mouse IFN-γ antibody (AN18, Mabtech). Single cell suspensions isolated from cervical lymph nodes and spleens were inoculated in IMDM containing 7.5% FCS with or without 5μg / ml synthetic peptides. IFN-γ spots were then detected with 2 μg / ml biotinylated anti-mouse IFN-γ antibody (R4-6A2, Mabtech) followed by horseradish peroxidase-coupled streptavidin (Cell Signaling) in PBS-T supplemented with 0.05% BSA. Color was developed with filtered "ready-to-use" TMB substrate (Mabtech) and analyzed using an Immunopost S6 Core Analyzer.

[0080] Figure 4 : AIM Bio replacement molecules loaded with aSyn prevent neuronal death in PD models and may improve endurance.

[0081] Compared with haSyn PD mice treated with vehicle, weekly treatment with a peptide containing a peptide (here aSyn peptide), an MHC class 1 antigen presenting domain, and an HLA-Gα3 domain (AIM Bio) for a total of 9 cycles resulted in significant dose-dependent neuroprotection of dopaminergic neurons in haSyn PD mice, with a significant decrease in tyrosine hydroxylase (TH) in the substantia nigra of the treated group. +The number of neurons was significantly higher. Immunohistochemical staining was performed using 40 μm PFA-fixed frozen sections that were processed to obtain unbiased stereology (TH) covering the entire SN. After 4% PFA fixation, sections were incubated with rabbit anti-mouse TH (1:1000) antibody and then with biotinylated goat anti-rabbit secondary antibody (Vector Labs). Development was performed using (DAB)-HCl-peroxidase (Vector Labs). The number of dopaminergic neurons in the substantia nigra was estimated by unbiased stereology using the Stereo Investigator software package (v11.07, MicroBrightField Biosciences).

[0082] The endurance of treated and untreated mice was tested by comparing pre- and post-operative latencies in the Rotarod test as described (Ip CW et al., 2017).

[0083] A: Images (scale bar = 100 μm); B: Quantification. C: Delay relative to preoperative assessment by Rotarod test.

[0084] Figure 5 : AIM Bio surrogate molecules loaded with aSyn prevent CD11b in PD models + Myeloid cell (microglia) infiltration, but induction of CD8 + T cells infiltrate the striatum.

[0085] CD11b in mouse tissue was analyzed using 10 μm fresh coronal striatal cryosections. + Myeloid cells and CD8 + T cells were stained with immunohistochemistry. After fixation with 4% PFA or acetone, sections were incubated with rat anti-mouse CD11b (1:100, Serotec) or rat anti-CD8 MCA609G 1:500 antibodies, followed by incubation with biotinylated rabbit anti-rat secondary antibody (Vector Labs). (DAB)-HCl-peroxidase (Vector Labs) was used for development. Microglia in the SN and striatum regions depicted by TH-stained serial sections were quantified at 200x magnification on a BH2 light microscope (Olympus).

[0086] A: Image (scale bars: 50 microns for the entire image; 20 microns for the lower left magnified area) showing CD11b+ microglia in the striatum; B: Quantification. C: Image (scale bars: 100 microns for the entire image; microns for the lower left magnified area) showing CD8+ T cells in the striatum. D: Quantification.

[0087] Figure 6 : AIM Bio surrogate molecules loaded with aSyn reduce aSyn accumulation in the substantia nigra in a PD model. Scale bar = 100 μm.

[0088] Neurons were fixed with 4% PFA, blocked with 5% NGS and 0.3% Triton X100, stained with rabbit anti-human α-synuclein (1:30,000, Sigma) and goat anti-rabbit Cy3 (1:300, Jackson Laboratory, Cat. No. 111–165-003) secondary antibodies, and counterstained with DAPI. Lower aSyn staining intensity was observed in mice treated with the aSyn-loaded AIM Bio alternative molecule.

[0089] Figure 7 AIM Bio loaded with aSyn protects tyrosine hydroxylase in striatal and substantia nigra neurons + fiber

[0090] Methods Figure 4 .

[0091] Tyrosine hydroxylase + There was a significant reduction in fibers, especially in the upper part of the striatum on the side injected with A53T aSyn AAV (A53T) (here, the right side). Injection of AIM Bio loaded with aSyn significantly reduced unilateral tyrosine hydroxylase + Fiber loss was dose-dependent.

[0092] A: Image; Scale bar = 1000 μm. B: Quantification. C: Repeat using alternative Nissl staining. Figure 4 Experiments to show neurons.

[0093] Figure 8 : AIM Bio loaded with aSyn induces CD8 + CD122 + Regulatory T cells

[0094] After treatment with aSyn-loaded AIM Bio, CD8 + CD122 +Significant induction of regulatory T cells. The frozen sections were fixed with acetone at -20 ° C for 10 minutes, then washed and blocked with PBS containing 5% BSA, 5% NGS, 0.2% Triton-X100 for 1 hour. In 1% BSA, 1% NGS, 0.2% Triton-X100, CD122 was stained overnight with anti-CD122i rabbit polyclonal antibody (MyBiosource, 1: 100) and anti-CD8i clone YTS191.1 rat (Biorad, 1: 200). Anti-rabbit Cy3 (Dianova, 1: 300) and anti-rat AF488 (Invitrogen, 1: 300) secondary antibodies were used for staining for 1 hour.

[0095] Fig. 9 : Stability of purified single-chain MHC Ib molecules. After purification, the stability of single-chain MHC Ib molecules was analyzed after 1 and 3 freeze-thaw cycles, storage at room temperature for 5 days, and heating to 50°C for 30 minutes. For this purpose, aHLA-G protein blotting (Western Blot) was performed under non-reducing conditions using A) 2 μg of AIM Bio in 12% polyacrylamide gels with Coomassie staining and B) 1 μg of protein with 2A12 aHLA-G antibody (1:1000). Both monomers and dimers were detected.

[0096] Fig.10 :Single-chain MHC Ib molecules are thermally stable. For thermal shift assay (TSA), 3 μg of the corresponding single-chain MHC Ib molecule or Motavizumab as a control molecule was diluted to a volume of 25 μl with PBS and 5×SYPRO Orange dye (stock solution 5000×, final concentration: 5×). The melting curve program was set up on the StepOnePlus instrument using StepOnePlus software 2.3. The starting temperature was 25°C for one minute, and then the temperature was increased to a final temperature of 95°C at a rate of 1°C per minute for 2 minutes, thereby measuring autofluorescence in arbitrary units. The data was exported and graphed in Prism V7.04. To determine the melting temperature (Tm), the Boltzman sigmoid function was used.

