MHC Ib-mediated myelin-specific immunosuppression as a novel therapy for multiple sclerosis and anti-MOG antibody-driven diseases

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 myelin-associated peptide antigens for the treatment of multiple sclerosis (MS), anti-MOG antibody-driven diseases, and anti-MOG antibody-positive neuromyelitis optica. The present invention more particularly 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 same, and their use for treating multiple sclerosis (MS), anti-MOG antibody-driven diseases, and anti-MOG antibody-positive neuromyelitis optica.

Description

[Technical field]

[0001] 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 multiple sclerosis (MS), anti-MOG antibody disease, and anti-MOG antibody-positive neuromyelitis optica. The present invention more particularly 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 same, and their use for treating multiple sclerosis (MS), anti-MOG antibody disease, and anti-MOG antibody-positive neuromyelitis optica. [Background technology]

[0002] Multiple sclerosis (MS) and MOG encephalomyelitis (also known as myelin oligodendrocyte glycoprotein antibody-mediated disease, anti-MOG antibody-mediated disease, or MOGAD) are autoimmune diseases in which T cells of the immune system attack the myelin sheaths of the central nervous system, causing slowly progressive neurodegeneration. The sporadic relapses of disease activity that characterize MS can be effectively suppressed by immunomodulatory treatments. There is no approved treatment for the rare MOGAD, which has long been considered a variant of MS, so treatment consists of watching and waiting for the progression of the disease in the early stages. In both diseases, progressive neurodegeneration occurs slowly. Some drugs that are effective in MS, such as the antibodies natalizumab or ocrelizumab, or the orally bioavailable S1P inhibitor fingolimod, even carry the risk of a persistent and usually harmless JC virus infection in the brain becoming uncontrollable, leading to the often fatal progressive multifocal leukoencephalopathy. In this regard, in both diseases, the problem of either insufficient immunomodulation or excessive immunomodulation with side effects remains unsolved, especially in the case of slow progression. Widely used biological drugs cannot cross the blood-brain barrier and therefore cannot have an anti-inflammatory effect in situ. In contrast, regulatory cells have been described to cross the blood-brain barrier (Schneider-Hohendorf et al., Eur J Immunol. 2010 Dec;40(12):3581-90).

[0003] Two approaches have been evaluated so far, at least in early clinical studies. Similar to hyposensitization therapy for allergies, large amounts of antigens are administered by different routes to induce antigen-specific tolerance. However, in autoimmune diseases, these approaches cause severe side effects and have not been clinically successful. A more promising approach is to induce tolerance by adoptive transfer of antigen-specific regulatory T cells or antigen-loaded tolerogenic dendritic cells. However, these approaches are very cumbersome and expensive, and require GMP-compliant production and quality control processes for each patient. Therefore, even if small-scale clinical trials are successful, it is questionable whether adoptive transfer therapy will be available to a large number of patients in the near future.

[0004] Immunosuppressive MHC class Ib molecules, such as HLA-G, are important for tolerance induction during pregnancy. They exert immunosuppressive effects on various immune cells via immunosuppressive receptors, such as ILT2, ILT4 and Kir2DL4. WO 2018 / 215340 relates to the combination of MHC class Ib molecules with peptides for targeted therapeutic immune modulation.

[0005] Overall, there remains a need for improved drugs for the treatment of multiple sclerosis (MS) and anti-MOG antibody-driven diseases. Similarly, there remains a need for improved drugs for the treatment of anti-MOG antibody-positive neuromyelitis optica (NMO). Summary of the Invention [Problem to be solved by the invention]

[0006] 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 therein. 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 similar structure and sequence to classical human MHC class Ia molecules, which induce antigenic 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 comprising 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.

[0007] Antigen-loaded HLA-G molecules can be unstable. Therefore, the inventors designed soluble recombinant polypeptides containing a peptide antigen, an MHC class Ib molecule such as HLA-G, and β2 microglobulin (b2m), and covalently linked these three components (e.g., via a covalent linker). Alternatively, the antigen-binding α1 and α2 domains of 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).

[0008] Surprisingly, the present inventors have found that the recombinant polypeptide of the present invention can be used to suppress immune responses against human myelin oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1). Thus, according to the present invention, multiple sclerosis (MS), anti-MOG antibody-induced disease, and anti-MOG antibody-positive neuromyelitis optica can be treated with the recombinant polypeptide of the present invention.

[0009] Our experimental data show that the presence of an appropriate peptide antigen and the [α]3 domain of an MHC class Ib molecule (e.g., HLA-G) is necessary to obtain the desired effect. Thus, this approach goes beyond previous methods that use antigen peptides without costimulation (leading to anergic rather than tolerogenic T cells) or MHC class Ib molecules in an antigen-nonselective context.

[0010] Furthermore, according to the present invention, the recombinant polypeptide of the present invention is expected to be highly effective in the immunotherapy of multiple sclerosis (MS), anti-MOG antibody-mediated diseases, and anti-MOG antibody-positive neuromyelitis optica, since it is expected to exhibit an improved safety profile compared to conventional drugs for these diseases which may be accompanied by serious side effects such as progressive multifocal leukoencephalopathy.

[0011] In addition, the present inventors have surprisingly found from model experiments that the polypeptide of the present invention not only regulates T cell responses but also prevents the formation of MOG-specific autoantibodies. Because MOG-specific autoantibodies are involved in these pathologies, this finding is expected to lead to clinical improvement in patients with multiple sclerosis (MS), anti-MOG antibody-induced disease, and MOG antibody-positive neuromyelitis optica. [Means for solving the problem]

[0012] Thus, the present invention relates to the following preferred embodiments: 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 myelin oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1); 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 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. 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. 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 the amino acid sequences of SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33. 4. 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 the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28 and SEQ ID NO: 30. 5. A recombinant polypeptide according to any one of the preceding claims, wherein the peptide antigen according to i) is a peptide of human myelin oligodendrocyte glycoprotein (MOG).

