Aquaporin 4 (AQP4)-specific immunosuppression mediated by MHC Ib as a novel treatment for neuromyelitis optica (NMO)

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 neuromyelitis optica (NMO). The present invention more specifically relates to recombinant polypeptides comprising a peptide antigen and one or more domains of a non-classical MHC class Ib molecule. The present invention also relates to methods for producing such recombinant polypeptides, pharmaceutical compositions comprising same, and their use for treating neuromyelitis optica (NMO).

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to the therapeutic use of non-classical human major histocompatibility complex (MHC) molecules (also referred to as MHC class Ib molecules) in combination with peptide antigens for the treatment of neuromyelitis optica (NMO). The present invention more specifically relates to recombinant polypeptides comprising a peptide antigen and one or more domains of a non-classical MHC class Ib molecule. The present invention also relates to methods for producing such recombinant polypeptides, pharmaceutical compositions comprising same, and their use for treating neuromyelitis optica (NMO). [Background technology]

[0002] background Neuromyelitis optica (NMO) is a demyelinating autoimmune disease of the central nervous system (CNS) with a prevalence of 0.05–0.4 / 100,000. NMO is characterized by symptoms similar to multiple sclerosis. However, whereas multiple sclerosis is often characterized by a relapsing-remitting course, NMO patients rarely experience remission. Furthermore, treatments that are effective in MS patients may be ineffective in NMO or may even worsen the disease. Thus, NMO is more difficult to treat than MS and usually progresses more rapidly. Current treatment strategies for NMO focus on preventing relapses or treating acute disease relapses. Acute relapses are usually treated with immunosuppressive medications such as glucocorticoids or with plasma exchange to remove autoreactive antibodies. More recently, therapeutic antibodies that inhibit the complement system (eculizumab) or inhibit inflammatory cytokines (satralizumab) have also been approved.

[0003] However, not only self-reactive but also protective immune functions are inhibited. These functions are essential to protect the patient against viruses, bacteria or tumors. Therefore, only partial inhibition can often be aimed for. Autoimmune diseases usually worsen after relapse, so great efforts are made to prevent relapse. However, the overall slow progression of this autoimmune disease can be slowed but not stopped. To circumvent this dilemma, attempts have been made for some time to induce immune tolerance to antigens attacked by autoreactive immune cells (antigen-specific immune suppression, ASI).

[0004] Two strategies have been evaluated so far, at least in early clinical studies. Similar to hyposensitization therapy in allergy, large amounts of antigens were administered by different routes to induce antigen-specific tolerance. However, in autoimmune diseases, these strategies cause severe side effects and have not been clinically successful. Attempts to induce tolerance by adoptive transfer of antigen-specific regulatory T cells or antigen-loaded tolerogenic dendritic cells seem more promising. However, these strategies are very complex and expensive, and require GMP-compliant manufacturing and quality control steps for each individual patient. Therefore, even if small-scale clinical trials are successful, it is highly questionable whether adoptive transfer therapy will be available to many patients in the foreseeable future.

[0005] WO 2018 / 215340 relates to combinations of MHC class Ib molecules and peptides for targeted therapeutic immune modulation.

[0006] Taken together, there remains a need for improved therapeutics for the treatment of neuromyelitis optica (NMO). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO 2018 / 215340 Summary of the Invention

[0008] Description of the Invention The inventors have found that human MHC class Ib molecules, such as HLA-G, have the ability to induce antigen-specific tolerance to peptide antigens presented. Thus, MHC class Ib molecules can be advantageously used according to the invention to suppress immune responses in an antigen-specific manner, despite having a structure and sequence similar to the classical human MHC class Ia molecules that induce antigen peptide-specific immune responses. Furthermore, the inventors have found that for the suppression of immune responses according to the invention, molecules other than naturally occurring MHC class Ib molecules can be used, in particular polypeptides that comprise at least one domain of an MHC class Ib molecule, preferably at least the [α]3 domain of an MHC class Ib molecule only: the [α]1 and [α]2 domains of variable class Ia molecules can be combined with the [α]3 domain of human MHC class Ib molecules to suppress immune responses to peptides presented by these antigens.

[0009] Antigen-loaded HLA-G molecules can be unstable. Therefore, the present inventors designed soluble recombinant polypeptides containing peptide antigens, MHC class Ib molecules such as HLA-G, and β2 microglobulin (b2m), and linked these three components covalently (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. Mouse H2-K b It was previously found that a construct containing the α1 and α2 domains of was able to 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).

[0010] Surprisingly, the inventors have found that the use of the recombinant polypeptides of the present invention can suppress immune responses to neuroinflammatory autoantigens and induce cells that induce tolerance to human aquaporin 4 (AQP4). Furthermore, using established models of neuroinflammatory disease, the inventors have shown that surrogates of these polypeptides (adapted for use in mice) can be used to treat neuroinflammatory disease. Thus, according to the present invention, neuromyelitis optica (NMO) can be treated by the recombinant polypeptides of the present invention.

[0011] 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 achieve the desired effect. Thus, this approach goes beyond the above-mentioned methods that use antigenic peptides in the absence of costimulation (leading to anergic rather than tolerogenic T cells) or MHC class Ib molecules in an antigen-nonspecific context.

[0012] Furthermore, according to the present invention, the recombinant polypeptide of the present invention not only regulates T cell responses but also prevents the formation of aquaporin 4 (AQP4)-specific autoantibodies, which are involved in the pathology of neuromyelitis optica (NMO), and it is expected that this benefit will lead to clinical improvement in human patients suffering from neuromyelitis optica (NMO).

[0013] 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 aquaporin 4; ii) optionally, a linker sequence; iii) optionally a sequence of a human polypeptide domain that includes a sequence of human β2 microglobulin, or an amino acid sequence that is at least 90% identical to the amino acid sequence of human β2 microglobulin represented by SEQ ID NO:5; iv) optionally, a linker sequence; v) optionally, the [α]1 domain of an MHC molecule; vi) optionally, the [α]2 domain of an MHC molecule; vii) an [α]3 domain of an MHC class Ib molecule, or a derivative of an [α]3 domain of an MHC class Ib molecule, which derivative is capable of binding to ILT2 or ILT4; viii) optionally, a protease cleavage site; ix) optionally, a spacer sequence; and x) Possibly, an affinity tag.

[0014] 1. 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.

[0015] 2. The recombinant polypeptide according to item 1 or 2, wherein the peptide antigen according to i) is composed of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 2, 22, 23 and 24.

[0016] 3. A recombinant polypeptide according to any one of the preceding claims, wherein the peptide antigen consists of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:22.

[0017] 4. The recombinant polypeptide according to any one of items 1 to 4, wherein the peptide antigen consists of the amino acid sequence of SEQ ID NO:2.

[0018] 5. The recombinant polypeptide according to any one of items 1 to 3, wherein the peptide antigen consists of the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24.

[0019] 6. 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.

[0020] 7. A recombinant polypeptide according to any one of the preceding claims, 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.

[0021] 8. 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.

[0022] 9. 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.

[0023] 10. The recombinant polypeptide according to item 10, wherein the [α]1 domain described in (v) and the [α]2 domain described in (vi) are derived from a human HLA-G molecule.

[0024] 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.

[0025] 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.

[0026] 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, to an [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 21.

[0027] 14. The recombinant polypeptide according to item 14, 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: 21.

[0028] 15. The recombinant polypeptide according to item 14, 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.

[0029] 16. The recombinant polypeptide according to item 14, 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: 21.

[0030] 17. The recombinant polypeptide according to item 14, 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.

[0031] 18. The recombinant polypeptide according to item 14, 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.

[0032] 19. The recombinant polypeptide according to item 14, 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.

[0033] 20. A recombinant polypeptide according to any one of the preceding paragraphs, wherein the linker sequence described in (ii) and / or the linker sequence described in (iv) comprises the amino acid sequence (GGGGS)n, where n is an integer equal to or greater than 1.