[0097] Fig.11 :Single-chain MHC Ib molecules induce Treg in a dose-dependent manner. OT-I mice were intraperitoneally injected with the indicated amount of single-chain H2_K bα1+2, HLA-Gα3 domain, human β2 microglobulin and the constructs of the indicated peptides, or vehicle (PBS). In these mice, Ova is the homologous peptide of the OT-I TCR and Gp34 is an unrelated virus-derived control peptide. After 14 days, the mice were sacrificed and IL10-secreting cells in splenocytes were detected in the memory immune mouse IL-10 ELISpot (200,000 cells per well, MabTech mouse IL-10 ELISpot kit, supplemented with 5 μg / ml of the indicated peptide or PBS alone, 48 hours). When 50 and 500 μg of mouse-adapted Ova_KbG were injected, a clear induction of IL-10-secreting cells reactive to the Ova peptide was observed.

[0098] Fig.12 :Single-chain MHC Ib molecules inhibit T cell lysis in a dose-dependent manner. OT1 / BL6 mice were sacrificed, spleen cells were collected and washed once in RPMI 5% FCS. Red blood cells were removed with 2ml 1× sterile RBC lysis buffer for 3 minutes. Cells were cultured at high density (10mio cells / ml) for 72 hours in RPMI 10% FCS medium containing GMCSF 20ng / mL, IL-2 20ng / ml and IL-4 10ng / ml and increasing doses of Ova_KbG. Cells were then scraped from the culture dish and CD8+ cells were purified by magnetic beads.

[0099] Use a sterile 96-well white plate. Load Panc02 target cells expressing luciferase with 20 μg / ml Ova peptide (SIINFEKL) and shake at 500 rpm for 60 minutes at 37°C. Add CD8+ effector T cells and luciferin at a ratio of 50:1. Measure luminescence after 0h, 24h, and 48h.

[0100] Fig.13: Single-chain MHC Ib molecules induce IL-10 expression in EAE-ODC Ova mice. Cytokines in EAE-ODC Ova mouse serum were measured using the Th1 / Th2 10plex Flowcytomix kit (eBioscience) according to the manufacturer's instructions. The kit was used to simultaneously detect mouse granulocyte-macrophage colony-stimulating factor (GMCSF), interleukin 1α (IL-1a), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 10 (IL-10), t-interleukin 17 (IL-17), and tumor necrosis factor α (TNF-α) in a single sample. Beads coated with ten specific capture antibodies were mixed. Subsequently, 25 μL of the mixed capture beads, 25 μL of unknown serum sample or standard dilution, and 25 μL of phycoerythrin (PE) detection reagent were added to each well of a 96V-bottom well plate in sequence and incubated at room temperature in the dark for 2 h. Wash the sample with 1 mL of wash buffer for 5 min and centrifuge. After discarding the supernatant, resuspend the bead pellet in 200 μL of buffer. TM The cells were measured on an NxT flow cytometer, and the samples were analyzed using Attune flow cytometer software (Thermo Fisher Scientific).

[0101] Fig.14 : The diagram shows the development process of the preferred polypeptide construct of the present invention.

[0102] Fig.15 : Thermal shift analysis. For thermal shift analysis (TSA), 3 μg of single-chain MHC Ib molecules were diluted to a volume of 25 μl with PBS and 5× SYPRO Orange dye (stock solution 5000×, final concentration: 5×). The melting curve program was set up on the StepOnePlus instrument using StepOnePlus software 2.3. The starting temperature was 25°C for one minute, then the temperature was increased at a rate of 1°C per minute to a final temperature of 95°C for 2 minutes, and autofluorescence was measured throughout the process. Data were exported and graphed in Prism V7.04. The melting temperature (Tm) was determined by the Boltzmann sigmoid function. A high melting temperature indicates good protein stability and can be used for therapeutic applications.

[0103] Fig.16 : Stability of the polypeptide constructs of the present invention (A, B).

[0104] sc HLA-A2 or A11α1-2HLA-Gα3 constructs with indicated peptides were purified by dot cap purification according to the manufacturer's protocol and then dialyzed ON. The constructs were then subjected to 3 freeze-thaw cycles, heated to 50°C for 30 minutes or stored at room temperature for 6 days. Degradation was then analyzed on Coomassie gels (12% gel, 4% concentrating gel, 1 μg purified protein, non-reducing conditions, 15 μl sample + 5 μl 4× Laemmli). Roti Mark TRICOLOR XTRA (Roth, 2244) was used for Coomassie gel staining for 5-6 hours. Destaining was performed with 12.5 ml methanol + 37.5 ml deionized water for 5 minutes and one more destaining cycle with deionized water.

[0105] Fig.17 : ELISpot results of PBMC from healthy donors. The figure shows the induction of IL-10 secreting regulatory cells in PBMC from healthy donors by the specified constructs of the present invention within 14 days.

[0106] Fig.18 : IL-10 ELISpot results of Parkinson's patients (A) and age-matched healthy individuals (B). The figure shows the regulatory cells that induced secretion of IL-10 by the specified constructs of the present invention and age-matched healthy donors (C1-C3) or normal healthy donors (B10, B12, B13) within 14 days. DETAILED DESCRIPTION OF THE INVENTION

[0108] Definition and general techniques

[0109] Unless otherwise defined below, the terms used in the present invention should be understood according to the common 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 cited herein can be cited by specifying a complete reference in the text, or by indicating the first author and publication date (e.g., "Brochard V et al., 2009"), and then citing the corresponding complete reference in the "References" section of this specification.

[0110] All proteins of the present invention, including the recombinant polypeptides of the present invention, can be obtained by methods known in the art. Such methods include methods for producing recombinant polypeptides. The recombinant polypeptides of the present invention can be expressed in the recombinant host cells of the present invention. The recombinant host cells of the present invention are preferably mammalian cells, such as CHO and HEK cells.

[0111] It should be understood that the recombinant polypeptide of the present invention is intended to optionally include a secretion signal peptide sequence. Similarly, the recombinant polypeptide of the present invention is intended to also optionally include an affinity tag, such as for the convenience of purification, and an optional protease cleavage site between the tag and the polypeptide, such as for the convenience of removing the tag by protease cleavage.

[0112] It should also be understood that any amino acid sequence mentioned herein is intended to cover not only the unmodified amino acid sequence, but also typical post-translational modifications of these amino acid sequences that occur in cell expression systems known in the art (e.g., glycosylation or deamidation of amino acids, cleavage of specific amino acids or other post-translational modifications), including mammalian cells, such as CHO and HEK cells.