[0013] 6. The recombinant polypeptide according to any one of items 1 to 2 and 4 to 5, wherein the peptide antigen consists of the amino acid sequence of SEQ ID NO: 2. 7. 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. 8. The recombinant polypeptide according to item 7, 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. 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 HLA-A2 molecule. 10. The recombinant polypeptide according to item 7, wherein the [α]1 domain described in (v) and the [α]2 domain described in (vi) are derived from an MHC class Ib molecule.

[0014] 11. A recombinant polypeptide described in any one of the preceding items, 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. 12. 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. 13. 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, with an [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 21. 14. The recombinant polypeptide according to item 13, 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. 15. A recombinant polypeptide according to item 13, 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: 21.

[0015] 16. The recombinant polypeptide according to item 13, 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. 17. A recombinant polypeptide according to item 13, 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: 21. 18. A recombinant polypeptide according to item 13, 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: 21. 19. The recombinant polypeptide according to item 13, 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: 21. 20. A recombinant polypeptide described in 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 of 1 or greater.

[0016] 21. The recombinant polypeptide according to item 20, 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. 22. A recombinant polypeptide according to item 20 or 21, 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. 23. 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. 24. A recombinant polypeptide according to any one of the preceding claims, wherein the polypeptide is a dimer or multimer. 25. A recombinant polypeptide according to any one of the preceding claims, wherein the polypeptide comprises or consists of all of components i) to vii).

[0017] 26. A recombinant polypeptide according to any one of the preceding claims, wherein the polypeptide does not comprise components viii) to x). 27. A recombinant polypeptide according to any one of items 1 to 25, wherein the polypeptide comprises or consists of all of components i) to x). 28. The recombinant polypeptide of any one of the preceding claims, further comprising an N-terminal secretory signal peptide sequence. 29. The recombinant polypeptide according to any one of items 1 to 27, wherein the recombinant polypeptide has 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:2, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33; and (b) The amino acid sequence of SEQ ID NO:16. 30. A recombinant polypeptide according to any one of the preceding paragraphs, wherein the recombinant polypeptide is soluble.

[0018] 31. A nucleic acid encoding one or more polypeptides according to any one of the preceding paragraphs. 32. The nucleic acid according to item 31, wherein the nucleic acid is a vector. 33. A pharmaceutical composition comprising at least one nucleic acid according to item 31 or 32. 34. A pharmaceutical composition or kit comprising at least one recombinant polypeptide according to any one of items 1 to 30. 35. The pharmaceutical composition or kit according to item 34, wherein the pharmaceutical composition or kit comprises at least two different recombinant polypeptides according to any one of items 1 to 30, each of the different polypeptides comprising a different peptide antigen as defined in any one of items 3 to 6.

[0019] 36. The pharmaceutical composition or kit according to any one of items 33 to 35, for use in treating multiple sclerosis (MS), anti-MOG antibody-mediated disease, or anti-MOG antibody-positive neuromyelitis optica in a human patient. 37. The pharmaceutical composition or kit for use according to item 36, wherein the treatment is treatment of multiple sclerosis (MS). 38. The pharmaceutical composition or kit for use according to item 36, wherein the treatment is treatment of an anti-MOG antibody-mediated disease. 39. The pharmaceutical composition or kit for use according to item 36, wherein the treatment is treatment of anti-MOG antibody-positive neuromyelitis optica. 40. The pharmaceutical composition or kit according to any one of items 36 to 39, wherein the treatment is an immunotherapy treatment.

[0020] 41. A pharmaceutical composition or kit for use according to any one of items 36 to 40, wherein the treatment is by inducing immune tolerance to myelin oligodendrocyte glycoprotein. 42. The pharmaceutical composition or kit according to any one of items 36 to 41, wherein the treatment is for reducing plasma or cerebrospinal fluid levels of autoantibodies against human myelin oligodendrocyte glycoprotein. 43. The pharmaceutical composition or kit for use according to any one of items 36 to 42, wherein the human patient is a patient who has plasma or cerebrospinal fluid autoantibodies against myelin oligodendrocyte glycoprotein before the start of treatment. 44. A pharmaceutical composition or kit for use according to any one of items 36 to 43, wherein the treatment is by inducing myelin-specific regulatory T cells. 45. A recombinant host cell comprising the nucleic acid or vector according to item 31 or 32 and expressing a recombinant polypeptide according to any one of items 1 to 30. 46. ​​A method for obtaining a pharmaceutical composition comprising a polypeptide according to any one of items 1 to 30, the method comprising the steps of: (a) culturing a recombinant host cell according to item 45 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]