[0034] 21. The recombinant polypeptide according to item 21, 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.

[0035] 22. The recombinant polypeptide according to item 21 or 22, 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.

[0036] 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.

[0037] 24. A recombinant polypeptide according to any one of the preceding claims, wherein the polypeptide is a dimer or multimer.

[0038] 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).

[0039] 26. A recombinant polypeptide according to any one of the preceding claims, wherein the polypeptide does not comprise components (viii) to (x).

[0040] 27. A recombinant polypeptide according to any one of items 1 to 26, wherein the polypeptide comprises or consists of all of components (i) to (x).

[0041] 28. The recombinant polypeptide of any one of the preceding claims, further comprising an N-terminal secretory signal peptide sequence.

[0042] 29. The recombinant polypeptide according to any one of items 1 to 28, wherein the recombinant polypeptide is composed of an amino acid sequence consisting of the following, in order from the N-terminus to the C-terminus: (a) and (b): (a) a peptide antigen selected from the group consisting of the amino acid sequences of SEQ ID NOs: 2, 22, 23 and 24, and (b) Amino acid sequence of SEQ ID NO:16.

[0043] 30. A recombinant polypeptide according to any one of the preceding paragraphs, wherein the recombinant polypeptide is soluble.

[0044] 31. A nucleic acid encoding one or more polypeptides according to any one of the preceding paragraphs.

[0045] 32. The nucleic acid according to item 32, wherein the nucleic acid is a vector.

[0046] 33. A pharmaceutical composition or kit comprising at least one nucleic acid according to item 32 or 33.

[0047] 34. A pharmaceutical composition or kit comprising at least one recombinant polypeptide according to any one of items 1 to 31.

[0048] 35. The pharmaceutical composition or kit according to item 35, wherein the pharmaceutical composition or kit comprises at least two different recombinant polypeptides according to any one of items 1 to 31, each of the different polypeptides comprising a different peptide antigen according to any one of items 3 to 6.

[0049] 36. A pharmaceutical composition or kit according to any one of items 34 to 36, for use in treating neuromyelitis optica in a human patient.

[0050] 37. The pharmaceutical composition or kit for use according to item 37, wherein the treatment is immunotherapy treatment.

[0051] 38. A pharmaceutical composition or kit for use according to any one of items 37 to 38, wherein the treatment is by inducing immune tolerance to human aquaporin 4.

[0052] 39. A pharmaceutical composition or kit for use according to any one of items 37 to 39, wherein the treatment is for reducing plasma or cerebrospinal fluid levels of autoantibodies against human aquaporin 4.

[0053] 40. The pharmaceutical composition or kit for use according to any one of items 37 to 40, wherein the human patient is a patient having plasma autoantibodies or cerebrospinal fluid autoantibodies against human aquaporin 4 before the start of treatment.

[0054] 41. A pharmaceutical composition or kit for use according to any one of items 37 to 41, wherein the treatment is by induction of myelin-specific regulatory T cells.

[0055] 42. A recombinant host cell comprising the nucleic acid or vector according to item 32 or 33 and expressing a recombinant polypeptide according to any one of items 1 to 31.

[0056] 43. A method for obtaining a pharmaceutical composition comprising a polypeptide according to any one of items 1 to 31, the method comprising the steps of: (a) culturing a recombinant host cell according to item 42 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]