[0113] Likewise, it should be understood that the recombinant polypeptides of the present invention may optionally include the corresponding propeptides.

[0114] It should also be understood that the recombinant polypeptides of the present invention may be in their soluble form or membrane-bound form. Whether a recombinant polypeptide is "soluble" under these conditions can be determined by methods known in the art, such as by measuring the turbidity of the recombinant polypeptide under the above-mentioned reference conditions. As used herein, soluble means that at least 95% of the recombinant polypeptide is determined to be soluble under these reference conditions.

[0115] Single-chain MHC molecules can be stored, for example, at -80°C in PBS (with or without 0.1% human albumin as a carrier, depending on the protein concentration), or at -20°C in 50% glycerol.

[0116] According to the present invention, the MHC molecule is preferably a human MHC molecule.

[0117] The recombinant polypeptide of the present invention is preferably an isolated recombinant polypeptide.

[0118] It should be understood how to prepare recombinant polypeptides according to the present invention that can bind and present peptide antigens. For example, peptide antigen binding domains such as α1 and α2 domains are well known, and these domains can be modified. The ability of peptide antigens to bind to polypeptides of the present invention and MHC molecules can be determined by techniques known in the art, including but not limited to exploratory methods, such as mass spectrometry and computer bioinformatics predictions after MHC peptide elution, and confirmatory methods, such as MHC peptide multimer binding methods and stimulation tests.

[0119] According to the present invention, the recombinant polypeptide, pharmaceutical composition and kit of the present invention are preferably suitable for use in human patients.

[0120] According to the present invention, the recombinant polypeptide, pharmaceutical composition and kit of the present invention are preferably suitable for treating Parkinson's disease in human patients.

[0121] According to the present invention, the recombinant polypeptide, pharmaceutical composition and kit of the present invention are preferably suitable for inducing immune tolerance against human α-synuclein, for example, in a human patient.

[0122] It should be understood that according to the present invention, the recombinant polypeptides, pharmaceutical compositions and kits of the present invention are stable.

[0123] It should be understood that for peptide antigens used according to the present invention, any length of these peptide antigens mentioned herein (e.g. "length of 7 to 11 amino acids") refers to the length of the peptide antigen itself. Therefore, the length of the peptide antigen mentioned herein does not include the length conferred by additional amino acids that do not belong to the peptide antigen, such as additional amino acids from possible linker sequences, etc.

[0124] According to the present invention, each occurrence of the term "comprising" may optionally be replaced by the term "consisting of".

[0125] Methods and techniques

[0126] Generally, unless otherwise defined herein, the methods used in the present invention (e.g., cloning methods or methods related to antibodies) are performed according to procedures known in the art, such as those 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.

[0127] Protein-protein binding, e.g. binding of an antibody to its respective target protein, can be assessed by methods known in the art. Protein-protein binding is preferably assessed by surface plasmon resonance spectroscopy measurements.

[0128] For example, the binding of the MHC class Ib molecule or recombinant polypeptide according to the present invention to its receptor, including ILT2 and ILT4, is preferably assessed by surface plasmon resonance spectroscopy. More preferably, the binding of the MHC class Ib molecule or recombinant polypeptide according to the present invention to its receptor is assessed by surface plasmon resonance measurement at 25°C. Shiroishi et al., Proc Natl Acad Sci US A. 2003 July 22; 100(15): 8856-8861 describes suitable conditions for such surface plasmon resonance measurements.

[0129] The sequence alignment 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 using the BLAST algorithm, which are suitable for peptide antigens according to 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. Therefore, terms such as "identity" or "identical" when used in connection with a sequence preferably refer to identity values ​​obtained using the BLAST algorithm.

[0130] Preparation of the pharmaceutical composition of the present invention

[0131] The pharmaceutical composition of the present invention is prepared according to known pharmaceutical composition preparation standards.

[0132] For example, the pharmaceutical composition should be prepared in such a way that it can be easily stored and administered.Thus, the pharmaceutical composition of the present invention may comprise pharmaceutically acceptable components, such as carriers, excipients and / or stabilizers.

[0133] These pharmaceutically acceptable components are non-toxic in the amounts used when the pharmaceutical composition is administered to human patients. The pharmaceutically acceptable components added to the pharmaceutical composition may depend on the chemical nature of the active ingredients present in the composition, the specific intended use of the pharmaceutical composition, and the route of administration.

[0134] In general, pharmaceutically acceptable components associated with the present invention are used according to existing knowledge in the art, such as from Remington's Pharmaceutical Sciences, Ed. AR Gennaro, 20th edition, 2000, Williams & Wilkins, PA, USA. Pharmaceutical compositions comprising nucleic acids (e.g., RNA) of the present invention can also be formulated according to existing knowledge in the art, such as using liposome formulations targeted to dendritic cells.

[0135] Peptide antigens according to the present invention

[0136] The peptide antigens that can be used according to the present invention include the peptide antigens defined above, and there is no particular limitation except that they can be presented on MHC molecules. It should be understood that the "peptide antigen presented by the recombinant polypeptide" related to the present invention refers to the peptide antigen presented by the recombinant polypeptide to human T cells (if such T cells exist) by binding to the T cell receptor on human T cells.

[0137] As is known in the art, peptides that can be presented on MHC molecules can be produced (see, e.g., Rammensee, Bachmann, Emmerich, Bachor, and Rock,Reits,Neefjes.PresentYourself! By MHC Class I and MHC Class II Molecules.Trends Immunol.2016Nov;37(11):724-737).

[0138] Peptide antigens are well known in the art. Generally, the peptide antigens according to the present invention are capable of binding to MHC class I proteins. It will be appreciated by those skilled in the art that for each MHC class Ib molecule or polypeptide capable of presenting a peptide according to the present invention, a peptide antigen capable of binding to the MHC class Ib molecule or recombinant polypeptide will preferably be used. These peptide antigens can be selected based on methods known in the art.

[0139] The binding of a peptide antigen to an MHC class Ib molecule according to the present invention or a polypeptide capable of binding to a peptide antigen can be assessed by methods known in the art, such as the following method:

[0140] Rammensee,Bachmann,Emmerich,Bachor, SYFPEITHI:database forMHC ligands and peptide motifs.Immunogenetics.1999Nov;50(3-4):213-9;

[0141] Pearson et al.MHC class I-associated peptides derive from selective regions of the human genome.J Clin Invest.2016Dec 1;126(12):4690-4701; and

[0142] Rock,Reits,Neefjes.Present Yourself! By MHC Class I and MHC Class II Molecules.Trends Immunol.2016Nov;37(11):724-737.