[0021] [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 created 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]Examples of vector constructs encoding single-chain MHC Ib molecules 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 I complex through non-covalent binding. To facilitate purification of the complex MHC Ib molecule, one or more protein tags (such as Spot tag, myc tag and / or His(6x) tag) may be introduced. These can be introduced for subsequent removal if necessary, by cleavage with optional factor Xa or Furin cleavage sites. Furthermore, the antigen peptide, β2 microglobulin and MHCIb[α] chain can be linked to increase stability. Vector maps are generated using Snapgene Viewer Software. [Figure 3A]Surrogates of recombinant polypeptides of the invention induce IL10-secreting Tregs in mice. In this experiment, 100 μg of surrogate molecules consisting of viral (Gp34) or ovalbumin (Ova) model peptide antigens, mouse H2-Kb-[α]1 and 2 domains, and human HLA-G[α]3 domain and β2 microglobulin were administered intraperitoneally to 12-week-old C57BL / 6 mice. After 14 days, mice were sacrificed and Ficoll-isolated splenocytes were rechallenged with 5 μg / ml of Gp34 or Ova peptides in a standard mouse IL10 ELISpot assay (Mabtech Mouse IL-10 HRP ELISpot Kit) for 48 hours (A). A significant increase in IL-10-secreting regulatory T cells was detected only upon rechallenge with peptides to which tolerance had been induced by injection of the surrogate molecules (B). [Figure 3B] Continued from Figure 3A. [Figure 4A]Surrogates of the recombinant polypeptides of the invention prevent CD8+ T cell-induced EAE in mice. In this mouse model of MS, the model antigen ovalbumin (OVA) is expressed in oligodendrocytes under the control of the myelin basic protein (MBP) promoter (ODC-OVA). This results in the presentation of the OVA257-264 peptide on the H-2Kb MHC molecule on oligodendrocytes. OT-I mice express a T cell receptor (OT-I) on CD8+ T cells that recognizes exactly this peptide-MHC combination. When CD8+ T cells from these mice are transferred into 10-day-old ODC-OVA mice, these mice develop experimental autoimmune encephalomyelitis (EAE), which in many ways resembles the pathology and symptoms of MS (Na et al., Brain, Volume 131, Issue 9, September 2008, Pages 2353-2365). In this experiment, 500 μg of virus (Gp34) or ovalbumin (Ova) model peptide antigens, mouse-derived H2-Kb-[α]1 and 2 domains, and surrogate molecules consisting of human HLA-G[α]3 domain and β2 microglobulin, or PBS alone were injected on the same day. EAE was scored according to Bittner et al., J Vis Exp. 2014 Apr 15;(86):51275. Only surrogate molecules inducing ovalbumin tolerance almost completely prevented EAE symptoms. (A) Experimental design, (B) Results. [Figure 4B] Continued from Figure 4A. [Figure 5A]Surrogates of recombinant polypeptides of the present invention can lead to effective bystander immune suppression. In this experiment, we tested whether Mog44 peptide surrogate molecules can induce protective T cells that can inhibit cytotoxic T cells targeting another peptide presented on the same cells. 250 μg of this molecule per mouse was injected into ODC-OVA mice together with OT-I cells as shown in FIG. 4. Although such a small amount of surrogate molecule did not completely prevent EAE symptoms, it was significantly reduced both by the molecule inducing tolerance to the directly targeted CD8 epitope (Ova_KbG) or to another epitope (Mog44) on the same cells (Mog44_DbG). (A) EAE score; (B) body weight. [Figure 5B] Continued from Figure 5A. [Figure 6A] Some of the surrogates of the recombinant polypeptides of the present invention selectively prevent CD4+ T cell-induced EAE in mice. (A) Experimental design: In this model, administration of MOG 35-55 peptide in combination with complete Freund's adjuvant, which activates CD4+ Th17 cells, and pertussis toxin, which increases the permeability of the blood-brain barrier, induces a strong myelin-specific autoimmune response (Protocol: Bittner et al., J Vis Exp. 2014 Apr 15; (86): 51275). Here, not only antibodies but also CD4+ cells play an important role in the development of EAE (Tigno-Aranjuez et al., J Immunol November 1, 2009, 183 (9) 5654-5661). In addition, 100 μg / mouse of surrogate molecules consisting of a viral antigen (Gp34) or two Mog peptide antigens (Mog37 or Mog44), mouse H2-Dbα1 and 2 domains, and human HLA-Gα3 domain and β2 microglobulin, or PBS alone were injected on the first day. The Mog44 peptide-containing surrogate molecules significantly reduced EAE symptoms (B) and weight loss (C). [Figure 6B] Continued from Figure 6A. [Figure 6C] Continued from Figure 6B. [Figure 7A] Mog44 surrogates of the recombinant polypeptides of the present invention prevented inflammation and CD8 T cell infiltration in the spinal cord. (A) Toluidine; (B) CD8-DAB. Fresh frozen sections of 10 μm were stained with a commercially available toluidine 1x staining reagent at room temperature for 1 hour. Strong infiltration of immune cells was detected in EAE, but was prevented by Mog44_Db_G. Fresh frozen sections of 10 μm were dried briefly at room temperature, fixed with acetone, blocked with 5% BSA 10% normal goat serum in PBS, and stained with 1:100 anti-CD8 antibody, HRP-conjugated secondary antibody, and DAB solution (detailed method: Karikari et al., Brain Behav Immun. 2022 Jan 12; 101: 194-210). EAE induced by Mog35-55 leads to strong infiltration of CD8+ cells into the spinal cord, but this was prevented by the MOG44_Db_G surrogate molecule. (A) Toluidine; (B) CD8-DAB. [Figure 7B] Continued from Figure 7A. [Figure 8] Detection of anti-MOG35-55 antibodies in Mog-EAE mice administered recombinant polypeptide surrogates of the invention ("AIM Bio"). After sacrificing the mice, mouse serum was collected by cardiac puncture and diluted 1:50 in PBS. Wells were coated overnight with 10 μg / ml Mog35-55 in PBS and then blocked with 1% BSA for 2 hours before adding diluted serum for 1 hour. Anti-Mog35-55 antibodies were detected using the indicated HRP-conjugated secondary antibodies (1:5000 dilution in PBS). Mog35-55-induced EAE correlated with high levels of Mog35-55-specific IgG autoantibodies, which were undetectable in animals administered 100 μg of MOG44_Db_G surrogate molecule. [Figure 9]List of human MS and MOGAD recombinant polypeptide candidates. Myelin (MAG, MBP, MOG, PLP) peptides and MHC class I presenting molecules are as follows. Furthermore, it is shown that combinations of myelin peptides and antigen presenting MHC class I α1 and 2 domains (HLAG=HLA-G, A2G=HLA-A2 presenting domain+HLA-Gα3 domain) give good results in terms of expression / production, ELISpot based prioritization or AlphaFold2 prediction in healthy blood donors (as shown in FIG. 10). His denotes a 6-histidine tag. Spt denotes a spot tag.

