Mhcb-mediated myelin-specific immunosuppression as a novel treatment for multiple sclerosis and moe antibody disease

Abstract

This invention relates to the therapeutic use of non-classical human major histocompatibility complex (MHC) molecules (also known as MHC class Ib molecules) in combination with myelin-associated peptide antigens for the treatment of multiple sclerosis (MS), MOG antibody disease, and MOG antibody-positive neuromyelitis optica. More specifically, this invention relates to recombinant polypeptides comprising a peptide antigen and one or more domains of a non-classical MHC class Ib molecule. The invention also relates to methods for preparing such recombinant polypeptides, pharmaceutical compositions comprising such recombinant polypeptides, and their use for the treatment of multiple sclerosis (MS), MOG antibody disease, and MOG antibody-positive neuromyelitis optica.

Description

Technical Field

[0001] This invention relates to the therapeutic use of non-classical human major histocompatibility complex (MHC) molecules (also known as MHC class Ib molecules) in combination with peptide antigens for the treatment of multiple sclerosis (MS), MOG antibody diseases, and MOG antibody-positive neuromyelitis optica. More specifically, this invention relates to recombinant polypeptides comprising a peptide antigen and one or more domains of a non-classical MHC class Ib molecule. The invention also relates to methods for preparing such recombinant polypeptides, pharmaceutical compositions comprising such recombinant polypeptides, and their use for the treatment of multiple sclerosis (MS), MOG antibody diseases, and MOG antibody-positive neuromyelitis optica. Background Technology

[0002] Multiple sclerosis (MS) and MOG encephalomyelitis (also known as myelin-oligodendrocyte glycoprotein antibody disease, MOG antibody disease, or MOGAD) are autoimmune diseases in which T cells of the immune system attack the myelin sheath of the central nervous system, leading to progressive neurodegeneration. Immunomodulatory therapy can effectively suppress the relapse of disease activity characteristic of MS. For the rare MOGAD (long considered a subtype of MS), there are currently no approved treatments, so treatment initially involves observation and waiting for disease progression. Slow, progressive neurodegeneration occurs in both diseases. Some treatments effective for MS, such as the antibodies natalizumab or ocrelizumab, or the oral bioavailable S1P inhibitor fingolimod, even carry the risk of uncontrolled, persistent, and usually harmless JC virus intracerebral infection leading to typically fatal progressive multifocal leukoencephalopathy. In this regard, the problems of insufficient or excessive immunomodulatory side effects remain unresolved in both diseases, especially in cases of slow disease progression. Common biologics cannot cross the blood-brain barrier and therefore cannot exert in situ anti-inflammatory effects. In contrast, regulatory cells have been described as being able to cross the blood-brain barrier well (Schneider-Hohendorf et al., Eur J Immunol. Dec 2010; 40(12): 3581-90).

[0003] To date, at least two strategies have been evaluated in early clinical studies. Similar to desensitization strategies in allergy, large doses of antigen are administered via different routes to induce antigen-specific tolerance. However, in autoimmune diseases, these strategies have caused serious side effects and have not been clinically successful. Attempts to induce tolerance through adoptive transfer of antigen-specific modulated T cells or antigen-loaded tolerating dendritic cells appear more promising. However, these strategies are extremely complex and expensive, requiring GMP-compliant manufacturing and quality control procedures for each patient. Therefore, even if small-scale clinical trials are successful, whether adoptive transfer therapy will be suitable for many patients in the foreseeable future remains a questionable issue.

[0004] Immunosuppressive MHC class Ib molecules (such as HLA-G) are crucial for the induction of tolerance during pregnancy. They exert immunosuppressive effects on various immune cells through immunosuppressive receptors such as ILT2, ILT4, and Kir2DL4. WO2018 / 215340 relates to combinations of immunomodulatory MHC class Ib molecules and peptides for targeted therapy.

[0005] In summary, there is still a need to improve drugs for treating multiple sclerosis (MS) and MOG antibody-related diseases. Similarly, there is a need to improve drugs for treating MOG antibody-positive neuromyelitis optica (NMO). Summary of the Invention

[0006] The inventors have discovered that human MHC class Ib molecules, such as HLA-G, can induce antigen-specific tolerance to presented peptide antigens. Therefore, although their structure and sequence are similar to those of classic human MHC class Ia molecules that induce antigen peptide-specific immune responses, according to the present invention, MHC class Ib molecules can be advantageously used in an antigen-specific manner to suppress immune responses. Furthermore, the inventors have discovered that for suppressing immune responses according to the present invention, molecules other than naturally occurring MHC class Ib molecules, particularly polypeptides containing only at least one domain of an MHC class Ib molecule, preferably at least one α3 domain of an MHC class Ib molecule, can be used: the α1 and α2 domains of a variable class Ia molecule can bind to the α3 domain of a human MHC class Ib molecule to suppress immune responses against these antigen-presented peptides.

[0007] HLA-G molecules carrying antigens can be unstable. Therefore, the inventors designed soluble recombinant peptides comprising a peptide antigen, an MHC class Ib molecule (e.g., HLA-G), and β2-microglobulin (b2m), and covalently linked these three components (e.g., via a covalent linker). Alternatively, the antigen-binding α1 and α2 domains of the MHC class Ib molecule (e.g., HLA-G) can be exchanged for corresponding domains of other MHC molecules to enhance the flexibility and versatility of these recombinant peptides (e.g., see [link to relevant documentation]). Figure 2 These alternative recombinant peptides can be engineered to bind with the antigen-binding domains of other human HLA molecules. Previously, it was found that peptides containing mouse H2-K... b The constructs of the α1 and α2 domains can present the ovalbumin-derived peptide SIINFEKL (SEQ ID NO: 66) to OT-1 T cells. (OT-1 T cells express a transgenic T cell receptor that specifically recognizes this antigen) (WO2018 / 215340).

[0008] Surprisingly, the inventors discovered that by using the recombinant peptides of the present invention, immune responses against human myelin-oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG), or human myelin lipoprotein (PLP1) can be suppressed. Therefore, according to the present invention, multiple sclerosis (MS), myelin-oligodendrocyte glycoprotein antibody disease (MOG antibody disease), and MOG antibody-positive neuromyelitis optica can be treated with the recombinant peptides of the present invention.

[0009] The inventors' experimental data show that the desired effect requires the presence of a suitable peptide antigen and the α3 domain of an MHC class Ib molecule (such as HLA-G). Therefore, this approach goes beyond previously described strategies, namely, using antigenic peptides in the absence of co-stimulation (leading to anergic rather than tolerant T cells) or using MHC class Ib molecules in an antigen-nonspecific environment.

[0010] Furthermore, according to the present invention, since the recombinant peptides of the present invention are expected to exhibit better safety compared to conventional drugs for treating these diseases, which may cause serious side effects (e.g., progressive multifocal leukoencephalopathy), the recombinant peptides of the present invention are expected to have significant advantages in the immunotherapy of multiple sclerosis (MS), MOG antibody diseases, and MOG antibody-positive neuromyelitis optica.

[0011] Furthermore, the inventors were surprised to find that the recombinant peptide of this invention not only regulates T-cell responses but also prevents the formation of MOG-specific autoantibodies in model experiments. Since MOG-specific autoantibodies are associated with the pathology of these diseases, this discovery is expected to translate into clinical improvements in patients with multiple sclerosis (MS), MOG antibody disease, and MOG antibody-positive neuromyelitis optica.

[0012] Therefore, the present invention relates to the following preferred embodiments:

[0013] 1. A recombinant polypeptide capable of presenting a peptide antigen, said recombinant polypeptide comprising, in the order from N-terminus to C-terminus:

[0014] i) A peptide antigen presented by the recombinant polypeptide, wherein the peptide antigen is a peptide of human myelin oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG), or human myelin lipoprotein (PLP1).

[0015] ii) Optionally, connector sequence;

[0016] iii) A human polypeptide domain sequence comprising a human β2 microglobulin sequence or an amino acid sequence that is at least 90% identical to the amino acid sequence of the human β2 microglobulin represented by SEQ ID NO: 5;

[0017] iv) Optionally, connector sequence;

[0018] v) The α1 domain of the MHC molecule;

[0019] vi) The α2 domain of the MHC molecule;

[0020] vii) The α3 domain of an MHC Ib molecule or a derivative thereof, wherein the derivative is capable of binding to ILT2 or ILT4.