[0143] These methods include experimental approaches and methods for predicting peptide antigen binding.

[0144] Anchor residues for anchoring peptide antigens to MHC class I molecules and ensuring binding of peptide antigens to MHC class I molecules are known in the art.

[0145] In a preferred embodiment according to all embodiments of the present invention, the peptide antigen used according to the present invention comprises any anchor or preferred amino acid residues at the predicted positions for MHC class I molecules.

[0146] Preferably, such predictions are performed as described in any of the following publications:

[0147] -Rammensee et al, SYFPEITHI: database for MHCligands and peptidemotifs. Immunogenetics (1999) 50:213-219

[0148] -Nielsen et al,Protein Sci(2003)12:1007-1017

[0149] -Neefjes et al. Nat Rev Immunol.2011Nov 11;11(12):823-36

[0150] -Diehl et al.Curr Biol.1996Mar 1;6(3):305-14,

[0151] -Lee et al.Immunity.1995Nov;3(5):591-600.

[0152] -Desai&Kulkarni-Kale,T-cell epitope prediction methods:anoverview.Methods Mol Biol. 2014;1184:333-64.

[0153] -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

[0154] In the present invention, the peptide antigen is derived from human α-synuclein.

[0155] It should be understood that the non-anchor amino acid residues of the peptide antigen of the present invention may or may not contain conservative substitutions, preferably no more than two conservative substitutions, more preferably one conservative substitution, relative to the corresponding amino acid sequence of the peptide antigen from human α-synuclein.

[0156] The peptide antigens of the present invention are preferably composed of natural amino acids. However, non-natural amino acids, such as modified amino acids, may also be used. For example, in one embodiment, the peptide antigens of the present invention include a peptidomimetic of the amino acid sequence of the peptide antigen of human α-synuclein.

[0157] Methods for the synthesis of peptide antigens, including peptide antigens according to the present invention, are well known in the art.

[0158] sequence

[0159] The preferred amino acid sequences mentioned in the present application can be independently selected from the following sequences. These sequences are represented in the order from N-terminus to C-terminus; and are represented by single-letter amino acid codes.

[0160] Exemplary sequences that are part of a recombinant polypeptide of the invention:

[0161] Leader peptide (deleted from the recombinant polypeptide due to processing during cell expression): e.g. MSRSVALAVLALLSLSGLEA (SEQ ID No: 1)

[0162] Peptide antigen: any MHC class I peptide corresponding to the MHC class I α1 & 2 domains, such as GAPQEGIL (SEQ ID No: 20)

[0163] First linker: for example, GGGGSGGGGSGGGGS (SEQ ID No: 3) or GCGASGGGGSGGGGS (SEQ ID No: 4)

[0164] beta-2 microglobulin, such as:

[0165] IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKD WSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID No: 5, human β2 microglobulin)

[0166] Second connector, for example:

[0167] GGGGSGGGGSGGGGSGGGGS(SEQ ID No:6)

[0168] The α1 and 2 domains are derived from human HLA-G or any other MHC class I α1 & 2 domains suitable for presenting the selected antigenic peptide. In the DT variant, Y84 can be C

[0169] For example, the α1 & 2 domains from human HLA-G:

[0170] GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRA(SEQ ID No:7)

[0171] Or: Human HLA-A2 α1&2 domains: For example:

[0172] GSHSMRYFYTSVSRPGRGEPRIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGCYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRT(SEQ ID No:8)

[0173] Human HLA-G α3 domain (or any MHC Ib α3 domain such as HLA-F, which also interacts with ILT2 and ILT4 receptors), for example:

[0174] DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDL (SEQ ID No: 9; sequence of HLA-Gα3).

[0175] Note that the following underlined amino acids in this sequence are involved in ILT2 or ILT4 receptor interaction:

[0176] DPPKTHVTHH PVFDYE ATLRCWALGFYPAEIILTWQRDGEDQTQD V ELVETRPAGDG TFQKWAAV V VPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDL

[0177] In addition, a shorter form of the human HLA-Gα3 domain may be used that lacks the alternative C-terminal amino acid sequence from intron 4 (SKEGDGGIMSVRESRSLSEDL; SEQ ID No: 47), namely:

[0178] DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDG TFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW(SEQ ID No:2),

[0179] Factor Xa restriction site: IEGRTGTKLGP (SEQ ID No: 10)

[0180] SpotTag: PDRVRAVSHWSSC(SEQ ID No:11)

[0181] Myc tag: EQKLISEEDL (SEQ ID No: 12)

[0182] His tag: HHHHHH* (SEQ ID No: 13)

[0183] Spacer sequence: for example NSAVD (SEQ ID No: 14) or GS

[0184] Other (alternative) exemplary peptide antigens that may be part of the recombinant polypeptide of the present invention are as follows:

[0185] Most preferred:

[0186] GAPQEGIL (SEQ ID No: 20), preferably used for recombinant polypeptides containing human HLA-Gα1&2 domains;

[0187] KTKEGVLYV (SEQ ID No: 28), preferably used for recombinant polypeptides containing human HLA-A2 α1&2 domains;

[0188] AVVTGVTAV (SEQ ID No: 33), preferably used for recombinant polypeptides containing human HLA-A2 α1&2 domains;

[0189] GVVHGVTTV (SEQ ID No: 31), preferably used for recombinant polypeptides containing human HLA-A2 α1&2 domains;

[0190] MDVFMKGLSK (SEQ ID No: 22), preferably used for recombinant polypeptides containing human HLA-A11α1&2 domains;

[0191] GVVAAAEKTK (SEQ ID No: 25) is preferably used for recombinant polypeptides containing human HLA-A11α1&2 domains.

[0192] Preferred:

[0193] MPVDPDNEAY (SEQ ID No: 21), preferably used for recombinant polypeptides containing human HLA-A1α1&2 domains;

[0194] PVDPDNEAY (SEQ ID No: 44), preferably for recombinant polypeptides containing human HLA-A1 α1&2 domains;

[0195] SIAAATGFV (SEQ ID No: 37), preferably used for recombinant polypeptides containing human HLA-A2 α1&2 domains;

[0196] VVTGVTAVA (SEQ ID No: 34), preferably used for recombinant polypeptides containing human HLA-A2 α1&2 domains;

[0197] VVAAAEKTK (SEQ ID No: 26), preferably used for recombinant polypeptides containing human HLA-A11α1&2 domains;

[0198] VFMKGLSKAK (SEQ ID No: 24), preferably used for recombinant polypeptides containing human HLA-A11α1&2 domains;

[0199] AAATGFVKKD (SEQ ID No: 42), preferably used for recombinant polypeptides containing human HLA-A11α1&2 domains;

[0200] AATGFVKK (SEQ ID No: 43) is preferably used for recombinant polypeptides containing human HLA-A11α1&2 domains.