[0057] [Figure 1] Schematic diagram of peptide-loaded soluble MHC Ib molecules suitable for achieving therapeutically effective antigen-specific immune modulation. The antigenic peptide is represented as a dotted sphere, the HLA-Gα1-3 domain is in light grey and the β2 microglobulin domain is in dark grey. The optional linker linking the antigenic peptide to the β2 microglobulin molecule is shown in grey stick form and the optional disulfide trap is depicted as a black sphere. The diagram was generated using Pymol and is based on structures published in Clements et al., Proc Natl Acad Sci US A. 2005 Mar 1;102(9):3360-5 and Hansen et al., Trends Immunol. 2010 Oct;31(10):363-9. [Diagram 2]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 association. To facilitate purification of the complex MHC Ib molecule, one or more protein tags (such as SpotTag, myc tag and / or His(6x) tag) may be introduced for subsequent optional removal by cleavage using an optional factor Xa cleavage site. Furthermore, the antigen peptide, β2 microglobulin and MHC Ib [α] chain can be linked to increase stability. Vector maps were generated using Snapgene Viewer Software. [Figure 3-1]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-Kbα1 and 2 domains, and human HLA-Gα3 domain and β2 microglobulin were injected intraperitoneally into 12-week-old C57BL / 6 mice. After 14 days, mice were sacrificed and splenocytes were rechallenged with 5 μg / ml of Gp34 or Ova peptides, and 48 hours later a standard mouse IL-10 ELIspot assay (Mabtech mouse IL-10 HRP ELISpot kit, cells cultured in RPMI 10% FCS 10 ng / ml IL2) was performed. A significant increase in IL-10-secreting regulatory T cells was detected only upon rechallenge with peptides to which tolerance was induced by injection of the surrogate molecules. (A) Experimental design; (B) Results [Figure 3-2] 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-Kbα1 and 2 domains, and human HLA-Gα3 domain and β2 microglobulin were injected intraperitoneally into 12-week-old C57BL / 6 mice. After 14 days, mice were sacrificed and splenocytes were rechallenged with 5 μg / ml of Gp34 or Ova peptides, and 48 hours later a standard mouse IL-10 ELIspot assay (Mabtech mouse IL-10 HRP ELISpot kit, cells cultured in RPMI 10% FCS 10 ng / ml IL2) was performed. A significant increase in IL-10-secreting regulatory T cells was detected only upon rechallenge with peptides to which tolerance was induced by injection of the surrogate molecules. (A) Experimental design; (B) Results [Figure 4-1]Surrogates of the recombinant polypeptides of the invention prevent CD8+ T cell-induced EAE in mice. In this mouse model of EAE, 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 H2-Kbα1 and 2 domains, and surrogate molecules consisting of human HLA-Gα3 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 4-2]Surrogates of the recombinant polypeptides of the invention prevent CD8+ T cell-induced EAE in mice. In this mouse model of EAE, 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 H2-Kbα1 and 2 domains, and surrogate molecules consisting of human HLA-Gα3 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 5-1]Some of the surrogates of the recombinant polypeptide of the present invention selectively prevent CD4+ T cell-induced EAE in mice. 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). CD4+ cells as well as antibodies 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 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. Mog44 peptide-containing surrogate molecules significantly reduced EAE symptoms and weight loss. (A) Experimental design; (B) EAE score; (C) Body weight [Figure 5-2]Some of the surrogates of the recombinant polypeptide of the present invention selectively prevent CD4+ T cell-induced EAE in mice. 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). CD4+ cells as well as antibodies 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 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. Mog44 peptide-containing surrogate molecules significantly reduced EAE symptoms and weight loss. (A) Experimental design; (B) EAE score; (C) Body weight [Figure 5-3]Some of the surrogates of the recombinant polypeptides of the present invention selectively prevent CD4+ T cell-induced EAE in mice. 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 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. Mog44 peptide-containing surrogate molecules significantly reduced EAE symptoms and weight loss. (A) Experimental design; (B) EAE score; (C) Body weight [Figure 6-1] Mog44 surrogate of the recombinant polypeptide of the present invention prevented inflammation and CD8 T cell infiltration in the spinal cord. Fresh frozen sections of 10 μm were stained with 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 MOG44_Db_G surrogate molecule. (A) Toluidine; (B) CD8-DAB [Figure 6-2]Mog44 surrogate of the recombinant polypeptide of the present invention prevented inflammation and CD8 T cell infiltration in the spinal cord. Fresh frozen sections of 10 μm were stained with 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 MOG44_Db_G surrogate molecule. (A) Toluidine; (B) CD8-DAB [Figure 7] Detection of anti-MOG35-55 antibodies in Mog-EAE mice administered a surrogate of the recombinant polypeptide of the invention ("AIM Bio") After sacrificing the mice, mouse serum was collected by cardiac puncture. After coating overnight with 10 μg / ml Mog35-55, wells were blocked with 1% BSA, and anti-Mog35-55 antibodies were detected with the indicated HRP-conjugated secondary antibody. Mog35-55-induced EAE correlated with high levels of Mog35-55-specific IgG autoantibodies, which were undetectable in animals administered 100 μg of the MOG44_Db_G surrogate molecule. [Figure 8]List of human NMO recombinant polypeptide candidates. The peptide antigen sequences of the candidate NMO recombinant polypeptides shown in the figure are as follows: Construct Peptide Antigen SEQ ID NO: hAQP4 42-50_HLAG Myc / His tag FLAMLIFVL SEQ ID NO: 31 hAQP4 45-53_HLAG Myc / His tag MLIFVLLSL SEQ ID NO: 32 hAQP4 65-72_HLAG Myc / His tag PLPVDMVL SEQ ID NO: 33 hAQP4 71-79_HLAG Myc / His tag VLISLCFGL SEQ ID NO: 22 hAQP4 126-135_HLAG Myc / His tag AIIGAGILYL SEQ ID NO: 34 hAQP4 127-135_HLAG Myc / His tag IIGAGILYL SEQ ID NO: 23 hAQP4 45-53_A2G Myc / His tag MLIFVLLSL SEQ ID NO: 32 hAQP4 65-72_A2G Myc / His tag PLPVDMVL SEQ ID NO:33 hAQP4 71-79_A2G Myc / His tag VLISLCFGL SEQ ID NO:22 hAQP4 126-135_A2G Myc / His tag AIIGAGILYL SEQ ID NO:34 hAQP4 127-135_A2G Myc / His tag IIGAGILYL SEQ ID NO:23 hAQP4 156-164_A2G Myc / His tag AGHGLLVEL SEQ ID NO:35 hAQP4 238-247_A2G Myc / His tag IIGAVLAGGL SEQ ID NO:24 hAQP4 45-HLAG SPOTtag MLIFVLLSL SEQ ID NO:32 hAQP4 36-43_HLAG SPOTtag KAVTAEFL SEQ ID NO:36 hAQP4 hAQP4 71-79_HLAG SPOTtag VLISLCFGL SEQ ID NO:22 hAQP4 64-72_HLAG SPOTtag KPLPVDMVL SEQ ID NO:2 hAQP4 71-79_A2G SPOTtag VLISLCFGL SEQ ID NO:22 hAQP4 127-A2G SPOTtag IIGAGILYL SEQ ID NO:23 hAQP4 238-A2G SPOTtag IIGAVLAGGL SEQ ID NO:24 hAQP4 137-145_HLAG SPOTtag VTPPSVVGGL SEQ ID NO:37Figure 8 further shows which combinations of AQP4 peptides with antigen-presenting MHC class I α1 and 2 domains (HLAg = HLA-G, A2G = HLA-A2 presenting domain + HLA-G α3 domain) give effective (high), intermediate (average, av) or unfavorable (low) results in terms of production, quality control and prioritization in healthy blood donors (described in Figure 9). [Figure 9-1]Upregulation of CD8 Tregs in Healthy Blood Donors by Recombinant Polypeptide of the Invention Containing KPLPVDMVL Antigen ("AQP_64") In vitro Treg induction via peptide-HLA-G containing constructs (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 1200 xg for 20 min with the brake off, after which the interphase was collected and washed with 1x PBS (5 min, 300 xg). PBMCs were frozen and stored until further use. PBMCs were thawed one day (d-1) before 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. 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, followed by washing 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 washed 5 times with 200 μl PBS, after which 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 seeded per well on ELISpot plates in duplicate with negative control (cells + PBS) and positive control (e.g. LPS) for 48 hours. Secondary antibodies were prepared: 1 μg / ml aIL-10 biotinylated antibody (1:1000 dilution in 0.5% BSA / 1x PBS) and horseradish peroxidase-labeled streptavidin (1:750 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. Final excess buffer was removed with paper towel. 25 μl diluted HRP-streptavidin (1:750) was added per well and incubated for 1 hour at room temperature in the dark, followed by 5 washes with sterile 1x PBS. 100 μl of filtered TMB substrate was added per well and left for 15-25 min until blue spots developed. The reaction was stopped by thoroughly washing the wells with water. The plastic underdrains of the plates were removed and the bottom and sides of the plates were also washed with tap water and allowed to dry. AQP4_64_G_Spt induced at least 30% more IL-10-secreting Tregs in 65% of all healthy blood donors. [Figure 9-2] FIG. 9 continued: % increase in IL10 spots in PBMCs of HLA-A2+ and HLA-A2- donors upon treatment with AQP4_64_G_Spt. [Figure 10] Control experiments of samples shown in Figure 7 show that total IgG is not reduced by treatment with MOG47_Db_G surrogate molecules. Total IgG was quantified using the Easy-Titer™ Human IgG (γ Chain) Assay Kit (Thermo Fisher) according to the manufacturer's instructions. This experiment, taken together with Figure 7, shows that single chain MHC Ib molecules can be used to suppress selective antibody responses. [Figure 11-1]Stability of purified single-chain MHC Ib molecules. After purification, single-chain MHC Ib molecules were analyzed for stability after one and three freeze-thaw cycles, after storage at room temperature for 5 days, and after heating at 50° C. for 30 min. For this, A) Coomassie gel staining of a 12% polyacrylamide gel with 2 μg AIM Bio and B) aHLA-G Western blot with 2A12aHLA-G antibody (1:1000) blot using 1 μg protein was performed under non-reducing conditions. Both monomers and dimers are detectable. [Figure 11-2] Stability of purified single-chain MHC Ib molecules. After purification, single-chain MHC Ib molecules were analyzed for stability after one and three freeze-thaw cycles, after storage at room temperature for 5 days, and after heating at 50° C. for 30 min. For this, A) Coomassie gel staining of a 12% polyacrylamide gel with 2 μg AIM Bio and B) aHLA-G Western blot with 2A12aHLA-G antibody (1:1000) blot using 1 μg protein was performed under non-reducing conditions. Both monomers and dimers are detectable. [Figure 12] Single-chain MHC Ib molecules are heat stable. For the Thermal Shift Assay (TSA), 3 μg of each single-chain MHC Ib molecule or Motavizumab as a control molecule was diluted in PBS and 5x SYPRO Orange dye (stock solution 5000x, final concentration: 5x) to a volume of 25 