[0021] viii) Optionally, protease cleavage sites;

[0022] ix) Optionally, an interval sequence; and

[0023] x) Optional, affinity label.

[0024] 2. The recombinant polypeptide according to claim 1, wherein the length of the peptide antigen according to i) is 7 to 11 amino acids, preferably 8 to 10 amino acids.

[0025] 3. The recombinant polypeptide according to claim 1 or 2, wherein the peptide antigen according to claim i) consists of the amino acid sequence of SEQ ID NO: 2.

[0026] 4. The recombinant polypeptide according to claim 1 or 2, wherein the peptide antigen according to claim i) consists of the amino acid sequence of SEQ ID NO: 53.

[0027] 5. The recombinant polypeptide according to any one of the preceding claims, wherein the peptide antigen according to i) is a peptide of human myelin oligodendrocyte glycoprotein (MOG).

[0028] 6. The recombinant polypeptide according to any of the preceding claims, wherein the α1 domain according to (v) and the α2 domain according to (vi) are derived from human MHC class Ia molecules or from human MHC class Ib molecules.

[0029] 7. The recombinant polypeptide according to item 6, wherein the α1 domain according to (v) and the α2 domain according to (vi) are derived from human MHC class Ia molecules.

[0030] 8. The recombinant polypeptide according to claim 7, wherein the α1 domain according to claim (v) and the α2 domain according to claim (vi) are derived from a human HLA-A2 molecule.

[0031] 9. The recombinant polypeptide according to claim 6, wherein the α1 domain according to (v) and the α2 domain according to (vi) are derived from human MHC class Ib molecules.

[0032] 10. The recombinant polypeptide according to any of the preceding claims, wherein the α3 domain of the MHC class Ib molecule according to (vii) is the α3 domain of human HLA-E, human HLA-F or human HLA-G.

[0033] 11. The recombinant polypeptide according to any of the preceding claims, wherein the α3 domain of the MHC class Ib molecule according to (vii) is the α3 domain of human HLA-G.

[0034] 12. The recombinant polypeptide according to any of the preceding claims, wherein the α3 domain or derivative according to (vii) has the same amino acid sequence as the α3 domain having the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 21, or SEQ ID NO: 54, or has at least 80% amino acid sequence identity, preferably at least 90% amino acid sequence identity, with the α3 domain having the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 21, or SEQ ID NO: 54.

[0035] 13. The recombinant polypeptide according to claim 12, wherein the α3 domain or derivative according to (vii) is identical to, or has at least 92% amino acid sequence identity with, the α3 domain having the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 21, or SEQ ID NO: 54.

[0036] 14. The recombinant polypeptide according to claim 12, wherein the α3 domain or derivative according to (vii) is identical to, or has at least 94% amino acid sequence identity with, the α3 domain having the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 21, or SEQ ID NO: 54.

[0037] 15. The recombinant polypeptide according to claim 12, wherein the α3 domain or derivative according to (vii) is identical to, or has at least 96% amino acid sequence identity with, the α3 domain having the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 21, or SEQ ID NO: 54.

[0038] 16. The recombinant polypeptide according to claim 12, wherein the α3 domain or derivative according to (vii) is identical in amino acid sequence to the α3 domain having the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 21, or SEQ ID NO: 54, or has at least 98% amino acid sequence identity with the α3 domain having the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 21, or SEQ ID NO: 54.

[0039] 17. The recombinant polypeptide according to claim 12, wherein the α3 domain or derivative according to (vii) is identical to, or has at least 99% amino acid sequence identity with, the α3 domain having the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 21, or SEQ ID NO: 54.

[0040] 18. The recombinant polypeptide according to claim 12, wherein the α3 domain according to (vii) is identical to the α3 domain having the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 21 or SEQ ID NO: 54.

[0041] 19. The recombinant polypeptide according to claim 12, wherein the α3 domain according to (vii) is identical to the α3 domain having the amino acid sequence shown in SEQ ID NO: 9.

[0042] 20. The recombinant polypeptide according to claim 12, wherein the α3 domain according to (vii) is identical to the α3 domain having the amino acid sequence shown in SEQ ID NO: 21.

[0043] 21. The recombinant polypeptide according to claim 12, wherein the α3 domain according to (vii) is identical to the α3 domain having the amino acid sequence shown in SEQ ID NO: 54.

[0044] 22. The recombinant polypeptide according to any of the preceding claims, wherein the adapter sequence according to (ii) and / or the adapter sequence according to (iv) comprises an amino acid sequence (GGGGS)n, where n is an integer equal to or greater than 1.

[0045] 23. The recombinant polypeptide according to claim 22, wherein the linker sequence according to (ii) comprises an amino acid sequence (GGGGS)n, wherein n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, and preferably selected from 2, 3, 4 and 5.

[0046] 24. The recombinant polypeptide according to claim 22 or 23, wherein the linker sequence according to (iv) comprises an amino acid sequence (GGGGS)n, wherein n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, and preferably selected from 2, 3, 4 and 5.

[0047] 25. The recombinant polypeptide according to any one of the preceding claims, wherein the sequence of the human polypeptide domain according to (iii) is at least 95% identical to the amino acid sequence of SEQ ID NO: 5, preferably at least 98% identical to the amino acid sequence of SEQ ID NO: 5, and more preferably identical to the amino acid sequence of SEQ ID NO: 5.

[0048] 26. The recombinant polypeptide according to any of the preceding claims, wherein the polypeptide is a dimer or a polymer.

[0049] 27. The recombinant polypeptide according to any of the preceding claims, wherein the polypeptide comprises components i) to vii) or consists of all components i) to vii).

[0050] 28. The recombinant polypeptide according to any of the preceding claims, wherein the polypeptide does not contain components (viii) to (x).

[0051] 29. The recombinant polypeptide according to any one of claims 1-27, wherein the polypeptide comprises components i) to x) or consists of all components i) to x).

[0052] 30. The recombinant polypeptide according to any of the preceding claims further comprises an N-terminal secretion signal peptide sequence.

[0053] 31. A recombinant polypeptide according to any one of claims 1-29, wherein the recombinant polypeptide consists of an amino acid sequence arranged from N-terminus to C-terminus consisting of the following ((a) and (b)):

[0054] (a) A peptide antigen selected from the amino acid sequence group of SEQ ID NO: 2 or 53;

[0055] (b) The amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 56-57.

[0056] 32. The recombinant polypeptide according to any of the preceding claims, wherein the recombinant polypeptide is soluble.

[0057] 33. A nucleic acid encoding one or more polypeptides as described in any of the preceding claims.

[0058] 34. The nucleic acid according to claim 33, wherein the nucleic acid is a vector.

[0059] 35. A pharmaceutical composition comprising at least one nucleic acid as described in claim 33 or 34.

[0060] 36. A pharmaceutical composition or kit comprising at least one recombinant polypeptide according to any one of claims 1-32.

[0061] 37. The pharmaceutical composition or kit according to claim 36, wherein the pharmaceutical composition or kit comprises at least two different recombinant polypeptides according to any one of claims 1-32, wherein each different polypeptide comprises a different peptide antigen as defined in any one of claims 3-5.

[0062] 38. The pharmaceutical composition or kit according to any one of claims 35-37 for treating multiple sclerosis (MS), MOG antibody disease, or MOG antibody-positive neuromyelitis optica in human patients.

[0063] 39. A pharmaceutical composition or kit for the use described in claim 38, wherein the treatment is for multiple sclerosis (MS).

[0064] 40. A pharmaceutical composition or kit for the use described in claim 38, wherein the treatment is a treatment for MOG antibody disease.

[0065] 41. A pharmaceutical composition or kit for the use described in claim 38, wherein the treatment is for MOG antibody-positive neuromyelitis optica.

[0066] 42. A pharmaceutical composition or kit for use according to any one of claims 38-41, wherein the treatment is performed via immunotherapy.