[0022] TIFF2025513720000001.tif204116 [Figure 10]Upregulation of CD8 Tregs in healthy blood donors by recombinant polypeptide of the invention ("Mog157_A2G") containing the VLLAVLPVL antigen. In vitro Treg induction mediated by AIM Biologicals was performed as follows: PBMCs from healthy donors were purified by density gradient centrifugation using Ficoll on white blood cells obtained from a leukapheresis chamber. Cells were centrifuged at 1200xg for 20 min without brake, after which the interphase was collected and washed with 1xPBS (5 min, 300xg). PBMCs were cryopreserved until further use. PBMCs were thawed one day (d-1) before PBMC pulsing and kept overnight at 37°C in 5 ml of X-VIVO 15 medium containing 5% human AB serum in wells of a 6-well plate. The next day (d0), cells were counted and resuspended at a cell density of 3x106 cells / ml in X-VIVO 15 complete medium (5% hAB serum and cytokine cocktail: 20ng / ml hIL-2, 20ng / ml hGM-CSF, 10ng / ml hIL-4 and 10ng / ml hTGF-b1). For the experiments, 3x106 cells were seeded in each well of a 12-well plate in a final volume of 1000μl of X-VIVO complete medium (containing cytokine cocktail and 5μg / ml AIM Bio molecule or respective control). On day 3, 1ml of complete medium (containing cytokines) was added and on day 6, a second pulse treatment was performed with 5μg / ml AIM Bio molecule (after removing the medium). On days 7, 10 and 12, 1ml of complete medium (containing cytokines) was added. On day 13, the PVDF membrane of the ELISPOT plate was activated with 50 μl / well EtOH (35% v / v) for 1 min and then washed 5 times with 200 μl sterile distilled water. The plate was coated overnight at 4 °C with 100 μl / well anti-hIL10 (clone 9D-7, 1:500 dilution in PBS, sterile filtered). 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 the plate was incubated at room temperature for 30 min to 2 h.On day 14, 200,000 cells were plated per well in duplicate on ELISPOT plates containing negative control (cells + PBS) and positive control (e.g. LPS). Secondary antibodies were prepared: 1 μg / ml aIL-10 biotinylated antibody in 0.5% BSA / 1xPBS (1:1000 dilution), and horseradish peroxidase-conjugated streptavidin (1:750 dilution in 0.5% BSA / PBS). Tetramethylbenzidine solution was filtered through a 0.45 μm filter and stored at 4°C until use. Cell supernatant was removed and washed 5 times with 100 μl PBS. Finally, excess buffer was removed with a paper towel. 25 μl of diluted HRP-streptavidin (1:750) was added to each well and incubated at room temperature in the dark for 1 h, followed by washing 5 times with sterile 1xPBS. 100 μl of filtered TMB substrate was added to each well for 15-25 minutes until blue spots were observed. The reaction was stopped by thoroughly washing the wells with tap water. The plastic underdrain of the plate was removed and the bottom and sides of the plate were washed with tap water and dried. MOG157_A2G_Spt induced at least 30% more IL-10-secreting Tregs in 75% of all healthy blood donors. [Figure 11] Control experiment for Figure 8 showing that total IgG is not reduced by MOG47_Db_G surrogate molecule treatment. Total IgG was quantified using the Easy-Titer™ Human IgG (γ Chain) Assay Kit (Thermo Fisher) according to the manufacturer's instructions. This experiment, together with Figure 8, shows that selective antibody responses can be suppressed by single chain MHCIb molecules. [Figure 12A]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 12B] Continued from Figure 12A. [Figure 13] 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 (5000x stock, 5x final concentration) 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 increased by 1°C per min, to a final temperature of 95°C for 2 min. 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 14]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 15] Single chain MHC Ib molecules inhibit T cell lysis in a dose-dependent manner. 10 mio OT-1 / ml in the presence of single chain MHC Ib molecules for 3 days, 2 hours at 37°C, shaking at 125 rpm, OT-1:Panc02 ratio 50:1. Methods: Isolation and processing of splenocytes. OT1 / BL6 mice were sacrificed and 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 minutes. Cells were cultured at high density (107 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 off the plates and CD8+ cells were purified with magnetic beads. Luciferase assay (48 hours). Sterile 96-well white plates were used. Panc02fluc+ target cells were loaded with 20μg / ml Ova peptide (SIINFEKL) and shaken at 500 rpm at 37℃ for 60 minutes. CD8+ effector T cells and PancO2 target cells (5000 target cells) were mixed at a ratio of 50:1 and luciferin was added. Luminescence was measured at 0, 24, and 48 hours. [Figure 16]Serum cytokines from EAE-ODC Ova mice. Serum cytokines from 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. This array kit provides a mixture of ten capture beads, each coated with a capture antibody specific to each cytokine, with a different fluorescence intensity. The ten specific capture antibody-coated beads 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 hours at room temperature in the dark. Samples were washed with 1 mL of wash buffer for 5 minutes 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 17A] Immunofluorescence analysis of the spinal cord of MOG-induced EAE model. A, B: Caspase 3 infiltration in MOG-induced EAE spinal cord, A: image, B: quantification. C, D: Lesions in MOG-induced EAE white matter in the spinal cord, C: image, D: quantification. E, F: CD3 infiltration in MOG-induced EAE spinal cord, E: image, F: quantification. [Figure 17B] Continued from Figure 17A. [Figure 17C] Continued from Figure 17B. [Figure 17D] Continued from Figure 17C. [Figure 17E] Continued from Figure 17D. [Figure 17F] Continued from Figure 17E. [Figure 18] Increase in IL10 spots following MOG157 A2G treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] 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 by reference in their entirety for all purposes. Publications mentioned herein may be cited by specifying the complete literature reference in the text.

[0024] 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 methods for producing recombinant polypeptides. The recombinant polypeptides of the present invention can be expressed in recombinant host cells according to the present invention. The recombinant host cells of the present invention are preferably mammalian cells, such as CHO cells and HEK cells.

[0025] 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.

[0026] 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.

[0027] Likewise, it will be understood that the recombinant polypeptides of the present invention are intended to optionally include the respective propeptides.

[0028] It will also be understood that the recombinant polypeptide of the present invention may be in a 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.