μl. A melting curve program was set up in the StepOnePlus Instrument using StepOnePlus Software 2.3. The starting temperature was 25°C for 1 min, then the temperature was increased by 1°C per min, to a final temperature of 95°C for 2 min, and autofluorescence was measured in arbitrary units. Data was exported and graphed in Prism V7.04. A Boltzmann sigmoid function was used to determine the melting temperature (Tm). [Figure 13]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 14] Single-chain MHC Ib molecules inhibit T cell lysis in a dose-dependent manner. OT1 / BL6 mice were sacrificed, splenocytes were harvested, and washed once in RPMI 5% FCS. Red blood cells were removed with 2 ml of 1x sterile red blood cell lysis buffer for 3 min. Cells were cultured at high density (107 cells / ml) for 72 h in RPMI 10% FCS medium containing 20 ng / ml GMCSF, 20 ng / ml IL-2, and 10 ng / ml IL-4, and increasing doses of Ova_KbG. Cells were then scraped off the plate and CD8+ cells were purified with magnetic beads. Sterile 96-well white plates were used. Luciferase-expressing Panc02 target cells were supplemented with 20 μg / ml Ova peptide (SIINFEKL) and shaken at 500 rpm for 60 min at 37 °C. CD8+ effector T cells as well as luciferin were added at a ratio of 50:1. Luminescence was measured after 24 and 48 hours. [Figure 15]Serum cytokines in EAE-ODC Ova mice. Serum cytokines in EAE-ODC Ova mice were measured using a Th1 / Th2 10plex Flowcytomix Kit (eBioscience) according to the manufacturer's instructions. This kit was used to simultaneously detect mouse granulocyte-macrophage colony-stimulating factor (GMCSF), interleukin 1α (IL-1a), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 17 (IL-17), and tumor necrosis factor (TNF) in one sample. This array kit provides a mixture of eight capture beads with different fluorescent intensities, each coated with a capture antibody specific for the respective cytokine. 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 h at room temperature in the dark. Samples were washed with 1 mL of wash buffer for 5 min and centrifuged. After removing the supernatant, the bead pellet was resuspended in 200 μL buffer. Samples were measured on an Attune™ NxT flow cytometer and analyzed with Attune Cytometric Software (Thermo Fisher Scientific). [Figure 16]The most preferred mouse-adapted AIM Bio molecular architecture map. The corresponding exemplary amino acid sequence is: MSRSVALAVLALLSLSGLEAVTTVHGNLGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQ KAKGNEQSFRVDLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRYLKNGNATLRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC (SEQ ID NO: 38) [Figure 17] Coomassie gel of purified and stability tested NMO surrogate 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 to 50° C. for 30 minutes. See FIG. 11 for method. [Figure 18] Western blot of purified and stability tested NMO surrogate single chain MHC Ib molecules (2A12 HLA-G5 antibody 1:5000). Both experiments showed that these molecules are very stable, although AQP203_H2KbG has a greater tendency to multimerize. [Figure 19] Thermal shift assay (TSA). The results obtained in the previous figure were confirmed by TSA, but the multimers give rise to high autofluorescence at low temperatures. [Figure 20]EAE scores. AQP147_KbG provided complete protection from EAE in treated 2D2 mice. Mice received injections of treated or control AIM Bios on D0, D15 and D30. Mice were evaluated daily based on the 0-10 EAE scoring system shown in Table 1. Untreated mice developed severe EAE symptoms during the experimental period. One untreated mouse reached a score of 6 on D14 and was sacrificed. Its score was extrapolated to 6 for the remaining days during the evaluation period. Control treatment with gp34_KbG did not provide the desired protection. (*p<0.05, **p<0.01, ***p<0.001) [Figure 21-1] Immune cell infiltration in the optic nerve of wild-type and 2D2 mice. AQP147_KbG treatment suppressed immune cell infiltration in the optic nerve. CD3+ and CD8+ fluorescent signals from each treatment group are shown in panels A (optic nerve) and B (spinal cord). DAPI images show the total nerve nuclei present in each section. The number of CD3+ and CD8+ T cells was significantly higher in the optic nerve (panel C) and spinal cord (panel D) of control and untreated mice. AQP203_KbG suppressed immune infiltration more than the control and untreated groups. Data are shown as mean + / - SEM. Scale bar 25 μm. One-tailed t-test. [Figure 21-2] Immune cell infiltration in the optic nerve of wild-type and 2D2 mice. AQP147_KbG treatment suppressed immune cell infiltration in the optic nerve. CD3+ and CD8+ fluorescent signals from each treatment group are shown in panels A (optic nerve) and B (spinal cord). DAPI images show the total nerve nuclei present in each section. The number of CD3+ and CD8+ T cells was significantly higher in the optic nerve (panel C) and spinal cord (panel D) of control and untreated mice. AQP203_KbG suppressed immune infiltration more than the control and untreated groups. Data are shown as mean + / - SEM. Scale bar 25 μm. One-tailed t-test. [Figure 21-3]Immune cell infiltration in the optic nerve of wild-type and 2D2 mice. AQP147_KbG treatment suppressed immune cell infiltration in the optic nerve. CD3+ and CD8+ fluorescent signals from each treatment group are shown in panels A (optic nerve) and B (spinal cord). DAPI images show the total nerve nuclei present in each section. The number of CD3+ and CD8+ T cells was significantly higher in the optic nerve (panel C) and spinal cord (panel D) of control and untreated mice. AQP203_KbG suppressed immune infiltration more than the control and untreated groups. Data are shown as mean + / - SEM. Scale bar 25 μm. One-tailed t-test. [Figure 21-4] Immune cell infiltration in the optic nerve of wild-type and 2D2 mice. AQP147_KbG treatment suppressed immune cell infiltration in the optic nerve. CD3+ and CD8+ fluorescent signals from each treatment group are shown in panels A (optic nerve) and B (spinal cord). DAPI images show the total nerve nuclei present in each section. The number of CD3+ and CD8+ T cells was significantly higher in the optic nerve (panel C) and spinal cord (panel D) of control and untreated mice. AQP203_KbG suppressed immune infiltration more than the control and untreated groups. Data are shown as mean + / - SEM. Scale bar 25 μm. One-tailed t-test. [Figure 22-1] Quantification of cleaved caspase 3 in the optic nerve, spinal cord and retina of wild type and 2D2 mice. AQP147_KbG completely inhibits apoptosis in the optic nerve, spinal cord and retina. Fluorescent signals obtained from cleaved caspase 3 (apoptosis marker) staining in the optic nerve (panels A, B), spinal cord (panels C, D) and retina (panels E, F) are shown. Untreated and control mice had the highest levels of caspase 3 and therefore the highest levels of apoptosis. Data are presented as mean + / - SEM. Scale bar 25 μm. One-tailed t-test [Figure 22-2]Quantification of cleaved caspase 3 in the optic nerve, spinal cord and retina of wild type and 2D2 mice. AQP147_KbG completely inhibits apoptosis in the optic nerve, spinal cord and retina. Fluorescent signals obtained from cleaved caspase 3 (apoptosis marker) staining in the optic nerve (panels A, B), spinal cord (panels C, D) and retina (panels E, F) are shown. Untreated and control mice had the highest levels of caspase 3 and therefore the highest levels of apoptosis. Data are presented as mean + / - SEM. Scale bar 25 μm. One-tailed t-test [Figure 22-3] Quantification of cleaved caspase 3 in the optic nerve, spinal cord and retina of wild type and 2D2 mice. AQP147_KbG completely inhibits apoptosis in the optic nerve, spinal cord and retina. Fluorescent signals obtained from cleaved caspase 3 (apoptosis marker) staining in the optic nerve (panels A, B), spinal cord (panels C, D) and retina (panels E, F) are shown. Untreated and control mice had the highest levels of caspase 3 and therefore the highest levels of apoptosis. Data are presented as mean + / - SEM. Scale bar 25 μm. One-tailed t-test [Figure 22-4] Quantification of cleaved caspase 3 in the optic nerve, spinal cord and retina of wild type and 2D2 mice. AQP147_KbG completely inhibits apoptosis in the optic nerve, spinal cord and retina. Fluorescent signals obtained from cleaved caspase 3 (apoptosis marker) staining in the optic nerve (panels A, B), spinal cord (panels C, D) and retina (panels E, F) are shown. Untreated and control mice had the highest levels of caspase 3 and therefore the highest levels of apoptosis. Data are presented as mean + / - SEM. Scale bar 25 μm. One-tailed t-test [Figure 22-5]Quantification of cleaved caspase 3 in the optic nerve, spinal cord and retina of wild type and 2D2 mice. AQP147_KbG completely inhibits apoptosis in the optic nerve, spinal cord and retina. Fluorescent signals obtained from cleaved caspase 3 (apoptosis marker) staining in the optic nerve (panels A, B), spinal cord (panels C, D) and retina (panels E, F) are shown. Untreated and control mice had the highest levels of caspase 3 and therefore the highest levels of apoptosis. Data are presented as mean + / - SEM. Scale bar 25 μm. One-tailed t-test [Figure 22-6] Quantification of cleaved caspase 3 in the optic nerve, spinal cord and retina of wild type and 2D2 mice. AQP147_KbG completely inhibits apoptosis in the optic nerve, spinal cord and retina. Fluorescent signals obtained from cleaved caspase 3 (apoptosis marker) staining in the optic nerve (panels A, B), spinal cord (panels C, D) and retina (panels E, F) are shown. Untreated and control mice had the highest levels of caspase 3 and therefore the highest levels of apoptosis. Data are presented as mean + / - SEM. Scale bar 25 μm. One-tailed t-test [Figure 23-1] CD8+CD122+ and CD4+FoxP3+ regulatory T cells in the optic nerve. This figure shows that AIM-treated mice have fewer CD8 T cells, but fewer but still significantly more regulatory T cells in the optic nerve (using one-tailed T-test). [Figure 23-2] CD8+CD122+ and CD4+FoxP3+ regulatory T cells in the optic nerve. This figure shows that AIM-treated mice have fewer CD8 T cells, but fewer but still significantly more regulatory T cells in the optic nerve (using one-tailed T-test). [Figure 24-1]Quantification of inner retinal thickness in wild-type and 2D2 mice. AQP147_KbG completely rescues IRL cells from degradation. Hematoxylin-eosin staining showed that the inner retinal thickness was preserved in AQP147_KbG, whereas the IRL was thinned in untreated and control mice. Data are shown as mean + / - SEM. Scale bar 25 μm. Two-tailed t-test. [Figure 24-2] Quantification of inner retinal thickness in wild-type and 2D2 mice. AQP147_KbG completely rescues IRL cells from degradation. Hematoxylin-eosin staining showed that the inner retinal thickness was preserved in AQP147_KbG, whereas the IRL was thinned in untreated and control mice. Data are shown as mean + / - SEM. Scale bar 25 μm. Two-tailed t-test.