[0067] 43. A pharmaceutical composition or kit for use according to any one of claims 38-42, wherein the treatment is performed by inducing immune tolerance against human myelin-oligodendrocyte glycoproteins.

[0068] 44. A pharmaceutical composition or kit for use according to any one of claims 38-43, wherein the treatment is for reducing the level of autoantibodies against human myelin-oligodendrocyte glycoprotein in plasma or cerebrospinal fluid.

[0069] 45. A pharmaceutical composition or kit for use according to any one of items 38-44, wherein the human patient is a patient who has antimyelin-oligodendrocyte glycoprotein autoantibodies in plasma or cerebrospinal fluid prior to the start of treatment.

[0070] 46. ​​A pharmaceutical composition or kit for use according to any one of claims 38-45, wherein the treatment is performed by inducing myelin-specific regulatory T cells.

[0071] 47. A recombinant host cell comprising a nucleic acid or vector according to claim 33 or 34 and expressing a recombinant polypeptide according to any one of claims 1-32.

[0072] 48. A method for obtaining a pharmaceutical composition comprising a polypeptide according to any one of claims 1-32, the method comprising the steps of: (a) culturing a recombinant host cell of claim 47 under conditions allowing expression of the recombinant polypeptide from a nucleic acid molecule, (b) recovering the recombinant polypeptide, (c) purifying the recombinant polypeptide, and (d) formulating the recombinant polypeptide into a pharmaceutical composition. Attached Figure Description

[0073] Figure 1 : Description of soluble MHC Ib molecules of loaded peptides suitable for achieving therapeutic antigen-specific immunomodulation.

[0074] The presented peptide antigens are represented by dashed spheres, the HLA-G α-3 domain is shown in light gray, and the β2-microglobulin domain is shown in dark gray. Optional linkers connecting the antigenic peptide and the β2-microglobulin molecule are represented by gray bars, while optional disulfide traps are represented by black spheres. This figure was generated using Pymol and modified based on structures published in Clements et al., ProcNatl Acad Sci US A. 2005 Mar 1;102(9):3360-5 and Hansen et al., TrendsImmunol. 2010 Oct;31(10):363-9.

[0075] Figure 2 Examples of vector-based constructs encoding single-chain MHC Ib molecules suitable for therapeutic peptide-specific immunomodulation.

[0076] HLA-G1 and HLA-G5 are each composed of three α-domains (shown in black), one non-covalently linked β2-microglobulin subunit (shown in dark gray), and an antigenic peptide presented on HLA-G (short black arrows). HLA-G1 also contains a transmembrane domain and a short intracellular chain (not shown). As shown in the figure, the α-3 domain can bind to receptor ILT2 (see Shiroishi et al., Proc Natl Acad Sci US A. 2003 July 22;100(15):8856-8861) and receptor ILT4 (see Shiroishi et al., Proc Natl Acad Sci US A. 2006 Oct 31;103(44):16412-7) on immune cells. Physiologically, these sequences form a non-covalently linked MHC class 1 complex. To simplify the purification of complex MHC Ib molecules, one or more protein tags (e.g., SpotTag, myc tag, and / or His(6x) tag) can be introduced. These can be introduced in such a way that they can be optionally removed later by cleavage at an optional factor Xa or furin protease cleavage site. Furthermore, the antigenic peptide, β2-microglobulin, and MHC Ib α chain can be linked to improve stability. Vector maps were generated using Snapgene Viewer software.

[0077] Figure 3 The alternative molecules of the recombinant polypeptide of the present invention induce Treg secretion of IL10 in mice.

[0078] In this experiment, 100 µg of the substitution molecule (made from viral (Gp34) or ovalbumin (Ova) model peptide antigen, mouse H2-K) was used.b The α1 and α2 domains, along with the human HLA-G α3 domain and β-2-microglobulin, were injected intraperitoneally (ip) into 12-week-old C57BL / 6 mice. Fourteen days later, the mice were sacrificed, and splenocytes isolated via a Ficoll gradient were again challenged with 5 µg / ml Gp34 or Ova peptide in a 48-hour standard mouse IL-10 ELISpot assay (Mabtech Mouse IL-10 HRP ELISpot Kit).

[0079] A significant increase in IL-10-secreting regulatory T cells was detected, which secrete IL-10 only upon induction of tolerance by alternative molecular injection and in response to restimulation by peptides targeting them (B).

[0080] Figure 4 The recombinant peptide of this invention provides a molecule that can prevent experimental autoimmune encephalomyelitis (EAE) induced by CD8+ T cells in mice. In this MS mouse model, 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 onto the H-2Kb MHC molecule on oligodendrocytes. OT-I mice express the T cell receptor (OT-I) on their CD8+ T cells, which accurately recognizes this peptide-MHC combination. When the CD8+ T cells of these mice are transferred to 10-day-old ODC-OVA mice, these mice develop experimental autoimmune encephalomyelitis (EAE), which is similar in many ways to the pathogenesis and symptoms of MS (Na et al., Brain, Volume 131, Issue 9, September 2008, Pages 2353–2365). In this experiment, 500µg of the alternative molecule (composed of viral (Gp34) or ovalbumin (Ova) model peptide antigen, mouse H2-K) was injected on the same day. b (Composed of α1 and α2 domains, as well as human HLA-G α3 domain and β-2-microglobulin) or injected with PBS alone. EAE was scored according to Bittner et al., J VisExp . 2014 Apr 15;(86):51275.

[0081] Only alternative molecules that induce ovalbumin tolerance can almost completely prevent EAE symptoms.

[0082] (A) Experimental design; (B) Results.

[0083] Figure 5 The alternative molecules of the recombinant polypeptide of the present invention can produce effective bystander immunosuppression.

[0084] This experiment tested whether a Mog44 peptide substitute could induce protective T cells that inhibit cytotoxic T cells targeting a different peptide presented by the same cells. Figure 4 The molecule described in the text was injected into ODC-OVA mice at a dose of 250 µg / mouse along with OT-I cells, as... Figure 4 As shown.

[0085] While this lower dose of alternative molecules cannot completely prevent EAE symptoms, it significantly alleviates EAE symptoms by inducing tolerance to molecules that directly target the CD8 epitope (Ova_KbG) or to another epitope (Mog44) expressed on the same cells (Mog44_DbG).

[0086] (A) EAE score; (B) Body weight.

[0087] Figure 6 The present invention provides alternative molecules of some recombinant peptides that can selectively prevent CD4 in mice. + EAE caused by T cells.

[0088] (A) Experimental Design: In this model, a strong myelin-specific autoimmune response was induced by administration of MOG 35-55 peptide combined with complete Freund's adjuvant (which activates CD4+ Th17 cells) and pertussis toxin (which increases the permeability of the blood-brain barrier) (Protocol: Bittner et al., J Vis Exp . 2014 Apr 15;(86):51275). Here, CD4+ cells and antibodies play a crucial role in the development of EAE (Tigno-Aranjuez et al., J Immunol November 1, 2009, 183 (9) 5654-5661). In addition, on the first day, each mouse was injected with 100 µg of virus (Gp34) or two Mog peptide antigens (Mog37 or Mog44), mouse H2-D b Alternative molecules consisting of α1 and α2 domains, as well as the human HLA-G α3 domain and β-2-microglobulin, or simply injected with PBS.

[0089] Mog44 peptide, which contains a substitute molecule, significantly alleviated EAE symptoms (B) and weight loss (C).

[0090] Figure 7 The Mog44-substituted molecule of the recombinant polypeptide of this invention can prevent spinal cord inflammation and CD8 T cell infiltration. (A) Toluidine; (B) CD8-DAB

[0091] Freshly frozen sections of 10 μm were stained with commercially available toluidine 1× staining reagent at room temperature for 1 h. Strong immune cell infiltration was detected in the EAE, but was blocked by Mog44_Db_G.

[0092] Freshly frozen sections of 10 µm were briefly dried at room temperature, fixed with acetone, blocked with 5% BSA and 10% normal goat serum in PBS, and stained with 1:100 anti-CD8 antibody and secondary antibody conjugated with HRP and DAB solution (detailed method: Karikari et al., Brain Behav Immun. 2022 Jan 12;101:194-210). Mog35-55-induced EAE leads to CD8... + Cells infiltrate the spinal cord extensively, and the MOG44_Db_G substitution molecule can block this process.