[0029] 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.

[0030] According to the present invention, the MHC molecule is preferably a human MHC molecule.

[0031] The recombinant polypeptide of the present invention is preferably an isolated recombinant polypeptide.

[0032] It will be understood how recombinant polypeptides capable of binding and presenting peptide antigens of the invention may 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 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.

[0033] According to the present invention, the recombinant polypeptides, pharmaceutical compositions and kits of the invention are preferably suitable for use in human patients.

[0034] 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 multiple sclerosis (MS), anti-MOG antibody-mediated disease and anti-MOG antibody-positive neuromyelitis optica in human patients.

[0035] According to the present invention, the recombinant polypeptides, pharmaceutical compositions and kits of the present invention are preferably suitable for inducing immune tolerance to human myelin oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin associated glycoprotein (MAG) or human myelin proteolipid protein (PLP1), e.g. in a human patient.

[0036] According to the present invention, it is understood that the recombinant polypeptides, pharmaceutical compositions and kits of the present invention are stable.

[0037] 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.

[0038] In accordance with the present invention, each occurrence of the term "comprising" may optionally be replaced with the term "consisting of."

[0039] 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.

[0040] 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.

[0041] For example, the binding of MHC class Ib molecules or recombinant polypeptides of the 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 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.

[0042] 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 identity values ​​obtained using the BLAST algorithm.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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).

[0049] Peptide antigens are widely known in the art. Generally, the peptide antigens of the present invention can bind to MHC class I proteins. 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.

[0050] 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 by 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; - Rock, Reits, Neefjes.Present Yourself! By MHC Class I and MHC Class II Molecules.Trends Immunol.2016 Nov;37(11):724-737.

[0051] Such methods include experimental methods and methods that predict peptide-antigen binding.

[0052] 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.

[0053] 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.

[0054] 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 et al.Highly accurate protein structure prediction with AlphaFold.Nature 596, 583-589(2021).

[0055] In the present invention, the peptide antigen is derived from human myelin oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1).

[0056] 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 myelin oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1).

[0057] The peptide antigens of the present invention are preferably composed of naturally occurring amino acids. However, it is possible to use non-naturally occurring amino acids, such as modified amino acids. For example, in one embodiment, the peptide antigens of the present invention include peptidomimetics of the indicated peptide antigen amino acid sequence of human myelin oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1).

[0058] Methods for the synthesis of peptidic antigens, including those of the present invention, are well known in the art.

[0059] Therapeutic Applications of the Invention The recombinant polypeptide of the present invention can be used to treat multiple sclerosis, anti-MOG antibody-induced disease, and anti-MOG antibody-positive neuromyelitis optica.

[0060] The treatment can be by inducing myelin-specific Tregs. Such Tregs (e.g., CD8+ Tregs) are activated in myelinated structures and provide target cell protection against cytotoxic T cells that recognize the same or different myelin antigens. Tregs (e.g., CD8+ Tregs) are known in the art and can be detected, for example, by secretion of IL-10.

[0061] CD8+ Tregs are less well known than CD4CD25Tregs, but have even been reported to be more potent. See, for example: Junfeng Liu, Dacan Chen, Golay D. Nie and Zhenhua Dai CD8+CD122+T-Cells:A Newly Emerging Regulator with Central Memory Cell Phenotypes.Front.Immunol.doi:10.3389 / fimmu.2015.00494; and Niederlova, V., Tsyklauri, O., Chadimova, T. and Stepanek, O. (2021), CD8 + Tregs revisited: A heterogeneous population with different phenotypes and properties. Eur. J. Immunol., 51:512-530. https: / / doi.org / 10.1002 / eji.202048614.

[0062] These (CD8 positive Tregs) are characterized by expression of CD122 and CD8 in mice, whereas the corresponding cells in humans have been reported to be CD8 and CXCR3 positive. Shi Z, Okuno Y, Rifa'i M, Endharti AT, Akane K, Isobe K, et al.Human CD8+CXCR3+T cells have the same function as murine CD8+CD122+Treg.Eur J Immunol(2009)39:2106-2119.doi:10.1002 / eji.200939314).

[0063] The treatment according to the invention may be a treatment for reducing plasma or cerebrospinal fluid levels of autoantibodies against human myelin oligodendrocyte glycoprotein. The human patient may be a patient who has plasma or cerebrospinal fluid autoantibodies against myelin oligodendrocyte glycoprotein before the start of the treatment.

[0064] According to the present invention, autoantibodies can be detected by various methods known in the art. A preferred approach is a cell-based assay (CBA) in which the antigen suspected to be the target of the autoantibody (e.g., myelin oligodendrocyte glycoprotein) is overexpressed in HEK293 or CHO cells, and the cells are then incubated with serum or cerebrospinal fluid, typically for 1 hour at room temperature. Mock-transfected sister cells serve as controls. Autoantibodies bound to the cells are detected with different fluorescently labeled anti-human specific secondary antibodies that recognize total human IgG (heavy and light chains), IgG-Fc (constant chain) or IgG1. Binding is quantified and often titrated by flow cytometry (CBA-FACS) or visual assessment of immunofluorescence by microscopy (CBA-IF). In the majority of established cell-based assays to detect MOG antibodies, the 218 amino acid A1 isoform of MOG is used, but an alternative approach, a truncated form of MOG with only the extracellular immunoglobulin and transmembrane domains, has also been tested. Other approaches such as enzyme-linked immunosorbent assay (ELISA) or Western blot are possible but often have lower sensitivity because conformationally sensitive antibodies may not be detected by these methods. Suitable approaches are described below: Waters, P., Pettingill, P. & Lang, B. Detection methods for neural autoantibodies. Handb. Clin. Neurol. 133, 147-163 (2016).

[0065] A detailed review of myelin oligodendrocyte glycoprotein (MOG) antibodies can be found in: Reindl M, Waters P.Myelin oligodendrocyte glycoprotein antibodies in neurological disease.Nat Rev Neurol.2019 Feb;15(2):89-102.doi: 10.1038 / s41582-018-0112-x.PMID:30559466.