[0058] Detailed Description of the Invention Definitions and General Techniques Unless otherwise defined below, the terms used in the present invention shall be understood according to the general meaning known to those skilled in the art. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. Publications mentioned herein may be cited by specifying the complete literature reference in the text.

[0059] All proteins of the present invention, including recombinant polypeptides of the present invention, can be obtained by methods known in the art. Such methods include the method of producing recombinant polypeptides. Recombinant polypeptides of the present invention can be expressed in recombinant host cells according to the present invention. Recombinant host cells of the present invention are preferably mammalian cells, such as CHO cells and HEK cells.

[0060] It is 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.

[0061] It is also understood that references to amino acid sequences referred to herein are 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.

[0062] Likewise, it will be understood that the recombinant polypeptides of the present invention are intended to optionally include the respective propeptides.

[0063] It is also understood that the recombinant polypeptide of the present invention can be in soluble or membrane-bound form.Whether a recombinant polypeptide is "soluble" under these conditions can be determined by methods known in the art, for example, by measuring the turbidity of the recombinant polypeptide under the standard conditions indicated above.As used herein, soluble means that at least 95% of the recombinant polypeptide is determined to be soluble under these standard conditions.

[0064] 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.

[0065] According to the present invention, the MHC molecule is preferably a human MHC molecule.

[0066] The recombinant polypeptide of the present invention is preferably an isolated recombinant polypeptide.

[0067] It will be understood how recombinant polypeptides capable of binding and presenting the peptide antigens of the present invention can be prepared. For example, peptide antigen binding domains such as [α]1 and [α]2 domains are well known and modifications of these domains can be made. The ability of the polypeptides of the present invention and peptide antigens to bind to MHC molecules can be determined by techniques known in the art, including but not limited to exploratory methods such as mass spectrometry after MHC peptide elution and in silico bioinformatics prediction, and confirmatory methods such as MHC peptide multimer binding and stimulation assays.

[0068] According to the present invention, the recombinant polypeptides, pharmaceutical compositions and kits of the invention are preferably suitable for use in human patients.

[0069] According to the present invention, the recombinant polypeptides, pharmaceutical compositions and kits of the invention are preferably suitable for use in the treatment of neuromyelitis optica in human patients.

[0070] According to the invention, the recombinant polypeptides, pharmaceutical compositions and kits of the invention are preferably suitable for inducing immune tolerance, for example against human aquaporin 4, in a human patient.

[0071] In accordance with the present invention, it is understood that the recombinant polypeptides, pharmaceutical compositions and kits of the present invention are stable.

[0072] It is 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 the length contributed by additional amino acids that are not part of the peptide antigen, such as additional amino acids from potential linker sequences, etc.

[0073] In accordance with the present invention, each occurrence of the term "comprising" may optionally be replaced with the term "consisting of."

[0074] 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.

[0075] 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.

[0076] For example, the binding of MHC class Ib molecules or recombinant polypeptides of the present invention to their receptors, such as ILT2 and ILT4, is preferably assessed by surface plasmon resonance spectroscopy. More preferably, the binding of MHC class Ib molecules or recombinant polypeptides of the present invention to their receptors is assessed by surface plasmon resonance at 25° C. Suitable conditions for such surface plasmon resonance measurements are described in Shiroishi et al., Proc Natl Acad Sci US A. 2003 July 22;100(15):8856-8861.

[0077] Sequence alignment of the sequences of the present invention is performed using the BLAST algorithm (see Altschul et al. (1990) "Basic local alignment search tool." Journal of Molecular Biology 215. p. 403-410.:Altschul et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402). Suitable parameters for sequence alignment of short peptides by the BLAST algorithm, suitable for the peptide antigens of the present invention, are known in the art. Most software tools using the BLAST algorithm automatically adjust the parameters for sequence alignment of short input sequences. In one embodiment, the following parameters are used: Max target sequences 10; Word size 3; BLOSUM 62 matrix; gap costs; existence 11, extension 1; conditional compositional score matrix adjustment. Thus, when used in the context of sequences, terms such as "identity" or "identical" preferably refer to the identity value obtained using the BLAST algorithm.

[0078] 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.

[0079] 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.

[0080] 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 will 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.

[0081] 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.

[0082] 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.

[0083] 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).

[0084] Peptide antigens are widely known in the art. Generally, the peptide antigen of the present invention can bind to MHC class I protein. Those skilled in the art will use, 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. These peptide antigens can be selected based on the methods known in the art.

[0085] Binding of a peptide antigen to an MHC class Ib molecule, or to a polypeptide capable of binding a peptide antigen of 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; and Rock, Reits, Neefjes. Present Yourself! By MHC Class I and MHC Class II Molecules. Trends Immunol. 2016 Nov;37(11):724-737.

[0086] Such methods include experimental methods and methods that predict peptide-antigen binding.

[0087] 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.

[0088] 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.

[0089] 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 2021;596:583-589.

[0090] In the present invention, the peptide antigen is derived from human aquaporin-4.

[0091] It is 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, with respect to the corresponding amino acid sequence of the human aquaporin 4-derived peptide antigen.

[0092] The peptide antigens of the present invention are preferably composed of naturally occurring amino acids. However, non-naturally occurring amino acids can also be used, 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 aquaporin 4.

[0093] Methods for the synthesis of peptidic antigens, including those of the present invention, are well known in the art.

[0094] Therapeutic Applications of the Invention The recombinant polypeptides of the present invention can be used to treat neuromyelitis optica.

[0095] The treatment can be by inducing myelin-specific regulatory T cells. Such regulatory T cells (e.g., CD8 positive regulatory T cells) are activated in myelinated structures and provide target cell protection against cytotoxic T cells that recognize the same or different myelin antigens. Regulatory T cells (e.g., CD8 positive regulatory T cells) are known in the art and can be detected, for example, by secretion of IL-10.

[0096] CD8+ regulatory T cells are less known than CD4CD25 regulatory T cells, 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.

[0097] These (CD8 positive regulatory T cells) 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).

[0098] The treatment according to the invention can be a treatment for reducing plasma or cerebrospinal fluid (CSF) levels of autoantibodies against human aquaporin 4. The human patient can be a patient who has plasma or cerebrospinal fluid (CSF) autoantibodies against human aquaporin 4 before the start of the treatment.