[0093] (A) Toluidine; (B) CD8-DAB

[0094] Figure 8 Detection of anti-MOG35-55 antibody (“AIM Bio”) in Mog-EAE mice treated with the recombinant peptide of this invention.

[0095] Mice were euthanized, and serum was collected via cardiac puncture and diluted 1:50 in PBS. The mice were coated overnight with 10 µg / ml Mog35-55 in PBS, followed by blocking with 1% BSA for 2 hours, then adding diluted serum and incubating for 1 hour. Anti-Mog35-55 antibodies were detected using the indicated secondary antibody HRP-conjugated antibody (diluted 1:5000 in PBS). Mog35-55-induced EAE was associated with high levels of Mog35-55-specific IgG autoantibodies, which were undetectable in animals treated with 100 µg MOG44_Db_G as a replacement molecule.

[0096] Figure 9 List of example human MS and MOGAD recombinant peptide candidates.

[0097] The following are myelin (MAG, MBP, MOG, PLP) peptides and MHC class I presenting molecules:

[0098] Recombinant peptides peptide antigen sequence SEQ ID NO: MAG160_A2G_His MVPDNCPEL 25 MAG160_G_His MVPDNCPEL 25 MAG237_G_Spt KYPPVIVEM 34 MOG104_A2G_Spt AIGEGKVTL 26 MOG104_A2G_Spt AIGEGKVTL 26 MBP29_G_Spt FLPRHRDTG 35 MBP75_G_Spt RSQPGLCNM 27 MBP169_G_Spt KGVDAQGTL 36 MBP244_A2G_Spt SLSRFSWGA 28 MOG38_G_Spt RHPIRALVG 31 MOG42_G_Spt RALVGDEVEL 32 MOG44_G_Spt FSRVVHLYRNG 37 MOG70_G_Spt RPPFSRVVHL 38 MOG102_G_Spt KDAIGEGKVTL 29 MOG104_A2G_Spt AIGEGKVTL 26 MOG157_A2G_Spt VLLAVLPVL 2 MOG157_G_Spt VLLAVLPVL 2 MOG163_G_Spt VSPGVLVLL 39 MOG185_G_His KLRAEIENL 40 MOG203_A2G_His RVPCWKITL 41 MOG203_G_His RVPCWKITL 41 PLP3_A2G_His LLLECCARCL 33 PLP3_G_His LLLECCARCL 33 PLP80_A2G_His FLYGALLLA 42 PLP105_G_Spt KTTICGKGL 43 PLP127_G_His RGQHQAHSL 44 PLP151_G_spt KFVGITYAL 45

[0099] Furthermore, it demonstrates which combinations of myelin peptides and antigen-presenting MHC class I α1 and α2 domains (HLAG = HLA-G, A2G = HLA-A2 presenting domain + HLA-G α3 domain) yielded favorable results in expression / production, ELISpot-based priority ranking of healthy blood donors (as shown in Figure 10), or AlphaFold2 prediction. His represents the 6-histidine tag, and Spt represents the Spot tag.

[0100] Figure 10 The recombinant polypeptide (“Mog157_A2G”) containing VLLAVLPVL antigen of this invention can upregulate CD8 Treg cells in healthy blood donors.

[0101] The in vitro Treg induction method mediated by AIM Biologicals is as follows:

[0102] Leukocytes from the leukocyte attenuation chamber were purified by density centrifugation using Ficoll to purify PBMCs from healthy donors. Cells were centrifuged continuously at 1200×g for 20 min, and then the intermediate phase loop was collected and washed with 1× PBS (5 min, 300×g). The PBMCs were frozen until further use.

[0103] One day before PBMC pulse (d-1), PBMCs were thawed and incubated overnight at 37°C in 5 ml of X-VIVO 15 medium containing 5% human AB serum in the wells of a 6-well plate.

[0104] On day 2 (d0), cells were counted and suspended in X-VIVO 15 complete medium (5% hAB serum and cytokine mixture: 20 ng / ml hIL-2, 20 ng / ml hGM-CSF, 10 ng / ml hIL-4 and 10 ng / ml hTGF-b1) at a cell density of 3 × 10⁶ cells / ml. 6 Cells / ml. In the experiment, 3 × 10⁻⁶ cells / ml were used. 6 Cells were seeded into the corresponding wells of a 12-well plate, with a final volume of 1000 µl of X-VIVO complete medium containing a cytokine mixture and 5 µg / ml AIM Bio molecules or a corresponding control.

[0105] On day 3, add 1 ml of complete culture medium (containing cytokines); on day 6, add 5 µg / ml of AIM Bio molecules for a second pulse treatment (after removing the culture medium). On days 7, 10, and 12, add 1 ml of complete culture medium (containing cytokines).

[0106] 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 of distilled sterile water. The plate was then coated overnight at 4°C with 100 µl / well anti-hIL10 (clone 9D-7, diluted 1:500 in PBS and sterile filtered). On day 2, unbound coating antibody was removed, and the plate was washed 5 times with 200 µl of PBS. 200 µl of blocking buffer (X-VIVO 15 5% hAB serum) was added, and the plate was incubated at room temperature for 30 min–2 h.

[0107] On day 14, 200,000 cells were seeded into each well of an ELISPOT plate, in duplicate, including a negative control (cells plus PBS) and a positive control (e.g., LPS).

[0108] Secondary antibody preparation: 1 µg / ml aIL-10-biotinylated antibody was dissolved in 0.5% BSA / 1× PBS (1:1000 dilution) and horseradish peroxidase-conjugated streptavidin (0.5% BSA / PBS, 1:750 dilution). Tetramethylbenzidine solution was filtered through a 0.45 µm filter and stored at 4 °C until use.

[0109] Remove the cell supernatant and wash five times with 100 µl PBS. Finally, remove excess buffer with paper.

[0110] Add 25 µl of diluted HRP-streptavidin (1:750) to each well, incubate at room temperature in the dark for 1 h, and then wash 5 times with sterile 1×PBS.

[0111] Add 100 µl of filtered TMB substrate to each well and let stand for 15–25 minutes until blue spots appear. Rinse the wells thoroughly with tap water to stop the reaction.

[0112] Remove the plastic drain pipe from the board and rinse the bottom and sides of the board with tap water and let it air dry.

[0113] MOG157_A2G_Spt induces at least a 30% increase in Tregs secreting IL-10 in 75% of healthy blood donors.

[0114] Figure 11 : Figure 8 Control experiments showed that total IgG was not reduced by MOG47_Db_G substitution molecule treatment. Total IgG was quantified using the Easy-Titer™ Human IgG (γ-chain) Detection Kit (Thermo Fisher) according to the manufacturer's instructions. This experiment was compared with... Figure 8 This combination suggests that single-chain MHC Ib molecules can be used to inhibit selective antibody responses.

[0115] Figure 12 Stability of purified single-chain MHC Ib molecules. After purification, the stability of single-chain MHC Ib molecules was analyzed after one and three freeze-thaw cycles, storage at room temperature for 5 days, and heating to 50°C for 30 min. For this purpose, under non-reducing conditions, A) Coomassie brilliant blue staining was performed using 2 µg AIM Bio gel with 12% polyacrylamide, and B) aHLA-G protein blotting was performed using 2A12aHLA-G antibody (1:1000) and 1 µg protein. Both monomers and dimers were detectable.

[0116] Figure 13 Single-chain MHC Ib molecules are thermally stable. For thermal shift analysis (TSA), 3 µg of the corresponding single-chain MHC Ib molecule or Motavizumab as a control molecule was diluted to a volume of 25 µl with PBS and 5× SYPRO Orange dye (stock solution 5000×, final concentration: 5×). A melting curve program was set on the StepOnePlus instrument using StepOnePlus software 2.3. The initial temperature was 25 °C for 1 min, then increased to a final temperature of 95 °C at a rate of 1 °C per minute for 2 min, allowing autofluorescence to be measured in arbitrary units. Data were exported and plotted in Prism V7.04. The Boltzmann sigmoid function was used to determine the melting temperature (Tm).