[0201] Examples of recombinant polypeptides of the present invention (without leader peptide):

[0202] GAPQEGILGCGASGGGGSGGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQP KIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGCYN QSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC* (SEQ ID No: 15; note, the asterisk indicates the position of the stop codon)

[0203] Note that the peptide antigen (here: GAPQEGIL) sequence of the above full-length protein can be replaced by any peptide antigen sequence of the present invention, that is, replaced by any peptide antigen presented by the recombinant polypeptide, wherein the peptide antigen is a peptide of human α-synuclein. That is, the recombinant polypeptide of the present invention can be composed of the following sequence, which consists of a peptide antigen, which is a peptide of human α-synuclein (e.g., any one of the peptide antigens of SEQ ID No: 20-46), followed by the following sequence:

[0204] GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDR DMGGGGSGGGGSGGGGSGGGGSGSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQETRNVKAQSQTDRVDLGTLRGCYNQSEDG SHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC* (SEQ ID No: 16; note, the asterisk indicates the position of the stop codon)

[0205] The receptors ILT2 (also known as LILRB1) and ILT4 (also known as LILRB2) are known in the art. According to the present invention, the preferred sequences of these receptors are as follows:

[0206] ILT2:

[0207] MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEETLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH(SEQ ID No:17)

[0208] ILT4:

[0209] (SEQ ID No: 18) The sequence of human α-synuclein is known in the art. Preferred human α-synuclein is as follows:

[0210] MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTKEQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSEEGYQDYEPEA(SEQ ID No:19)

[0211] Therapeutic applications of the invention:

[0212] Parkinson's disease (PD), including prodromal-PD and its classifications, is known in the art.

[0213] Prodromal PD refers to the stage in which an individual does not meet diagnostic criteria for PD (i.e., bradykinesia and at least 1 other motor sign) but exhibits signs and symptoms that indicate a higher-than-average risk of future motor symptoms and a diagnosis of PD. Most prodromal symptoms are nonmotor and have a significant impact on the quality of life of patients with prodromal PD and those whose disease stage has progressed to motor PD. Therefore, early detection and treatment of these prodromal symptoms are critical for high-quality care. The most typical symptoms of prodromal PD include hyposmia, constipation, mood disturbances, and rapid eye movement sleep behavior disorder (RBD).

[0214] The present invention is further illustrated by the following non-limiting examples: Example

[0215] Reference Example: Screening of target antigens in mice

[0216] 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 computer-predicted A53T-aSyn peptides (e.g., by NetMHC). By screening for aSyn-specific immune responses in C57BL / 6 mice, the inventors 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 of cervical lymph node and brain-derived T cells to the haSyn peptide is also shown.

[0217] Example 1 : Prevented neuronal death in an in vivo model of PD

[0218] The inventors reasoned that, given the critical role of T cells in neurodegenerative processes (as demonstrated by depletion / reconstitution experiments), this haSyn PD mouse model, which closely resembles the human disease, would be ideally suited to assess the efficacy of immunomodulation in the context of PD-like α-synuclein (aSyn) pathology, as well as to assess the suitability of the recombinant polypeptides of the present invention for the treatment of PD.

[0219] The inventors further reasoned that inducing immune tolerance to aSyn would induce tissue-wide protection through local antigen-specific activation of Tregs. After determining that the A53T-aSyn-derived aSyn 68-78 peptide was immunogenic in mice, the inventors developed a recombinant polypeptide corresponding to the recombinant polypeptide of the present invention but carrying the peptide antigen that is immunogenic in mice and mouse H2-D b α1 and 2 domains (hereinafter also referred to as the "mouse AIM Bio replacement molecule" or "AIM Bio aSynIn a pilot experiment, haSyn PD mice were injected weekly with 0.5 μg / g AIM Bio starting 3 days after AAV injection. aSyn or 2μg / g AIM Bio aSyn or off-target unrelated control peptide (gp34). The Gp34 peptide is a well-characterized T cell epitope derived from the lymphocytic choriomeningitis virus (LCMV) glycoprotein. Although this antigen was traditionally referred to as Gp33, it was later discovered that H2-K b The epitope presented on H2-K d The epitope presented on the H2-K b The epitope is called Gp34, which is in line with the latest recommendations. However, the use of the nomenclature of Gp33 and Gp34 in the literature remains unclear. The peptide sequence used here is AVYNFATM (SEQ ID No:48). Control mice were injected with vector only (PBS). Ten weeks after AAV delivery, the inventors found that in haSyn PD mice injected with PBS or injected with Gp34_KbG, their substantia nigra neurons lost about 45% compared to animals injected with non-pathogenic empty vector control (EV). Surprisingly, low doses (0.5μg / g) and high doses (2μg / g) of AIM Bio aSyn This neuronal loss was attenuated in a dose-dependent manner ( Figure 4 ). In addition, the inventors found that in the PD model, AIM Bio replacement molecules loaded with aSyn can prevent CD11b + Myeloid cells infiltrate the striatum ( Figure 5 ), aSyn-loaded AIM Bio alternative molecules reduce aSyn accumulation in the substantia nigra ( Figure 6 ), AIM Bio loaded with aSyn protects tyrosine hydroxylase in the striatum + fiber( Figure 7 ).

[0220] Embodiment 2: aSyn-specific MHC class I-based therapies for the treatment of PD in human patients

[0221] The inventors' findings indicate that single-chain proteins containing aSyn peptide antigens and HLA-Gα3 domains can largely prevent neuronal death in animal models of Parkinson's disease, that is, they can be used to treat PD. Based on these findings, the inventors have prepared aSyn peptide and MHC class I fusion molecules for treating human PD patients.