[0099] According to the invention, autoantibodies can be detected by various methods known in the art. A preferred approach is the cellular assay (CBA) in which the antigen suspected to be the target of the autoantibody (e.g., aquaporin 4) 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 titers are determined by flow cytometry (CBA-FACS) or microscopic immunofluorescence evaluation (CBA-IF). Other approaches such as enzyme-linked immunosorbent assay (ELISA) or Western blot are possible, but are often less sensitive, since 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).

[0100] Methods for detecting Aqp4-specific autoantibodies are described in: Lennon, VA, Kryzer, TJ, Pittock, SJ, Verkman, AS & Hinson, SR IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel. J. Exp. Med.202, 473-477 (2005).

[0101] 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.

[0102] 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., KPLPVDMVL (SEQ ID NO: 2) First linker: for example, GGGGSGGGSGGGGS (SEQ ID NO: 3) or GCGASGGGGSGGGGS (SEQ ID NO: 4)

[0103] β2 microglobulin, for example: IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 5, human beta 2 microglobulin) A second linker, for example: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 6)

[0104] [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 or A in the DT variant For example, the [α] 1 and 2 domains from human HLA-G: GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRA (SEQ ID NO: 7) Or: human HLA-A2 [α] 1 and 2 domains: e.g. GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRT (SEQ ID NO: 8)

[0105] Human HLA-G [α]3 domain (or any MHC Ib [α]3 domain, such as HLA-F, which also interacts with the ILT2 and ILT4 receptors), for example: DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDL (SEQ ID NO: 9; sequence of HLA-G[alpha]3).

[0106] Of note, the following underlined amino acids in this sequence are relevant for ILT2 or ILT4 receptor interaction: TIFF2025510786000001.tif14161

[0107] 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.: DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO: 21), 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

[0108] Further examples of (alternative) peptide antigens which can be part of the recombinant polypeptide of the invention are: VLISLCFGL (SEQ ID NO: 22), preferably contained in an HLA-Gα1 and 2 domain-containing recombinant polypeptide IIGAGILYL (SEQ ID NO: 23), preferably contained in an HLA-A2 α1 and 2 domain-containing recombinant polypeptide IIGAVLAGGL (SEQ ID NO: 24), preferably contained in an HLA-A2 α1 and 2 domain-containing recombinant polypeptide

[0109] Examples of recombinant polypeptides of the present invention (optionally including a leader peptide): MSRSVALAVLALLSLSGLEAKPLPVDMVLGCGASGGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRV NHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGCYNQSEA SSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC* (SEQ ID NO: 15; note that the asterisk represents the stop codon)

[0110] It should be noted that the peptide antigen sequence of the full-length recombinant polypeptide above (here: KPLPVDMVL) can be replaced with any peptide antigen sequence of the present invention, i.e., any peptide antigen presented by said recombinant polypeptide, where the peptide antigen is a peptide of human aquaporin 4. That is, the recombinant polypeptide of the present invention can be composed of a sequence consisting of a peptide antigen that is a peptide of human aquaporin 4 (e.g., any one of the peptide antigens of SEQ ID NOs: 2, 22, 23, and 24), followed by the following sequence: GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGS GGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGS DGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC* (SEQ ID NO: 16; note that the asterisk represents the stop codon)

[0111] These recombinant polypeptides of the present invention can also optionally contain a leader peptide, as exemplified above.

[0112] The receptors ILT2 (also known as LILRB1) and ILT4 (also known as LILRB2) are known in the art. Preferred sequences of the receptors of the present invention are as follows: ILT2: MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRL YREKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTG AYIKPTLSAQPSPVVNSGGNVILQCDSQVAFDGFSLKEGEDEHPQCLNSQPHARGSSRA IFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEE TLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGA HNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKE GAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVS GPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLF LILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQ PEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMD TEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (SEQ ID NO: 17)

[0113] ILT4: MTPIVTVLICLGLSLGPRTHVQTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRL YREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGA YPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEEEHPQCLNSQPHARGSSRAI FSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPGVSKKPSLSVQPGPVVAPGES LTLQCVSDVGYDRFVLYKEGERDLRQLPGRQPQAGLSQANFTLGPVSRSYGGQYRCYGAH NLSSECSAPSDPLDILITGQIRGTPFISVQPGPTVASGENVTLLCQSWRQFHTFLLTKAG AADAPLRLRSIHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSDPYLLSHPSEPLELVVSG PSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVVLLLLLLLLLF LILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKDTQ PEDGVEMDTRAAASEAPQDVTYAQLHSLTLRRKATEPPPSQEREPPAEPSIYATLAIH (SEQ ID NO: 18)

[0114] The sequence of human aquaporin 4 is known in the art. The preferred amino acid sequence of human aquaporin 4 is as follows: >sp|P55087-2|AQP4_HUMAN Isoform 1 of Aquaporin-4 OS=Homo sapiens OX=9606 GN=AQP4 MVAFKGVWTQAFWKAVTAEFLAMLIFVLLSLGSTINWGGTEKPLPVDMVLISLCFGLSIA TMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVV GGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDSKRTDVTGSIALAIGFSVAIGHL FAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRF KEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSS V (SEQ ID NO: 19)

[0115] >sp|P55087|AQP4_HUMAN Aquaporin-4 Isoform 2 OS=Homo sapiens OX=9606 GN=AQP4 PE=1 SV=2 MSDRPTARRWGKCGPLCTRENIMVAFKGVWTQAFWKAVTAEFLAMLIFVLLSLGSTINWG GTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIA AQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDS KRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIG AVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVH VIDVDRGEEKKGKDQSGEVLSSV (SEQ ID NO: 29)

[0116] >NP_001304313.1 aquaporin-4 isoform M1x [Homo sapiens] MSDRPTARRWGKCGPLCTRENIMVAFKGVWTQAFWKAVTAEFLAMLIFVLLSLGSTINWGGTEKPLPVDM VLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPS VVGGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGA SMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEV EDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSVXLEDRTESRQDSLELSSDFLPPIKETD LL (SEQ ID NO:30; X in the sequence can be tryptophan, cysteine, arginine or serine, or a stop codon)

[0117] The present invention is further illustrated by the following non-limiting examples: (Example) Example 1

[0118] Methods for Producing Recombinant Polypeptides of the Invention Expi-293F cells (Thermo Fisher), grown in Expi-293™ Expression Medium (Thermo Fisher): Opti-MEM (Thermo Fisher) for DNA complex formation with Expifectamine, 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 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. Add the required amount 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. Prepare 500 μM Spot peptide solution in PBS, remove supernatant, incubate with ⅓ volume of Spot peptide solution for 5-10 min.

[0119] 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.

[0120] Amicon Ultra-4 centrifugal filters (15 kDa cutoff) are rinsed with PBS followed by 0.1 N NaOH (centrifuged at 4000 g, 4° C.) to remove traces of glycerol.

[0121] 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 minutes with the brake off, after which the ring between the layers was collected and washed with 1x PBS (5 minutes, 300xg). PBMCs were stored frozen until further use.

[0122] B) PBMC pulsing (under a 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.

[0123] The next day (d0), cells were counted and cultured at 3 × 10 6 The cells were resuspended at a cell density of 1000 cells / ml.

[0124] 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.

[0125] On day 3, 1 ml of complete medium (containing cytokines) was added, and on day 6, a second pulse treatment (after removing the medium) was performed 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. What is needed is the following: X-VIVO 15 medium + 5% human AB serum X-VIVO 15 complete medium: X-VIVO 15 medium + 2% human AB serum (with cytokine cocktail added): 10 ng / ml TGF-b1, 10 ng / ml IL-4, 20 ng / ml IL-2, 20 ng / ml GM-CSF

[0126] 2) ELISPOT Laminar flow hood On day 13, ELISPOT plates were coated with anti-hIL10 (clone 9D-7, 1:500 dilution in PBS, sterile filtered) and aIL10 (10G8-biotin), and on day 14, 200,000 cells were plated per well in duplicate on ELISPOT plates, including a negative control (cells + PBS) and a positive control (e.g., LPS).