[0117] Figure 14 Single-chain MHC Ib molecules induce Tregs in a dose-dependent manner. OT-I mice were intraperitoneally injected with a specified amount of single-chain H2_K. b The α1+2 and HLA-G alpha3 domains, human β-2-microglobulin, and the specified peptide were constructed or carried in PBS. Ova was a homologous peptide of the OT-I TCR in these mice, and Gp34 was an unrelated virus-derived control peptide. After 14 days, mice were sacrificed, and IL-10-secreting cells in splenocytes were tested in a recalled mouse IL-10 ELISpot (200,000 cells per well, MabTech Mouse IL-10 ELISpot Kit, with 5 µg / ml of the specified peptide or PBS alone, 48 h). Significant induction of IL-10-secreting cells responsive to the Ova peptide was observed upon injection of 50 µg and 500 µg of mouse-adapted Ova_KbG.

[0118] Figure 15Single-chain MHC Ib molecules inhibit T cell lysis in a dose-dependent manner. Panc02 cells loaded with Ova peptide (SIINFEKL (SEQ ID NO: 66)) were cultured at 10 mIO OT-1 / ml for 3 days in the presence of single-chain MHC Ib molecules and then added at a 50:1 ratio. 25 µg / ml of the Ova-presenting AIM Bio substitute molecule almost completely prevented the lysis of target cells loaded with Ova peptide.

[0119] Methods: OT1 / BL6 mice were sacrificed, and spleen cells were collected and washed once in RPMI 5% FCS. Red blood cells were removed by treatment with 2 ml of 1× sterile RBC lysis buffer for 3 min. Cells were cultured at high density (10 mIo cells / ml) for 72 h in RPMI 10% FCS (containing GMCSF 20 ng / mL, IL-2 20 ng / mL, and IL-4 10 ng / mL, with escalating doses of Ova_KbG). Cells were then scraped from the culture dish and purified as CD8+ cells using magnetic beads.

[0120] Luciferase assays were performed using sterile 96-well white plates. PancO2 fluc+ target cells were loaded with 20 µg / ml Ova peptide (SIINFEKL (SEQ ID NO: 66)) and shaken at 500 rpm for 60 minutes at 37°C. Effector CD8+ T cells were mixed with PancO2 target cells (5000 target cells) at a ratio of 50:1, and Promega firefly luciferin was added. Luminescence intensity was measured at 0, 24, and 48 hours.

[0121] Figure 16Serum cytokines in EAE-ODC Ova mice. Serum cytokines in EAE-ODC Ova mice were measured using the Th1 / Th210plex Flowcytomix Kit (eBioscience) according to the manufacturer's instructions. This kit is 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 a single sample. The array kit provides a mixture of ten capture microbeads with different fluorescence intensities, each coated with a specific capture antibody against each cytokine. The magnetic beads coated with the ten specific capture antibodies were mixed. Subsequently, 25 μL of the mixed capture beads, 25 μL of unknown serum sample or standard dilution, and 25 μL of phycoerythrin (PE) detection reagent were sequentially added to each well of a 96-V plate and incubated at room temperature in the dark for 2 h. The samples were washed with 1 mL of washing buffer for 5 min, followed by centrifugation. The supernatant was discarded, and the beads were resuspended in 200 μL of buffer. Attune was used as the measuring medium. TM Samples were measured using an NxT flow cytometer and analyzed using Attune flow cytometer software (Thermo Fisher Scientific).

[0122] Figure 17 Immunofluorescence of spinal cord in MOG-induced EAE model

[0123] A, B: MOG-induced Caspase 3 infiltration in the spinal cord of EAE. A: Image; B: Quantitative analysis.

[0124] C, D: MOG-induced EAE spinal cord white matter lesions. C: Image; D: Quantitative analysis.

[0125] E, F: MOG-induced CD3 infiltration in the spinal cord of EAE. E: Image; F: Quantitative analysis.

[0126] Figure 18 IL10 spots increased after MOG157_A2G treatment.

[0127] Figure 19 A: Simple Western blotting results for AIM Bio expression and polymerization. All compounds were found to be monomers (~50 kDa); B: Expression levels in the supernatant were quantified by ELISA.

[0128] The constructs used in this figure are described in the "Sequence" section below and in Example 9. Simple Western blotting was performed as described in Example 9; sandwich ELISA was performed as described in Example 9.

[0129] Figure 20 ELISpot assay was performed using peripheral blood mononuclear cells from the patient. A: Image of the wells in the assay; duplicate wells are always arranged in a row. The image shows the number of spots counted. B: Quantitative analysis.

[0130] The ELISpot was performed as described in Example 9, except that only 1.2 mi of PBMC was used to seed the cells in 24-well plates, and the volume of all culture media was reduced accordingly.

[0131] In response to AIM Bio AQP65 P65K Of the 5 NMO patients treated with _G, 4 had increased IL-10 spots in their PBMCs compared to untreated PBMCs.

[0132] Figure 21 AIM Bios inhibits EAE and optic neuritis in a 2D2 transgenic spontaneous optic neuritis model. a) 2D2 transgenic mice expressing MOG-targeting TCRs were treated with 3 µg / g of MOG-specific AIM Bios or a control molecule at days 0, 15, and 30. Ocular inflammation, EAE scores, and body weight were collected for a total of 42 days. Blood and tissue samples were collected after 42 days for further analysis (n=3). b) Ocular inflammation scores are expressed as mean ± standard error. Statistical differences were determined using one-way ANOVA and Tukey's multiple comparison test (last time point). c) Daily EAE disease scores are expressed as mean ± standard error. Statistical differences were determined using two-way ANOVA and multiple comparisons. df) Apoptosis in the retina (d), spinal cord (e), and optic nerve (f) as measured by IHC staining with anti-caspase 3 antibody. All data are expressed as mean ± standard error (SEM). Unpaired t-tests were used to determine statistical differences.

[0133] Figure 22To characterize the purified proteins, size exclusion chromatography was performed at room temperature using an Äkta FPLC (Amersham Biosciences) Superdex 200 increase 10 / 300 column and Unicorn 5.31 software (GE Healthcare). Before run, the buffer was replaced from 20% EtOH (stock solution) with 2 CV H2O. During analysis, the column was washed with 2 CV PBS (Sigma, D8537-500 ml) at a flow rate of 0.5 ml / min. Subsequently, 10–20 µg of AIM Bio (500 µl) was loaded onto the Superdex 200 increase 10 / 300 column (Cytiva), and the column was run for one column volume at a flow rate of 0.5 ml / min to collect the protein fraction.

[0134] Figure 23 Thermal displacement analysis (TSA)

[0135] Figure 24 ILT2-Fc combined with ELISA

[0136] Figure 25 ILT4-Fc combined with ELISA

[0137] Figure 26 ELISpot detection

[0138] Figure 27 In addition, the upregulation of other potentially dangerous cytokines (such as IFNγ or TNFα) in the PBMC supernatant of MOGAD patients was analyzed using the Biolegend LEGENDplex™ HU Th cytokine panel (12-fold) according to the manufacturer’s instructions.

[0139] Figure 28 : Dextran staining. Invention Details

[0141] Definitions and General Techniques

[0142] Unless otherwise stated below, the terminology used in this invention should be understood in the manner of its common meaning as known to those skilled in the art. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety. Publications mentioned herein may be cited by explicitly listing their full reference numbers in the text.

[0143] All proteins described in this invention, including the recombinant polypeptides of this invention, can be obtained by methods known in the art. These methods include methods for preparing recombinant polypeptides. According to this invention, the recombinant polypeptides of this invention can be expressed in recombinant host cells. The recombinant host cells of this invention are preferably mammalian cells, such as CHO cells and HEK cells.

[0144] It should be understood that the recombinant peptides of the present invention are intended to selectively include a secretion signal peptide sequence. Similarly, the recombinant peptides of the present invention may also selectively include an affinity tag (e.g., for ease of purification) and an optional protease cleavage site between the tag and the peptide (e.g., for ease of removal of the tag by protease cleavage).