[0222] Target sequence (human α-synuclein):

[0223] MDVFMKGLSK AKEGVVAAAE KTKQGVAEAA GKTKEGVLYV GSKTKEGVVH GVATVAEKTKEQVTNVGGAV VTGVTAVAQK TVEGAGSIAA ATGFVKKDQL GKNEEGAPQE GILEDMPVDP DNEAYEMPSEEGYQDYEPEA(SEQ ID No:19)

[0224] Related peptides:

[0225] GAPQEGIL (SEQ ID No: 20)

[0226] MPVDPDNEAY (SEQ ID No: 21)

[0227] MDVFMKGLSK (SEQ ID No: 22)

[0228] DVFMKGLSK (SEQ ID No: 23)

[0229] VFMKGLSKAK (SEQ ID No: 24)

[0230] GVVAAAEKTK (SEQ ID No: 25)

[0231] VVAAAEKTK (SEQ ID No: 26)

[0232] GVAEAAGKTK (SEQ ID No: 27)

[0233] KTKEGVLYV (SEQ ID No: 28)

[0234] GVLYVGSKTK (SEQ ID No: 29)

[0235] VLYVGSKTK (SEQ ID No: 30)

[0236] GVVHGVTTV (SEQ ID No: 31) (based on A53T mutant)

[0237] GVTTVAEKTK (SEQ ID No: 32) (based on A53T mutant)

[0238] AVVTGVTAV (SEQ ID No: 33)

[0239] VVTGVTAVA (SEQ ID No: 34)

[0240] VTGVTAVAQK (SEQ ID No: 35)

[0241] GSIAATGF ​​(SEQ ID No: 36)

[0242] SIAAATGFV (SEQ ID No: 37)

[0243] SIAAATGFVK (SEQ ID No: 38)

[0244] IAAATGFVK (SEQ ID No: 39)

[0245] GSIAAATGFVK (SEQ ID No: 40)

[0246] AAATGFVKK (SEQ ID No:41)

[0247] AAATGFVKKD (SEQ ID No:42)

[0248] AATGFVKK (SEQ ID No:43)

[0249] PVDPDNEAY (SEQ ID No: 44)

[0250] MPSEEGYQDY (SEQ ID No: 45)

[0251] PSEEGYQDY (SEQ ID No: 46)

[0252] Most preferably:

[0253] SIAAATGFV (SEQ ID No: 37) + HLA-A2 presentation domain

[0254] KTKEGVLYV (SEQ ID No: 28) + HLA-A2 presentation domain

[0255] 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 is preferred:

[0256] MPVDPDNEAY (SEQ ID No: 21) + HLA-A1 presentation domain

[0257] PVDPDNEAY (SEQ ID No: 44) + HLA-A1 presentation domain

[0258] VVAAAEKTK((SEQ ID No:26)+HLA-A11 presentation domain

[0259] VFMKGLSKAK (SEQ ID No: 24) + HLA-A11 presentation domain

[0260] AAATGFVKKD (SEQ ID No: 42) + HLA-A11 presentation domain

[0261] AATGFVKK (SEQ ID No: 43) + HLA-A11 presentation domain

[0262] MDVFMKGLSK (SEQ ID No: 22) + HLA-A11 presentation domain

[0263] GVVAAAEKTK (SEQ ID No: 25) + HLA-A11 presentation domain

[0264] GAPQEGIL (SEQ ID No: 20) + HLA-G presentation domain

[0265] The most preferred and preferred peptides mentioned above are preferred because the inventors found that based on online prediction algorithms (Syfpeithi, NetMHC), literature reviews on immunodominant regions, 3D protein folding predictions (AlphaFold2) and protein expression data as well as the experimental results reported in this specification, these epitopes are very likely to induce tolerance effects in Parkinson's patients.

[0266] Embodiment 3: Method used in the present invention

[0267] Method for producing the recombinant polypeptide of the present invention

[0268] -Expi-293F cells (Thermo Fisher), in Expi-293 TM Growth in expression medium (Thermo Fisher): using Expifectamine TM 293 transfection kit (Thermo Fisher) was used to transfect 1 μg of DNA into 2.5 × 10 6Cells / ml were added, DNA was complexed with Expifectamine using Opti-MEM (Thermo Fisher), and enhancers were added according to the protocol after 18-20 hours. Supernatants were harvested 4-6 days later (37°C, 8% CO2, humidified incubator) and 19 mm 2 Orbital shaker 125 rpm.

[0269] -Spot-tag protein purification: Spot-Cap resin equilibration: Transfer the required amount of slurry to an appropriate tube, pellet the beads by centrifugation (4°C, 4 min, 2500g), remove and discard the supernatant, add 10 bed volumes of PBS (cold) to the beads, invert to mix, pellet the beads by centrifugation (4°C, 4 min, 2500g), remove and discard the supernatant, repeat 2 times.

[0270] - Add the required volume of beads to the supernatant, incubate at 4°C on a rotator, wash the beads by repeated centrifugation (4°C, 4 min, 2500 g), and then remove the supernatant.

[0271] - Prepare 500 μM Spot peptide solution in PBS, remove supernatant, and incubate with 1 / 3 Spot peptide solution for 5-10 minutes.

[0272] Beads were pelleted by centrifugation. Protein concentration was performed using an Amicon Ultra-4 centrifugal filter (15 kDa cutoff) and Spot peptides were removed using a 15 kDa Amicon cutoff column.

[0273] Amicon Ultra-4 centrifugal filters (15 kDa cut-off) were rinsed with PBS and then with 0.1 N NaOH (centrifugation at 4000 g, 4°C) to remove traces of glycerol.

[0274] ELISPOT:

[0275] 1) Cell culture

[0276] A) PBMC isolation (under laminar flow hood)

[0277] To isolate peripheral blood mononuclear cells (PBMCs), density centrifugation was performed using leukocytes from a leukocyte reduction chamber and a density gradient medium (e.g., Ficoll or ROTI Sep 1077). Cells were centrifuged at 1200×g for 20 minutes without stopping, and then the interphase layer was collected and washed with 1×PBS (5 minutes, 300×g). PBMCs were frozen until further use.

[0278] B) PBMC pulse (under laminar flow hood)

[0279] PBMCs were thawed 1 day before PBMC pulse (d-1) and stored in 5 ml X-VIVO 15 medium containing 5% human AB serum in wells of 6-well plates at 37°C overnight.

[0280] The next day (d0), the cells were counted and suspended in X-VIVO 15 complete medium (5% hAB serum and cytokine mixture: 20 ng / ml hIL-2, 20 ng / ml hGM-CSF, 10 ng / ml hIL-4 and 10 ng / ml hTGF-b1) at a cell density of 3 × 10 6 cells / ml.

[0281] In the experiment, 3×10 6 Cells were seeded into the corresponding wells of a 12-well plate in a final volume of 1000 μl X-VIVO complete medium containing a cytokine mixture and

[0282] 5 μg / ml AIM Bio molecule (recombinant polypeptide of the present invention) or corresponding control.