[0066] 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.

[0067] Examples of sequences which are part of the recombinant polypeptides of the invention: Optional 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., VLLAVLPVL (SEQ ID NO: 2) (most preferred) First linker: for example, GGGGSGGGSGGGGS (SEQ ID NO: 3) or GCGASGGGGSGGGGS (SEQ ID NO: 4)

[0068] β2 microglobulin, for example: TIFF2025513720000002.tif14158 (SEQ ID NO:5, human β2 microglobulin) A second linker, for example: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 6)

[0069] [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: TIFF2025513720000003.tif22159 (SEQ ID NO: 7) Or: human HLA-A2[α] 1 and 2 domains: e.g. TIFF2025513720000004.tif22159 (SEQ ID NO:8)

[0070] 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: TIFF2025513720000005.tif15158 (sequence number 9; sequence of HLA-G[α]3).

[0071] Of note, the following underlined amino acids in this sequence are relevant for ILT2 or ILT4 receptor interaction: TIFF2025513720000006.tif15158

[0072] 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: 20), i.e.: TIFF2025513720000007.tif14158 (SEQ ID NO: 21),

[0073] 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.

[0074] Further (alternative) examples of peptide antigens which can be part of the recombinant polypeptide of the invention are:

[0075] [Table 1] TIFF2025513720000009.tif60155

[0076] Examples of recombinant polypeptides of the present invention (including optional leader peptides): TIFF2025513720000010.tif48159 (SEQ ID NO: 15; note that the asterisk represents the position of the stop codon).

[0077] It should be noted that the peptide antigen sequence of the full-length recombinant polypeptide described above (here: VLLAVLPVL) 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 myelin oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1). That is, the recombinant polypeptide of the present invention may be comprised of a sequence consisting of a peptide antigen that is a peptide of human myelin oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1) (e.g., any one of the peptide antigens of SEQ ID NO:2, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33), followed by the following sequence: TIFF2025513720000011.tif35160TIFF2025513720000012.tif15159 (SEQ ID NO: 16; note that the asterisk represents the stop codon). These recombinant polypeptides of the invention may include the optional leader peptides shown in the examples above.

[0078] 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: TIFF2025513720000013.tif75139 (SEQ ID NO: 17)

[0079] ILT4: TIFF2025513720000014.tif68138 (SEQ ID NO: 18)

[0080] The sequences of human myelin oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1) are known in the art. The preferred amino acid sequences of human myelin oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1) are as follows:

[0081] Human myelin oligodendrocyte glycoprotein (MOG): >NP_996532.2 myelin-oligodendrocyte glycoprotein isoform alpha1 precursor [Homo sapiens] TIFF2025513720000015.tif29151 (SEQ ID NO: 19)

[0082] Human myelin basic protein (MBP): >NP_001020252.1 myelin basic protein isoform 1 [Homo sapiens] TIFF2025513720000016.tif22154 (SEQ ID NO: 22)

[0083] Human Myelin Associated Glycoprotein (MAG): >NP_002352.1 myelin-associated glycoprotein isoform a precursor [Homo sapiens] TIFF2025513720000017.tif65155 (SEQ ID NO: 23)

[0084] Human myelin proteolipid protein (PLP1): >NP_000524.3 myelin proteolipid protein isoform 1 [Homo sapiens] TIFF2025513720000018.tif15152TIFF2025513720000019.tif14150(SEQ ID NO:24)

[0085] The present invention is further illustrated by the following non-limiting examples: (Example) EXAMPLES

[0086] 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.

[0087] 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.

[0088] 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.

[0089] Prepare 500 μM Spot peptide solution in PBS, remove supernatant, incubate with ⅓ volume of Spot peptide solution for 5-10 min.

[0090] The beads are pelleted by centrifugation. Proteins are concentrated and Spot peptides are removed using an Amicon Ultra-4 centrifugal filter (15 kDa cutoff) over a 15 kDa Amicon cutoff column.

[0091] 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.

[0092] 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 without brake, after which the interphase ring was collected and washed with 1xPBS (5 min, 300xg). PBMCs were stored frozen until further use.

[0093] 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.

[0094] 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). 6 The cells were resuspended at a cell density of 1000 cells / ml.

[0095] For the experiments, 3 × 10 cells were plated in each well of a 12-well plate in a final volume of 1000 μl of X-VIVO complete medium (containing cytokine cocktail and 5 μg / ml of AIM Biomolecules or respective controls). 6 cells were seeded.

[0096] 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.

[0097] Test materials: 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 and 12 well plates.

[0098] 2) ELISPOT 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 plated per well in duplicate on ELISPOT plates, including a negative control (cells + PBS) and a positive control (e.g., LPS).

[0099] 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.

[0100] The respective antigen peptide (e.g. MOG157) in DMSO or DMSO as a control was 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.

[0101] Outside the laminar flow hood Secondary antibodies were prepared: 1 μg / ml aIL-10 biotinylated antibody in 0.5% BSA / 1x PBS (1:1000 dilution) and horseradish peroxidase-conjugated streptavidin (1:750 in 0.5% BSA / PBS), tetramethylbenzidine solution was filtered using a 0.45 μm filter and stored at 4° C. until use.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] The plastic underdrain of the plate was removed and the bottom and sides of the plate were washed with tap water and dried.

[0106] Plates were read using an ImmunoSpot S6 Ultra-V Analyzer (Cellular Technology Limited), analyzed in Excel, and graphs / statistics were performed in Graphad Prism.