[0127] 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.

[0128] The respective antigen peptides in DMSO, or DMSO as control, were prepared and a final amount of 5 μg peptide / ml was added to a final volume of 100 μl / well. 150,000 cells were seeded per well in X-VIVO 15 medium containing 5% human AB serum. The blocking buffer (X-VIVO 15 medium + 5% human AB serum) was carefully removed and medium containing PBS as a negative control and stimuli (5 μg / ml total per well) were added to other wells and incubated at 37°C overnight.

[0129] Outside the laminar flow hood Secondary antibodies were prepared: 1 μg / ml aIL-10 biotinylated antibody (1:1000 dilution) in 0.5% BSA / 1x PBS and horseradish peroxidase-conjugated streptavidin (1:750 in 0.5% BSA / PBS), tetramethylbenzidine solution was filtered using a 0.45 μm filter and stored at 4° C. until use.

[0130] 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.

[0131] 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.

[0132] 100 μl of filtered TMB substrate was added per well and left for 15-25 min until blue spots developed. The reaction was stopped by thoroughly washing the wells with tap water.

[0133] The plastic underdrain of the plate was removed and the bottom and sides of the plate were washed with tap water and dried.

[0134] Plates were read using an ImmunoSpot S6 Ultra-V Analyzer (Cellular Technology Limited), analyzed in Excel, and graphs / statistics were performed in Graphad Prism. What is needed is the following: Capture antibodies: anti-hIL10 (clone: ​​9D-7, Mabtech #3430-3-250; 1:500 dilution), anti-hIL10 biotinylated (Mabtech #3430-6-250) 1x PBS (sterile) 35% EtOH (v / v) Blocking buffer: X-vivo 5% hAB serum (sterile) [blocking was performed in the same medium as cell culture] Dilution buffer: 0.5% BAS in PBS Washing buffer: 1x PBS Culture medium: For T cells, X-VIVO 15 medium (Lonza) Filter syringe: Millex GV ELISPOT PVDF plate (#MSIP4510, Millipore) TMB substrate Example 2

[0135] 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 100 μg of a recombinant polypeptide (also called "AIMBio") having the following sequence: Ova_KbG SIINFEKLGCGASGGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKI VKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGCYNQS KGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC (SEQ ID NO: 25), and Gp34_KbG AVYNFATMGCGASGGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVT LSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRV DLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDL GSPDRVRAVSHWSSC (sequence number 26). The Gp34 peptide is a well-characterized T cell epitope derived from the glycoprotein of lymphocytic choriomeningitis virus (LCMV). This antigen was previously designated Gp33, but was later renamed H2-K. b The epitope presented above was found to include only amino acids 34-41 (in contrast to the epitope beginning at amino acid 33, which is not associated with H2-K d (Presented above.) Therefore, we propose that H2-K b The epitope is called Gp34, which is in line with the latest recommendations. Nevertheless, the use of the nomenclature Gp33 and Gp34 in the literature is ambiguous. The first 8 amino acids of SEQ ID NO: 26 show the correct sequence. After 2 weeks, 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. Example 3

[0136] 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.), mice expressing ovalbumin in oligodendrocytes were treated with H2-K b Adoptive transfer of CD8+ OT-I T cells that recognize ovalbumin epitopes in EAE leads to experimental autoimmune encephalomyelitis that reproduces many MS and end-stage NMO 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 showed no significant preventive effect (FIG. 4). The sequence of the recombinant polypeptide surrogate molecule is shown in Example 2. Example 4

[0137] Some of the surrogates of the recombinant polypeptide of the present invention bind to CD4 + Selectively preventing T cell-induced EAE On the day of intraperitoneal injection of 33 μg or 100 μg of a surrogate molecule of a recombinant polypeptide of the present invention ("AIM Bio"), 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 into 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_Kb_G) 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. 5). In this model, Mog44 AIM Bio also prevented inflammation in the spinal cord and infiltration of CD8 T cells (Figure 6). The sequences of the recombinant polypeptide surrogate molecules are described in Example 2 or below:

[0138] Mog44_DbG FSRVVHLYRNGGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQ PKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSMRYFETAVSRPGLEEPRYISVGYVDNKEFVRFDSDAENPRYEPRAPWMEQEGPEYWERETQKAKGQEQWFRVSLRNLLGCYN QSAGGSHTLQQMSGCDLGSDWRLLRGYLQFAYEGRDYIALNEDLKTWTAADMAAQITRRKWEQSGAAEHYKAYLEGECVEWLHRYLKNGNATLLRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC (SEQ ID NO: 27)

[0139] Mog37_DbG VGWYRSPFSRGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQP KIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSMRYFETAVSRPGLEEPRYISVGYVDNKEFVRFDSDAENPRYEPRAPWMEQEGPEYWERETQKAKGQEQWFRVSLRNLLGCYNQ SAGGSHTLQQMSGCDLGSDWRLLRGYLQFAYEGRDYIALNEDLKTWTAADMAAQITRRKWEQSGAAEHYKAYLEGECVEWLHRYLKNGNATLLRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC (SEQ ID NO: 28)

[0140] In this model, Mog44 AIM Bio also completely prevented the formation of MOG-specific autoantibodies in serum, as verified by ELISA (Figure 7; see also confirmation in Figure 10). This strongly indicates that the recombinant polypeptides of the present invention are effective therapeutic agents for NMO, which is often characterized by an antibody response against human aquaporin 4. Thus, patient populations are defined by common autoimmune-associated antigens. Certain MHC molecules are also associated with NMO.

[0141] Mog-reactive antibodies in serum from mice treated with AIM Bio (33 or 100 μg) were detected by a 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 then loaded with mouse serum diluted 1:25 in PBS 1% BSA for 1 h. Detection was performed with anti-mouse IgG-HRP or anti-mouse heavy and light chain HRP antibodies diluted 1:5000. Example 5

[0142] Candidate human recombinant polypeptides of the present invention for NMO 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 regulatory T cells in vitro.

[0143] FIG. 8 shows a list of human MS&MOGAD recombinant polypeptide candidates.

[0144] According to our findings, a single-chain protein containing the AQP4 peptide antigen and the HLA-Gα3 domain can induce tolerogenic T cells in healthy donors. Thus, CD8 Tregs were upregulated by at least 30% in 65% of all healthy blood donors (Figure 9). Example 6

[0145] Further proof of principle regarding the stability and efficacy of recombinant polypeptides of the invention In addition to the above, the inventors set out to obtain and test recombinant polypeptides having the general structure of the recombinant polypeptides of the present invention but containing a variety of different peptide antigens, to further provide proof of principle that the recombinant polypeptides of the present invention and their surrogates are stable and effective. As shown in Figure 11 and Figure 12, respectively, the recombinant polypeptides examined are stable during freeze-thaw and storage, and are heat stable. Furthermore, these recombinant polypeptides induce Tregs in a dose-dependent manner (Figure 13) and inhibit T cell lysis in a dose-dependent manner (Figure 14). The effect of the recombinant polypeptides on serum cytokine profile in EAE-ODC Ova mice is shown in Figure 15. 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. Example 7

[0146] Therapeutic effects of recombinant peptides containing mouse aquaporin antigen peptides in 2D2 TCR transgenic mice material and method Experimental model 2D2 TCR transgenic mice were divided into four cohorts. Each cohort consisted of at least five animals. Litter-matched WT (C57BL / 6) mice were the fifth cohort. The experimental period began when 2D2 mice spontaneously developed optic neuritis. Disease onset was determined by ocular abnormalities or elevated anti-MOG IgG levels in serum. The day after disease onset, mice were injected intravenously with either single-chain MHC Ib molecules AQP147_KbG (antigen peptide: VTTVHGNL; SEQ ID NO: 39) / AQP203_KbG (antigen peptide: FAINYTGASM; SEQ ID NO: 40) matched to the treated mice, or with the control AIM Bio Gp34_KbG. Control mice received PBS only. The dose was 5 mg / kg body weight in 250 μl. Control or treatment injections were administered intravenously (D0). Naive mice received PBS. Injections were repeated on D15 and D30. Mice were weighed daily and monitored for symptoms of EAE (Table 1). Mice that reached an EAE score of 6 were sacrificed before the end of the study period. All animals were sacrificed by CO2 on D42. Optic nerves, eyes, spinal cord, brain, serum, and lymph nodes were harvested and stored frozen at -20°C. Cardiac blood was collected and processed for serum analysis.