[0283] On day 3, 1 ml of complete medium (containing cytokines) was added, and on day 6, a second pulse (after removal of medium) was performed with 5 μg / ml of the recombinant polypeptide of the present invention or its surrogate molecule (collectively referred to as "AIM Bio" molecules). On days 7, 10, and 12, 1 ml of complete medium (containing cytokines) was added.

[0284] Required:

[0285] X-VIVO 15 medium + 5% human AB serum

[0286] X-VIVO 15 complete medium: X-VIVO 15 medium + 2% human AB serum, supplemented with cytokine mixture: 10ng / ml TGF-b1, 10ng / ml IL-4, 20ng / ml IL-2, 20ng / ml GM-CSF

[0287] 6-well plate

[0288] 12-well plate

[0289] 2) ELISPOT

[0290] Laminar flow hood

[0291] On day 13, ELISpot plates were coated with anti-hIL10 (clone 9D-7, diluted 1:500 in PBS, sterile filtered) and aIL10 (10G8-biotin), and on day 14, 200,000 cells were seeded per well on the ELISpot plates in duplicate, including a negative control (cells plus PBS) and a positive control (e.g., LPS).

[0292] The PFDF membrane was activated with 50 μl / well EtOH (35% v / v) for 1 minute, and then washed 5 times with 200 μl distilled sterile water. The plate was coated with 100 μl / well antibody solution at 4°C overnight. The next day, unbound coating antibody was removed, washed 5 times with 200 μl PBS, and 200 μl blocking buffer (X-VIVO 15 5% hAB serum) was added, and the plate was incubated at room temperature for 30 minutes to 2 hours.

[0293] Prepare DMSO or DMSO containing the corresponding antigen peptide as a control, the final amount of peptide added is 5 μg peptide / ml, and the final volume is 100 μl / well. 150,000 cells are seeded per well in X-VIVO 15 medium containing 5% human AB serum. Carefully remove the blocking buffer (X VIVO 15 medium + 5% hAB serum), use a medium containing PBS as a negative control, and add the stimulator (total volume per well is 5 μg / ml) to other wells and incubate overnight at 37°C.

[0294] Outside the laminar flow hood

[0295] Prepare secondary antibodies: 1 μg / ml aIL-10-biotinylated antibody and horseradish peroxidase-conjugated streptavidin in 0.5% BSA / 1× PBS (1:1000 dilution) and filter the tetramethylbenzidine solution using a 0.45 μm filter and store at 4°C until use.

[0296] Remove the cell supernatant and wash five times with 100 μl PBS. Finally, wipe off the excess buffer with a paper towel.

[0297] 25 μl of diluted HRP-streptavidin (1:750) was added to each well and incubated for 1 hour at room temperature in the dark, followed by 5 washes with sterile 1× PBS.

[0298] Add 100 μl of filtered TMB substrate to each well and react for 15-25 minutes until blue spots appear. Rinse the wells thoroughly with tap water to terminate the reaction.

[0299] Remove the board's plastic drain and rinse the bottom and sides of the board with tap water and allow to dry.

[0300] Plate data were read using an ImmunoSpot S6 Ultra-V analyzer (Cellular Technology Limited), analyzed in Excel, and graphs / statistics were plotted in Graphad Prism.

[0301] Required:

[0302] Capture antibodies: anti-hIL10 (clone: ​​9D-7, Mabtech #3430-3-250; 1:500 dilution), anti-hIL10-biotinylated (Mabtech, #3430-6-250)

[0303] 1× PBS (sterile)

[0304] 35% ethanol (v / v)

[0305] Blocking buffer: X-vivo 5% hAB serum (sterile) [Block in the same medium as the cell culture medium]

[0306] Dilution buffer: 0.5% BAS in PBS

[0307] Washing buffer: 1× PBS

[0308] Culture medium: For T cells, X-VIVO 15 medium (Lonza)

[0309] Filter syringe: Millex GV

[0310] ELISPOT PVDF Plate (#MSIP4510, Millipore)

[0311] TMB substrate

[0312] Embodiment 4: Further proof of concept of the stability and efficacy of the recombinant polypeptides of the present invention.

[0313] In addition, the inventors set out to obtain and test recombinant polypeptides having the general structure of the recombinant polypeptides of the present invention but containing various different peptide antigens in order to obtain further proof of concept that the recombinant polypeptides of the present invention and their surrogate molecules are stable and effective. Fig. 9 and 10 As shown in Figure 2, the tested recombinant polypeptides were stable during freeze-thaw and storage and were thermostable. In addition, they induced Tregs ( Fig.11 ) and inhibited T cell lysis in a dose-dependent manner ( Fig.12 ). Fig.13The effects of recombinant peptides on the serum cytokine profile of EAE-ODC Ova mice are shown. IL-10 and possibly IL-4 are induced, both known immunosuppressive cytokines that downregulate immune responses in an inflammatory setting. This requires the HLA-G α3 domain plus the cognate peptide. The induction of IL-2 appears to result from presentation of the cognate peptide to the cells independent of the α3 domain. IL-2 is required for T cell activation and survival.

[0314] Fig.15 and 16 Further evidence of the stability of the recombinant polypeptides of the invention is also provided.

[0315] Example 5 : ELISpot results from PBMC of healthy donors ( Fig.17 ), and from Parkinson's patients ( Fig.18 A) and age-matched healthy individuals ( Fig.18 B) IL-10 ELISpot results. These experiments show that a significant induction of immunosuppressive cells caused by the constructs of the invention can be observed in vitro after 14 days both in healthy donor PBMCs and in patients PBMCs.

[0316] References:

[0317] 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.

[0318] 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.

[0319] Bas J,Calopa M,Mestre M,Mollevi DG,Cutillas B,Ambrosio S,Buendia E(2001),Lymphocytepopulations in Parkinson's disease and in rat models ofparkinsonism.J Neuroimmunol 113:146-152.

[0320] Brochard V,Combadiere B,Prigent A,Laouar Y,Perrin A,Beray-Berthat V,Bonduelle O,Alvarez-Fischer D,et al.(2009),Infiltration of CD4+lymphocytesinto the brain contributes toneurodegeneration in a mouse model of Parkinsondisease.J Clin Invest 119:182-192.

[0321] 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 thesubstantia nigra results indegeneration of the nigrostriatal system withLewy-like pathology and motor impairment:a newmouse model for Parkinson'sdisease.Acta Neuropathol Commun 5:11.

[0322] Lacan G,Dang H,Middleton B,Horwitz MA,Tian J,Melega WP,Kaufman DL(2013),BacillusCalmette-Guerin vaccine-mediated neuroprotection is associatedwith regulatory T-cell induction inthe 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease.J NeurosciRes 91:1292-1302.