[0107] 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), 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, Wash buffer: 1x PBS, Media: for T cells, X-VIVO 15 medium (Lonza), Filter syringe: Millex GV, ELISPOT PVDF plate (#MSIP4510, Millipore), TMB substrate. EXAMPLES

[0108] Surrogates of the recombinant polypeptides of the present invention induce IL10-secreting Tregs in mice To induce immune tolerance to the OVA peptide or the viral Gp34 peptide, wild-type black 6 mice were injected with the OVA peptide or the viral Gp34 peptide, respectively. Ova_KbG TIFF2025513720000020.tif48159 (SEQ ID NO: 46), and Gp34_KbG The mice were injected with 100 μg of recombinant polypeptide (also called "AIMBio") having the sequence of TIFF2025513720000021.tif48159 (SEQ ID NO: 47). The Gp34 peptide is a well-characterized T cell epitope derived from the Lymphocytic Choriomeningitis Virus (LCMV) glycoprotein. This epitope was previously named Gp33, but the epitope presented on H2-Kb was later found to contain only amino acids 34-41. (In contrast, the epitope starting at amino acid 33 is presented on H2-Kd.) Therefore, we refer to the H2-Kb epitope as Gp34, which is in line with the latest recommendations. Nevertheless, the nomenclature of Gp33 and Gp34 is used ambiguously in the literature. The first 8 amino acids of SEQ ID NO: 47 indicate the individual peptide sequence. Two weeks later, mice were sacrificed and splenocytes were rechallenged with matched or mismatched peptides. IL-10-secreting cells were quantified by ELIspot. The results are shown in Figure 3. EXAMPLES

[0109] Surrogates of the recombinant polypeptides of the invention selectively prevent CD8+ T cell-induced EAE in mice As described in (Na et al, Brain. 2008 Sep;131(Pt 9):2353-65.), adoptive transfer of CD8+OT-I T cells that recognize the ovalbumin epitope in H2-Kb into mice expressing ovalbumin in oligodendrocytes induces experimental autoimmune encephalomyelitis that reproduces many MS and MOGAD symptoms. In this animal model, a single injection of 500 μg of a recombinant polypeptide surrogate molecule (also called "AIMBio") that induces tolerance to the targeted ovalbumin epitope almost completely prevented EAE symptoms, whereas a surrogate molecule presenting a control peptide had no significant preventive effect (FIG. 4). The sequence of the recombinant polypeptide surrogate molecule is shown in Example 2.

[0110] In the same model, we were further able to show that effective bystander immune suppression can be induced by using antigen peptides that are also presented in the tissues and cells of interest. Here, 250 μg of recombinant polypeptide surrogate molecule (also called "AimBio") was injected per mouse (FIG. 5). The sequence of the recombinant polypeptide surrogate molecule was as follows:

[0111] Mog44_DbG TIFF2025513720000022.tif48159 (SEQ ID NO: 48)

[0112] Mog37_DbG TIFF2025513720000023.tif48159 (SEQ ID NO: 49) EXAMPLES

[0113] Some surrogates of the recombinant polypeptides of the present invention selectively prevent CD4+ T cell-induced EAE in mice On day 0, 33 μg or 100 μg of a surrogate molecule of a recombinant polypeptide of the invention ("AIM Bio") was injected intraperitoneally, and 100 μl of MOG35-55 peptide / CFA (complete Freund's adjuvant; final concentration of M. tuberculosis H37RA and peptide 1 mg / ml each) emulsion was injected subcutaneously in the left and right flanks, respectively, and 250 ng pertussis toxin (in 200 μl PBS) was injected intraperitoneally. A second pertussis toxin injection was given 3 days later. In this animal model, a single injection of an AIM Bio surrogate molecule inducing tolerance to a Mog epitope (Mog44_DbG) significantly reduced the symptoms of EAE, whereas a surrogate molecule presenting a control peptide (Gp34) or a non-functional Mog peptide (Mog37) showed no significant protective effect (FIG. 6). In this model, Mog44 AIM Bio also prevented inflammation in the spinal cord and infiltration of CD8 T cells (FIG. 7). The sequence of the recombinant polypeptide surrogate molecule is shown in Example 3.

[0114] In this model, Mog44 AIM Bio also completely prevented the formation of MOG-specific autoantibodies in serum as verified by ELISA (Figures 8 and 11). This strongly indicates that the recombinant polypeptides of the present invention are effective therapeutic agents for MS and MOGAD, which are often characterized by antibody responses against MOG. Thus, patient populations are defined by common autoimmune-associated antigens.

[0115] Mog-reactive antibodies in serum from mice treated with AIM Bio (33 or 100 μg) were detected by standard ELISA protocol with three washes between each step. Briefly, ELISA plates were coated with 10 μg / ml Mog35-55 peptide, blocked with PBS 1% BSA, and loaded with mouse serum diluted 1:25 in PBS 1% BSA for 1 hour. Detection was performed with anti-mouse IgG-HRP or anti-mouse heavy and light chain HRP antibodies diluted 1:5000. EXAMPLES

[0116] Candidate human recombinant polypeptides of the present invention for MS and MOGAD The recombinant polypeptide of the present invention is a newly developed protein complex derived from the pregnancy-associated immunosuppressive MHC molecule HLA-G. HLA-G is believed to enable the embryo to influence the maternal immune system to tolerate embryonic antigens but to better antagonize pathogen-derived antigens. The recombinant polypeptide of the present invention containing variable peptides could selectively eliminate peptide-specific cytotoxic effector T cells and induce peptide-specific Treg in vitro.

[0117] FIG. 9 shows a list of human MS&MOGAD recombinant polypeptide candidates.