[0145] It should also be understood that any amino acid sequence mentioned herein is intended not only to cover unmodified amino acid sequences, but also to cover typical post-translational modifications of these amino acid sequences that occur in cellular expression systems known in the art, including mammalian cells such as CHO and HEK cells (e.g., glycosylation or deamidation of amino acids, splicing of specific amino acids, or other post-translational modifications).

[0146] Similarly, it should be understood that the recombinant polypeptides of the present invention are intended to optionally include the corresponding propeptides.

[0147] It should also be understood that the recombinant polypeptides of the present invention can be in their soluble form or their membrane-bound form. Whether the 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 above-described reference conditions. Hereinafter, "soluble" means that at least 95% of the recombinant polypeptide is determined to be soluble under these reference conditions.

[0148] For example, single-chain MHC molecules can be stored in PBS at -80°C (with or without 0.1% human serum albumin as a carrier, depending on the protein concentration), or in 50% glycerol at -20°C.

[0149] According to the present invention, the MHC molecule is preferably a human MHC molecule.

[0150] The recombinant polypeptide of the present invention is preferably an isolated recombinant polypeptide.

[0151] It is understood how to prepare recombinant peptides capable of binding and presenting peptide antigens according to the present invention. For example, peptide antigen-binding domains, such as α1 and α2 domains, are well known and can be modified. According to the present invention, the ability of peptide antigens to bind to peptides and MHC molecules can be determined by techniques known in the art, including but not limited to exploratory methods, such as MHC peptide elution followed by mass spectrometry and computer bioinformatics prediction, and confirmatory methods, such as MHC peptide multimer binding assays and stimulation assays.

[0152] According to the present invention, the recombinant peptides, pharmaceutical compositions and kits of the present invention are preferably suitable for human patients.

[0153] According to the present invention, the recombinant peptides, pharmaceutical compositions and kits of the present invention are preferably suitable for treating multiple sclerosis (MS), MOG antibody disease or MOG antibody-positive neuromyelitis optica in human patients.

[0154] According to the present invention, the recombinant peptides, pharmaceutical compositions and kits of the present invention are preferably suitable for, for example, inducing immune tolerance against human myelin-oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG) or human myelin lipoprotein (PLP1) in human patients.

[0155] According to the present invention, the recombinant peptides, pharmaceutical compositions, and kits of the present invention, wherein the α1 domain according to (v) and the α2 domain according to (vi) are derived from the human HLA-A2 molecule, are preferably used to treat multiple sclerosis (MS), MOG antibody disease, or MOG antibody-positive neuromyelitis optica in HLA-A2 positive human patients. Compared with HLA-A2 negative patients, this use is expected to be associated with more favorable (lower immunogenicity) immunogenicity characteristics and a longer half-life (slower clearance rate).

[0156] In contrast, HLA-G does not exhibit the high polymorphism of classic HLA alleles. The HLA-G-based peptides of this invention are expected to possess favorable immunogenicity and half-life in the human population.

[0157] It should be understood that, according to the present invention, the recombinant peptides, pharmaceutical compositions and kits of the present invention are stable.

[0158] It should be understood that, with respect to the peptide antigens used in this invention, the length of any peptide antigen mentioned herein (e.g., "7 to 11 amino acids") refers to the length of the peptide antigen itself. Therefore, the length of the peptide antigen referred to herein does not include the length imparted by additional amino acids that are not part of the peptide antigen (such as additional amino acids in possible linker sequences).

[0159] According to the present invention, each occurrence of the term "comprising" can be selectively replaced with the term "consisting of".

[0160] Methods and Techniques

[0161] Generally, unless otherwise defined herein, the methods used in this invention (e.g., cloning methods or antibody-related methods) are performed in accordance with procedures known in the art, such as those described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989), Ausubel et al. (Molecular Biology Laboratory Protocols, 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.

[0162] Protein-protein binding, such as the binding of an antibody to its respective target protein, can be assessed using methods known in the art. Protein-protein binding is preferably assessed by surface plasmon resonance spectroscopy measurements.

[0163] For example, the binding of MHC class b molecules or recombinant peptides according to the invention to their receptors, including ILT2 and ILT4, is preferably evaluated by surface plasmon resonance spectroscopy. More preferably, the binding of MHC class I b molecules or recombinant peptides according to the invention to their receptors is evaluated by surface plasmon resonance measurements at 25°C. Appropriate conditions for such surface plasmon resonance measurements have been described by Shiroishi et al. Proc Natl Acad Sci US A. July 22, 2003; 100(15): 8856-8861.

[0164] According to the present invention, sequence alignment is performed using the BLAST algorithm (see Altschul et al. (1990) “Basic local alignment search tool.” Journal of Molecular Biology 215.p. 403-410.; Altschul et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.). Suitable parameters for sequence alignment of short peptides using the BLAST algorithm are known in the art and are applicable to the peptide antigens according to the present invention. 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: maximum target sequence number 10; word length 3; BLOSUM 62 matrix; gap cost: 11 for presence, 1 for expansion; conditional combination score matrix adjustment. Therefore, when using sequence-related terms such as “identity” or “same,” the identity value obtained using the BLAST algorithm is preferred.

[0165] Preparation of the pharmaceutical composition of the present invention

[0166] The pharmaceutical compositions according to the present invention are prepared according to known standards for the preparation of pharmaceutical compositions.

[0167] For example, the pharmaceutical composition is prepared in a manner that enables it to be properly stored and used. Therefore, the pharmaceutical compositions of the present invention may include pharmaceutically acceptable components, such as carriers, excipients, and / or stabilizers.

[0168] When the pharmaceutical composition is administered to human patients, these pharmaceutically acceptable components are non-toxic at the dosage level. The pharmaceutically acceptable components added to the pharmaceutical composition may depend on the chemical nature of the active ingredient present in the composition, the specific use of the pharmaceutical composition, and the route of administration.

[0169] Typically, pharmaceutically acceptable components relevant to this invention are used based on knowledge known in the art, see, for example, Remington's Pharmaceutical Sciences, Ed. AR Gennaro, 20th edition, 2000, Williams & Wilkins, PA, USA. Pharmaceutical compositions comprising the nucleic acids (e.g., RNA) of this invention may also be formulated based on knowledge known in the art, for example, using liposome formulations targeting dendritic cells.

[0170] peptide antigens according to the present invention

[0171] Peptide antigens that can be used according to the present invention include peptide antigens as defined above, without particular limitation except that they can be presented on MHC molecules. It should be understood that "peptide antigen presented by the recombinant polypeptide" as used in the present invention refers to a peptide antigen presented to human T cells (if such T cells are present) by the recombinant polypeptide in a manner that binds to T cell receptors on human T cells.

[0172] As is known in the art, peptides capable of being presented on MHC molecules can be produced (see, for example, Rammensee, Bachmann, Emmerich, Bachor, Stevanović. 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).

[0173] Peptide antigens are well known in the art. Typically, peptide antigens according to the invention are capable of binding to MHC class I proteins. Those skilled in the art will understand that, for each MHC class Ib molecule or polypeptide capable of presenting a peptide according to the invention, a peptide antigen capable of binding to said MHC class Ib molecule or recombinant polypeptide will preferably be used. These peptide antigens can be selected based on methods known in the art.

[0174] The binding of peptide antigens to MHC class Ib molecules according to the present invention or polypeptides capable of binding to peptide antigens can be evaluated using methods known in the art, for example:

[0175] Rammensee, Bachmann, Emmerich, Bachor, Stevanović. SYFPEITHI:database for MHC ligands and peptide motifs.Immunogenetics.1999 Nov;50(3-4):213-9;

[0176] 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

[0177] Rock, Reits, Neefjes.Present Yourself! By MHC Class I and MHC ClassII Molecules.Trends Immunol.2016 Nov;37(11::724-737).

[0178] These methods include experimental methods and methods for predicting peptide-antigen binding.

[0179] Anchoring residues used to anchor peptide antigens to MHC class I molecules and ensure that peptide antigens bind to MHC class I molecules are known in the art.

[0180] In a preferred embodiment of all embodiments of the invention, the peptide antigen used according to the invention comprises any or preferred amino acid residues anchored at a position predicted for MHC class I molecules.