[0323] Liu J,Gong N,Huang X,Reynolds AD,Mosley RL,Gendelman HE(2009),Neuromodulatoryactivities of CD4+CD25+regulatory T cells in a murine model ofHIV-1-associatedneurodegeneration.J Immunol 182:3855-3865.

[0324] Magistrelli L,Comi C(2020),Beta2-Adrenoceptor Agonists in Parkinson'sDisease and OtherSynucleinopathies.J Neuroimmune Pharmacol 15:74-81.

[0325] McGeer PL,Itagaki S,Boyes BE,McGeer EG(1988),Reactive microglia arepositive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer'sdisease 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,epidermalgrowth factor and transforming growth factor-alpha are elevated inthe brainfrom parkinsonian patients.Neurosci Lett 180:147-150.

[0326] Mogi M,Harada M,Riederer P,Narabayashi H,Fujita K,Nagatsu T(1994),Tumor necrosisfactor-alpha(TNF-alpha)increases both in the brain and in thecerebrospinal fluid from parkinsonianpatients.Neurosci Lett 165:208-210.

[0327] Musacchio T,Rebenstorff M,Fluri F,Brotchie JM,Volkmann J,Koprich JB,Ip CW(2017),Subthalamic nucleus deep brain stimulation is neuroprotective inthe A53T alpha-synuclein Parkinson's disease rat model.Ann Neurol 81:825-836.

[0328] 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 AmongPatients With Inflammatory Bowel Disease.JAMA Neurol 75:939-946.

[0329] Racette BA,Gross A,Vouri SM,Camacho-Soto A,Willis AW,Searles NielsenS(2018),Immunosuppressants and risk of Parkinson disease.Ann Clin TranslNeurol 5:870-875.

[0330] Reynolds AD,Banerjee R,Liu J,Gendelman HE,Mosley RL(2007),Neuroprotective activities of CD4+CD25+regulatory T cells in an animal modelof Parkinson's disease.J Leukoc Biol82:1083-1094.

[0331] Reynolds AD,Stone DK,Hutter JA,Benner EJ,Mosley RL,Gendelman HE(2010),Regulatory T cells attenuate Th17 cell-mediated nigrostriataldopaminergic neurodegeneration in a model of Parkinson's disease.J Immunol184:2261-2271.

[0332] 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.

[0333] Saunders JA, Estes KA, Kosloski LM, Allen HE, Dempsey KM, Torres-RussottoDR, Meza JL, Santamaria PM, et al. (2012), CD4+regulatory and effector / memory Tcell subsets profile motor dysfunction in Parkinson's disease. J NeuroimmunePharmacol 7:927-938.

[0334] Storelli E, Cassina N, Rasini E, Marino F, Cosentino M (2019), Do Th17Lymphocytes and IL-17Contribute to Parkinson's Disease? A Systematic Review ofAvailable 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. Nature546:656-661.

[0335] Industrial Applicability

[0336] The pharmaceutical compositions, polypeptides, nucleic acids, cells and products used in the present invention have industrial application value, for example, they can be used to manufacture medicines or used 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 α-synuclein; ii) optionally, a linker sequence; iii) optionally, a human polypeptide domain sequence, the human polypeptide domain sequence comprising a human β2 microglobulin sequence, or an amino acid sequence that is at least 90% identical to the human β2 microglobulin amino acid sequence shown in SEQ ID No: 5; iv) optionally, a linker sequence; v) optionally, an α1 domain of an MHC molecule; vi) optionally, an α2 domain of an MHC molecule; vii) an α3 domain of an MHC class Ib molecule or a derivative of the α3 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) Optionally, an affinity tag.

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

3. The recombinant polypeptide according to claim 1 or 2, wherein the peptide antigen according to i) consists of an amino acid sequence selected from the group consisting 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 according to any one of the preceding claims, wherein the α1 domain according to (v) and the α2 domain according to (vi) are from a human MHC class Ia molecule or from a human MHC class Ib molecule. 5 . The recombinant polypeptide according to claim 4 , wherein the α1 domain according to (v) and the α2 domain according to (vi) are from a human MHC class Ib molecule, and wherein the human MHC class Ib molecule is a human HLA-G molecule.

6. The recombinant polypeptide according to claim 5, wherein the α1 domain according to (v) and the α2 domain according to (vi) are from human MHC class Ia molecules, and wherein the α1 domain according to (v) and the α2 domain according to (vi) are from human HLA-A2 molecules, human HLA-A1 molecules or human HLA-A11 molecules.

7. The recombinant polypeptide according to any one of the preceding claims, wherein the α3 domain of the MHC class Ib molecule according to (vii) is the α3 domain of human HLA-E, human HLA-F or human HLA-G, preferably human HLA-G.

8. A recombinant polypeptide according to any of the above claims, wherein the α3 domain or derivative according to (vii) is identical to the α3 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 with the α3 domain having the amino acid sequence of SEQ ID No: 9 or SEQ ID No: 2, or is identical to the α3 domain having the amino acid sequence of SEQ ID No: 9 or SEQ ID No:

2.

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

10. A recombinant polypeptide according to any one of the preceding claims, wherein the sequence of the human polypeptide domain according to (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 according to any one of the above claims, wherein the polypeptide is a dimer or a multimer.

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

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

14. The recombinant polypeptide according to any one of claims 1 to 12, wherein the recombinant polypeptide consists of the following amino acid sequence, which consists of the following ((a) and (b)) in order from N-terminus to C-terminus: (a) a peptide antigen selected from the group consisting 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, The group consisting of the amino acid sequences of 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; (b) The amino acid sequence of SEQ ID No:

16.

15. The recombinant polypeptide according to any one of the above claims, wherein the recombinant polypeptide is soluble.

16. A nucleic acid encoding one or more polypeptides according to any one of the preceding claims, 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 any one of claims 1-15.

19. The pharmaceutical composition or kit according to claim 18, wherein the pharmaceutical composition or kit comprises at least two different recombinant polypeptides according to any one of claims 1 to 15, and wherein each different polypeptide comprises a different peptide antigen as defined in claim 3.

20. The pharmaceutical composition or kit according to any one of claims 17 to 19, for use in treating Parkinson's disease in a human patient.

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

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

23. A recombinant host cell comprising the nucleic acid or vector according to claim 16, and expressing the recombinant polypeptide according to any one of claims 1-15.

24. A method for obtaining a pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 15, the method comprising the following steps: (a) culturing the recombinant host cell of claim 23 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.

Images