[0118] According to the findings of the present inventors, a single-chain protein containing myelin peptide antigen and HLA-Gα3 domain is able to induce tolerogenic T cells in healthy donors. Thus, the recombinant polypeptide of the present invention containing the VLLAVLPVL antigen (also called "Mog157_A2G"; see FIG. 10) upregulated CD8 Tregs by at least 30% in 75% of all healthy blood donors. EXAMPLES

[0119] Further proof of principle regarding the stability and efficacy of 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 substitutes are stable and effective. As shown in Figures 12 and 13, 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 14) and inhibit T cell lysis in a dose-dependent manner (Figure 15). The effect of the recombinant polypeptides on serum cytokine profile in EAE-ODC Ova mice is shown in Figure 16. 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. EXAMPLES

[0120] The recombinant polypeptide of the invention that induces MOG resistance prevents apoptosis (cleaved caspase 3), infiltration of CD3+ immune cells, and myelin lesions in the spinal cord. The mouse model is depicted in FIG. Immunofluorescence analysis of the spinal cord in MOG-induced EAE model material: Primary antibody αCD3i clone CD3-12 (1:100); rat monoclonal (cross-reactive with rat anti-human and mouse, Bio-Rad) αMBP (1:300, rabbit monoclonal, MBL) α-cleaved caspase 3 (1:400, rabbit monoclonal, Cell Signaling) DAPI (1:500, Sigma #D8417) secondary antibody α-RabbitCy3 (red, 1:300, Dianova #111-165-144), α-Rat AF488 (green, 1:300, Invitrogen), or α-RabbitCy3 (red, 1:300, Dianova #112-165-167)

[0121] protocol The frozen sections are warmed at room temperature for 5 minutes, the areas to be stained are marked with a Pap-pen, and then fixed in acetone for 10 minutes at -20°C. Wash 3 times with 1xPBS Block for 1 hour in 5% BSA + 0.2% Triton-X100 + 1% NGS in 1x PBS. Primary antibody: 1% BSA + 1% NGS + 0.2% Triton-X100, overnight at 4℃ Wash: 3 times with 1xPBS Secondary antibody: 1% BSA + 1% NGS + 0.2% Triton-X100, 1 hour at room temperature Wash: 3 times with 1xPBS DAPI: 1:300 in 1x PBS, 10 min, room temperature Wash: 2 times with 1xPBS Mount with Aquapolymount.

[0122] Detailed Results Figure 17A, B: Caspase 3 activation (apoptosis) can be suppressed in MOG-induced EAE spinal cord. A: Images, B: Quantification. Figure 17C, D: MOG-induced EAE in the spinal cord can prevent lesions in the white matter. C: Images, D: Quantification. Figure 17E, F: CD3 entry into the spinal cord can be prevented in MOG-induced EAE. E: images, F: quantification. EXAMPLES

[0123] Increase in IL10 spots after treatment with MOG157_A2G Figure 18 shows an increase in IL10 spots following treatment with MOG157_A2G indicating that tolerogenic Tregs were induced. This is an alternative depiction of Figure 10.

[0124] 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, the peptide antigen being a peptide of human myelin oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), myelin-associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1); 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 set forth in 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, wherein the 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 The recombinant polypeptide comprising:

2. 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:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, or 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:2, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, and SEQ ID NO:

30.

4. 2. The recombinant polypeptide of claim 1, wherein the peptide antigen described in (i) is a peptide of human myelin oligodendrocyte glycoprotein (MOG), preferably consisting of the amino acid sequence of SEQ ID NO:

2.

5. 2. 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, preferably a human HLA-A2 molecule, or from a human MHC class Ib molecule.

6. The 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 human HLA-G.

7. The [α]3 domain or derivative according to (vii) is identical to or has at least 80% amino acid sequence identity, preferably at least 90% amino acid sequence identity with the [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 21, or the [α]3 domain or derivative according to (vii) is identical to or has at least 92% amino acid sequence identity with the [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 21, or the [α]3 domain or derivative according to (vii) is identical to or has at least 94% amino acid sequence identity with the [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 21, or the [α]3 domain or derivative according to (vii) is identical to or has at least 94% amino acid sequence identity with the [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 21, or 9 or SEQ ID NO:21, or the [α]3 domain or derivative described in (vii) is identical to or has at least 96% amino acid sequence identity with the [α]3 domain having the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:21, or the [α]3 domain or derivative described in (vii) is identical to or has at least 98% amino acid sequence identity with the [α]3 domain having the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:21, or the [α]3 domain or derivative described in (vii) is identical to or has at least 99% amino acid sequence identity with the [α]3 domain having the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:21, or 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:

21.

8. 2. The recombinant polypeptide according to claim 1, 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 greater than or equal to 1, the linker sequence according to (ii) preferably 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, preferably an integer selected from the group consisting of 2, 3, 4 and 5, and / or the linker sequence according to (iv) preferably 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, preferably an integer selected from the group consisting of 2, 3, 4 and 5.

9. 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.

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

11. 2. The recombinant polypeptide of claim 1, wherein the polypeptide comprises or consists of all of (i) to (vii), and / or the polypeptide does not comprise (viii) to (x).

12. 2. The recombinant polypeptide of claim 1, wherein the polypeptide comprises or consists of all of (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:2, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33; 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. A nucleic acid encoding one or more polypeptides according to claim 1, wherein the nucleic acid is preferably a vector.

17. A pharmaceutical composition 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 described in claim 1, each of the different polypeptides comprising a different peptide antigen as defined in any one of claims 3-4.

20. 19. The pharmaceutical composition of claim 17 or the pharmaceutical composition or kit of claim 18 for use in the treatment of multiple sclerosis (MS), anti-MOG antibody-mediated disease, or MOG antibody-positive neuromyelitis optica in a human patient, wherein the treatment is preferably an immunotherapy treatment, and / or the treatment is preferably by inducing immune tolerance to human myelin oligodendrocyte glycoprotein, and / or the treatment is preferably for reducing the amount of autoantibodies to human myelin oligodendrocyte glycoprotein in plasma or cerebrospinal fluid, and / or the human patient is preferably a patient who had autoantibodies to myelin oligodendrocyte glycoprotein in plasma or cerebrospinal fluid before initiation of treatment, and / or the treatment is preferably by inducing myelin-specific regulatory T cells.

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

22. A method for obtaining a pharmaceutical composition comprising the polypeptide of claim 1, comprising the steps of: (a) culturing a recombinant host cell comprising the nucleic acid of claim 16 and expressing the recombinant polypeptide of 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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