[0181] Preferably, such predictions are made according to the descriptions in any of the following publications:

[0182] - Rammensee et al., SYFPEITHI: MHC ligand and peptide motif database. Immunogenetics (1999) 50: 213-219

[0183] - Nielsen et al, Protein Sci (2003) 12:1007-1017

[0184] - Neefjes et al. Nat Rev Immunol. 2011 Nov 11;11(12):823-36

[0185] - Diehl et al. Curr Biol. March 1, 1996; 6(3):305-14,

[0186] - Lee et al. Immunity. November 1995;3(5):591-600.

[0187] - Desai & Kulkarni-Kale, T-cell epitope prediction methods: anoverview. Methods Mol Biol. 2014;1184:333-64.

[0188] - Jumper et al. Highly accurate protein structure prediction withAlphaFold. Nature 2021; 596: 583–589

[0189] In this invention, the peptide antigen is derived from human myelin-oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG), or human myelin protein lipoprotein (PLP1).

[0190] It should be understood that the non-anchored amino acid residues of the peptide antigen of the present invention may contain or not contain conserved substitutions relative to the corresponding amino acid sequence of the peptide antigen from human myelin oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG) or human myelin lipoprotein (PLP1), preferably no more than two conserved substitutions, and more preferably only one conserved substitution.

[0191] The peptide antigens of the present invention are preferably composed of naturally occurring amino acids. However, non-naturally occurring amino acids, such as modified amino acids, may also be used. For example, in one embodiment, the peptide antigens of the present invention comprise peptide mimics of a specified peptide antigen amino acid sequence of human myelin oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG), or human myelin lipoprotein (PLP1).

[0192] Methods for synthesizing peptide antigens, including methods for synthesizing peptide antigens according to the present invention, are well known in the art.

[0193] Therapeutic applications of the present invention

[0194] The recombinant polypeptide of the present invention can be used to treat multiple sclerosis, MOG antibody disease, and MOG antibody-positive neuromyelitis optica.

[0195] This therapy can be achieved by inducing myelin-specific regulatory T cells. When regulatory T cells (e.g., CD8-positive regulatory T cells) are activated within the myelin sheath structure, they can protect target cells from attack by cytotoxic T cells that recognize the same or other myelin antigens. Regulatory T cells (e.g., CD8-positive regulatory T cells) are known in the art and can be detected, for example, by their secretion of IL-10.

[0196] Although CD8-positive regulatory T cells are not as well-known as CD4-CD25-positive regulatory T cells, they have been reported to be even more effective. For example, see: 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: Aheterogeneous population with different phenotypes and properties. Eur. J.Immunol., 51: 512-530. https: / / doi.org / 10.1002 / eji.202048614

[0197] Although these cells are characterized by the expression of CD122 and CD8 in mice, their human counterparts have been described as positive for CD8 and CXCR3. See, for example:

[0198] 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).

[0199] This therapy can be used to reduce the level of autoantibodies against human myelin oligodendrocyte glycoprotein in plasma or cerebrospinal fluid. Human patients are those who have autoantibodies against myelin oligodendrocyte glycoprotein in their plasma or cerebrospinal fluid before the start of treatment.

[0200] According to the present invention, autoantibodies can be detected by various methods known in the art. A preferred method is cell-based assay (CBA), in which a suspected target antigen of the autoantibody (e.g., myelin oligodendrocyte glycoprotein) is overexpressed in HEK293 or CHO cells, and these cells are then incubated with serum or cerebrospinal fluid, typically at room temperature for 1 hour. Sham-transfected sister cells serve as a control. The autoantibodies binding to the cells can be detected by different fluorescently labeled anti-human specific secondary antibodies that recognize total human IgG (heavy and light chains), IgG-Fc (constant chain), or IgG1. Binding can be quantified by visual scoring via flow cytometry (CBA-FACS) or by microscopic assessment of immunofluorescence (CBA-IF), typically requiring titration. In most established cell-based MOG antibody detection methods, the 218-amino acid MOG a1 isoform is used, although an alternative, a truncated version of MOG containing only extracellular immunoglobulins and transmembrane domains, has also been tested. Other methods, such as enzyme-linked immunosorbent assay (ELISA) or Western blotting, are also feasible, but are generally less sensitive because conformation-sensitive antibodies may not be detectable by these methods. Suitable methods have been described in the following aspects:

[0201] Waters, P., Pettingill, P., and Lang, B, Detection methods for neuralautoantibodies. Handb. Clin. Neurol. 133, 147–163 (2016).

[0202] For a detailed overview of myelin oligodendrocyte glycoprotein (MOG) antibodies, please see Reindl M, Waters P. Myelin oligodendrocyte glycoprotein antibodies in neurological disease, Nat Rev Neurol. 2019 Feb;15(2):89-102. doi: 10.1038 / s41582-018-0112-x. PMID: 30559466.

[0203] sequence

[0204] The preferred amino acid sequences mentioned in this application may be independently selected from the following sequences. The sequences are represented in N-terminal to C-terminal order; and they are represented by a one-letter amino acid code.

[0205] Exemplary sequences of the recombinant polypeptides of this invention:

[0206] Optional leader peptides (not present in recombinant peptides due to processing during cell expression): for example, MSRSVALAVLALLSLSGLEA (SEQ ID NO: 1), encoded by the DNA sequence ATGAGCAGATCTGTGGCCCTGGCTGTTCTGGCTCTGCTGTCTCTGTCTGGCCTGGAAGCC (SEQ ID NO: 50).

[0207] Peptide antigen: Any MHC class I peptide corresponding to the α1 and α2 domains of MHC class I, such as VLLAVLPVL (SEQ ID NO: 2), or any other peptide antigen according to the present invention:

[0208] Recombinant peptides peptide antigen sequence SEQ ID NO: MOG203-212 RVPCWKITLF 53

[0209] First connector: for example, GGGGSGGGGSGGGGS (SEQ ID NO: 3) or GCGASGGGSGGGGS (SEQ ID NO: 4)

[0210] β2 microglobulin, for example:

[0211] IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 5, human β2 microglobulin)

[0212] Second connector, for example:

[0213] GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 6)

[0214] The α1 and α2 domains are derived from human HLA-G or any other MHC class I α1 and α2 domains, suitable for presenting the selected antigenic peptide. Y84 in the DT variant may be C, for example, the α1 and α2 domains, derived from human HLA-G.

[0215] For example,

[0216] GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRA (SEQ ID NO: 7)

[0217] Alternatively: derived from human HLA-A2 α1 and α2 domains: for example,

[0218] GSHSMRYFFTSVSRPGRGEPRIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRT (SEQ ID NO: 8)

[0219] Human HLA-G α3 domain (or any MHC class Ib α3 domain, such as HLA-F, which also interacts with ILT2 and ILT4 receptors), for example: DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDL (SEQ ID NO: 9; HLA-G α3 sequence, wherein the furin cleavage site is present in the C-terminal amino acid sequence of the 4th intron of the human HLA-G gene), or

[0220] DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESGSLSEDL (SEQ ID NO: 54; HLA-G α3 sequence, wherein the furin cleavage site of the C-terminal amino acid sequence in human HLA-G intron 4 has been removed).

[0221] Note: The following underlined amino acids in this sequence are associated with interactions with ILT2 or ILT4 receptors:

[0222] DPPKTHVTHH PVFDYE ATLRCWALGFYPAEIILTWQRDGEDQTQD V ELVETRPAGDGTFQKWAAV V VPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDL, or

[0223] DPPKTHVTHH PVFDYE ATLRCWALGFYPAEIILTWQRDGEDQTQD V ELVETRPAGDGTFQKWAAV V VPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESGSLSEDL

[0224] Alternatively, a shorter human HLA-G [alpha]3 domain can be used, which lacks the optional C-terminal amino acid sequence from intron 4 (SKEGDGGIMSVRESRSLSEDL; SEQ ID NO: 20, or SKEGDGGIMSVRESGSLSEDL; SEQ ID NO: 55), i.e.:

[0225] DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO: 21).