[0147] EAE was scored according to a 10-point scoring system (Bittner et al., Journal of visualized experiments: JoVE, (86), 51275).

[0148] Immunohistochemistry Serial sections of 20 μm thickness were cut from the eyeballs and stained with hematoxylin and eosin. Briefly, the frozen sections were dried at room temperature for 10 min and stained in hematoxylin staining solution for 10 min. The tissue sections were washed in running water for 10 min and stained in eosin staining solution for 30 s. The sections were then dehydrated sequentially in 70%, 96%, and 100% ethanol for 30 s each. After this, the sections were incubated with xylol for 10 min and mounted with vitro cloud.

[0149] Immunofluorescence Serial longitudinal sections of optic nerves 10 μm thick were cut for immunohistochemistry. Tissue sections were fixed with 4% PFA in PBS for 10 min; then blocked with a buffer containing 5% BSA, 0.2% Triton-X100, and 5% NGS in 1X PBS for 2 h at room temperature. Tissues were stained with single primary antibodies or appropriate combinations of primary antibodies overnight at 4°C in staining buffer containing 1% BSA, 1% NGS, and 0.2% Triton-X100. Primary antibodies were: i) rat anti-mouse CD3 (1:200, Invitrogen); ii) rat anti-mouse CD8 (1:200, Biorad); iii) rabbit anti-cleaved caspase 3 (1:400, Cell signaling). Sections were washed three times with PBS and stained with the corresponding fluorescently labeled secondary antibodies: i) anti-rabbit-Cy3 (1:300, Dianova); ii) anti-rat-AF488 (1:300, Invitrogen) for 1 h at room temperature in the dark. Sections were washed with PBS and DNA was stained with DAPI (1:500, Sigma-Aldrich) for 10 min at room temperature in the dark. Finally, sections were washed and mounted with Aqua-poly / Mount (Polysciences). Optical sections were obtained at 20x or 40x magnification using a Zeiss Axiocam.

[0150] Quantification of immunostaining Immunofluorescence images were quantified using ImageJ-Fiji Version 1.53t. Three to four sections were analyzed for each animal. Images were split into individual channels and converted to 8-bit images. The threshold for 8-bit images for cleaved caspase-3 staining was set at 5 MFI and for DAPI staining at 100. Area covered by cleaved caspase-3 / area covered by DAPI indicates the percentage of cleaved caspase-3 in the section.

[0151] Quality Control and Stability After purification, the single-chain MHC Ib molecules were analyzed for their 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 of single-chain MHC-Ib molecules and B) HLA-G Western blot with 2A12a HLA-G antibody (1:1000) blot with 1 μg of protein were performed under non-reducing conditions. Both monomers and dimers are detectable.

[0152] result Quality Control and Stability Quality control and stability studies showed that all NMO surrogate single-chain MHC Ib molecules were very stable (Figures 17-19). However, AQP203_H2KbG_spt appeared to be more polymeric, which may explain why it was less effective in some assays.

[0153] Treatment effect Surprisingly, AQP147_KbG treatment provided complete protection from EAE in treated mice (Figure 20), suppressed immune cell infiltration in the optic nerve (Figure 21), and completely inhibited apoptosis in the optic nerve, spinal cord, and retina (Figure 22). Furthermore, although there were fewer CD8 T cells in AIM-treated mice, there were fewer but significantly more regulatory T cells in the optic nerve (Figure 23). In addition, AQP147_KbG completely rescued IRL (inner retinal layer) cells from degradation (Figure 24).

[0154] These data confirm that the recombinant polypeptides of the invention and their surrogates have therapeutic efficacy. Thus, in accordance with the invention, the recombinant polypeptides of the invention can be used to treat neuromyelitis optica in human patients. [Industrial Applicability]

[0155] The pharmaceutical compositions, polypeptides, nucleic acids, cells and products used in the present invention can be industrially applicable, for example, they can be used in the manufacture of medicines or as medicines.

Claims

1. A recombinant polypeptide capable of presenting a peptide antigen, the recombinant polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen presented by the recombinant polypeptide, wherein the peptide antigen is a peptide of human aquaporin 4; ii) optionally, a linker sequence; iii) optionally a sequence of a human polypeptide domain comprising the sequence of human β2 microglobulin, or an amino acid sequence that is at least 90% identical to the amino acid sequence of human β2 microglobulin represented by SEQ ID NO: 5; iv) optionally, a linker sequence; v) optionally the [α]1 domain of an MHC molecule; vi) optionally, the [α]2 domain of an MHC molecule; vii) an [α]3 domain of an MHC class Ib molecule, or a derivative of an [α]3 domain of an MHC class Ib molecule, which is capable of binding to ILT2 or ILT4; viii) optionally, a protease cleavage site; ix) optionally, a spacer sequence; and x) Possibly affinity tags The recombinant polypeptide comprising:

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

3. 2. The recombinant polypeptide of claim 1, wherein the peptide antigen described in i) consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs: 2, 22, 23 and 24.

4. 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 a human MHC class Ib molecule, preferably a human HLA-G molecule.

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

6. The recombinant polypeptide of claim 1, wherein the [α]3 domain or derivative described in (vii) is identical to an [α]3 domain having the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:21, or has at least 80% amino acid sequence identity, at least 90% amino acid sequence identity, at least 92% amino acid sequence identity, at least 94% amino acid sequence identity, at least 96% amino acid sequence identity, at least 98% amino acid sequence identity, or at least 99% amino acid sequence identity, or is identical to an [α]3 domain having the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:

21.

7. 2. The recombinant polypeptide of claim 1, wherein the linker sequence according to (ii) and / or the linker sequence according to (iv) comprises the amino acid sequence (GGGGS)n, where n is an integer equal to or greater than 1, and 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.

8. 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.

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

10. 2. The recombinant polypeptide of claim 1, wherein the polypeptide comprises or consists of all of components (i) through (vii); the polypeptide does not comprise components (viii) through (x); or the polypeptide comprises or consists of all of components (i) through (x).

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

12. 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 NOs: 2, 22, 23, and 24; 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:

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

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

15. A pharmaceutical composition or kit comprising at least one nucleic acid according to claim 14.

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

17. 17. The pharmaceutical composition or kit of claim 16, wherein the pharmaceutical composition or kit comprises at least two different recombinant polypeptides of claim 1, each of the different polypeptides comprising a different peptide antigen of claim 3.

18. 16. The pharmaceutical composition or kit of claim 15 for use in treating neuromyelitis optica in a human patient.

19. 19. The pharmaceutical composition or kit for use according to claim 18, wherein the treatment is an immunotherapeutic treatment, preferably the treatment is by inducing immune tolerance to human aquaporin 4.

20. The pharmaceutical composition or kit for use described in claim 18, wherein the treatment is for reducing plasma or cerebrospinal fluid levels of autoantibodies against human aquaporin 4 and the human patient is a patient who has plasma or cerebrospinal fluid autoantibodies against human aquaporin 4 before the start of treatment.

21. 19. The pharmaceutical composition or kit for use according to claim 18, wherein the treatment is by induction of myelin-specific regulatory T cells.

22. A recombinant host cell comprising the nucleic acid or vector of claim 14 and expressing the recombinant polypeptide of claim 1.

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

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