[0226] The inventors discovered that any of the above-mentioned HLA-G α3 sequences can be used to produce recombinant peptides. The recombinant peptides contain the sequence SEQ ID NO: 54, i.e., the HLA-G α3 sequence, in which the furin cleavage site of the C-terminal amino acid sequence in human HLA-G intron 4 has been removed. This is beneficial for improving the purification of the recombinant peptides, for example, for purifying recombinant peptides containing a C-terminal tag.

[0227] Xa factor restriction site: IEGRTGTKLGP (SEQ ID NO: 10)

[0228] SpotTag: PDRVRAVSHWSSC (SEQ ID NO: 11)

[0229] Myc tag: EQKLISEEDL (SEQ ID NO: 12)

[0230] His tag: HHHHHH* (SEQ ID NO: 13)

[0231] Spacer sequences: such as NSAPD (SEQ ID NO: 14) or GS

[0232] The following are exemplary reference peptide antigens that can serve as components of recombinant polypeptides:

[0233] Table 1: Reference peptide antigens

[0234] Peptide antigens are the following peptides: peptide sequence SEQ ID NO: Remark MAG MVPDNCPEL 25 Preferred for recombinant peptides containing human HLA-A2 α1&2 domains MOG AIGEGKVTL 26 Preferred for recombinant peptides containing human HLA-A2 α1&2 domains MBP RSQPGLCNM 27 Preferred for recombinant peptides containing α1&2 domains derived from human HLA-G MBP SLSRFSWGA 28 Preferred for recombinant peptides containing human HLA-A2 α1&2 domains MOG KDAIGEGKVTL 29 Preferred for recombinant peptides containing α1&2 domains derived from human HLA-G MOG AIGEGKVTL 30 Preferred for recombinant peptides containing human HLA-A2 α1&2 domains MOG RHPIRALVG 31 Preferred for recombinant peptides containing α1&2 domains derived from human HLA-G MOG RALVGDEVEL 32 Preferred for recombinant peptides containing α1&2 domains derived from human HLA-G PLP LLLECCARCL 33 Preferred for recombinant peptides containing α1&2 domains derived from human HLA-G

[0235] Exemplary peptide antigens that can be used as part of the recombinant polypeptides of the present invention are as follows:

[0236] Table 2: Exemplary peptide antigens according to the present invention

[0237] Peptide antigens are the following peptides: peptide sequence SEQ ID NO: Remark MOG RVPCWKITLF 53 Preferred for recombinant peptides containing α1&2 domains derived from human HLA-G MOG VLLAVLPVL 2 Preferred for recombinant peptides containing the α1&2 domains derived from human HLA-A2

[0238] Examples of recombinant polypeptides of the present invention (including optional leader peptides):

[0239] MSRSVALAVLALLSLSGLEAVLLAVLPVLGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTE KDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTD RMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHH PVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC* (SEQ ID NO: 15; note asterisk indicates stop codon)

[0240] Note that according to the present invention, the peptide antigen sequence of the above full-length recombinant polypeptide (here: VLLAVLPVL) can be replaced by any other peptide antigen sequence, that is, any peptide antigen presented by the recombinant polypeptide, where the peptide antigen is a peptide of human myelin oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1). That is, the recombinant polypeptide of the present invention can consist of the following sequence: a peptide antigen, which is a peptide of human myelin oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1) (for example, the peptide antigen of SEQ ID NO: 2 or 53 according to the present invention), followed by the following sequence

[0241] GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC* (SEQ ID NO: 16; note that the asterisk indicates a stop codon),

[0242] or followed by the following sequence:

[0243] GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGS

[0244] GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQ RRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESGSLSEDLGSPDRVRAVSHWSSC* (SEQ ID NO: 56; note asterisk indicates stop codon),

[0245] Alternatively, it may be followed by the following sequence, which does not contain the C-terminal amino acid sequence of intron 4 of the HLA-G α3 domain, the variable cleavage site, and the optional tag:

[0246] GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAH AQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO: 57).

[0247] These recombinant peptides of the present invention may also include any of the optional leader peptide, the C-terminal amino acid sequence of intron 4 of the HLA-G α3 domain sequence, and any of the optional tags shown above.

[0248] The following two examples of recombinant peptides according to the invention are particularly preferred. The corresponding sequences shown below lack an optional leader peptide sequence. The sequences shown below are followed by sequences of human HLA-G α3 domains, for example, the human HLA-G α3 domain of SEQ ID NO: 21 (i.e., the human HLA-G α3 domain with the C-terminal amino acid sequence of intron 4 removed), the human HLA-G α3 domain of SEQ ID NO: 9 (i.e., the human HLA-G α3 domain containing the C-terminal amino acid sequence of intron 4, which includes a furin cleavage site), or the human HLA-G α3 domain of SEQ ID NO: 54 (i.e., the human HLA-G α3 domain containing the C-terminal amino acid sequence of intron 4, but with the furin cleavage site removed):

[0249] MOG 157-165 _A2G (VLLAVLPVL containing the peptide sequence: SEQ ID NO: 2):

[0250] VLLAVLPVL GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVG YVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRT (SEQ ID NO: 58), followed by the sequence of the human HLA-G α3 domain (eg, SEQ ID NO: 9, 21, or 54).

[0251] Encoded by DNA sequences (note that the DNA sequences encoding the N-terminal leader peptide are not shown below):

[0252]

[0253] MOG 203-212 _G_fs-: (Contains the peptide sequence of SEQ ID NO: 53: RVPCWKITLF):

[0254] RVPCWKITLF GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMG YVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRA (SEQ ID NO: 59), followed by the sequence of the human HLA-G α3 domain (eg, SEQ ID NO: 9, 21, or 54).

[0255] These sequences may also contain variable cut sites, optional adapters (e.g., GS), and / or optional tags.

[0256] Receptors ILT2 (also known as LILRB1) and ILT4 (also known as LILRB2) are known in the art. According to the present invention, the preferred sequences of these receptors are as follows:

[0257] ILT2:

[0258] MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEETLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (SEQ ID NO: 17)

[0259] ILT4:

[0260] MTPIVTVLICLGLSLGPRTHVQTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGAYPKPTLSAQPSPVVT SGGRVTLQCESQVAFGGFILKEGEEEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPGVSKKPSLSVQPGPVVAPGESTLTLQCVSDVGYDRFVLYKEGERDLRQLPGR QPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSECSAPSDPLDILITGQIRGTPFISVQPGPTVASGENVTLLCQSWRQFHTFLLTKAGAADAPLRLRSIHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSDPY LLSHPSEPLELVVSGPSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVVLLLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKDTQ PEDGVEMDTRAAASEAPQDVTYAQLHSLTLRRKATEPPPSQEREPPAEPSIYATLAIH (SEQ ID NO: 18)

[0261] The sequences of human myelin-oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG), or human myelin lipoprotein (PLP1) are known in the art. Preferred amino acid sequences of MOG, MBP, MAG, or PLP1 are as follows:

[0262] Human myelin oligodendrocyte glycoprotein (MOG):

[0263] NP_996532.2 Myelin oligodendrocyte glycoprotein subtype α1 precursor [Homo sapiens]

[0264] MASLRSRPSLPSCLCSFLLLLLLQVSSSYAGQFRVIGPRHPIRALVGDEVELPCRISPGKNATGMEVGWYRPPFSRVVHLYRNGKDQDGDQAPEYRGRTELLKDAIGEGKVTLRIRNVRFSDEG GFTCFFRDHSYQEEAAMELKVEDPFYWVSPGVLVLLAVLPVLLLQITVGLIFLCLQYRLRGKLRAEIENLHRTFDPHFLRVPCWKITLFVIVPVLGPLVALIICYNWLHRRLAGQFLEELRNPF

[0265] (SEQ ID NO: 19)

[0266] Human myelin basic protein (MBP):

[0267] >NP_001020252.1 myelin basic protein isoform 1 [Homo sapiens]

[0268] MASQKRPSQRHGSKYLATASTMDHARHGFLPRHRDTGILDSIGRFFGGDRGAPKRGSGKVPWLKPGRSPLPSHARSQPGLCNMYKDSHHPARTAHYGSLPQKSHGRTQDENPVVHFFKNIVTPRTPPPSQGKGRGLLSRFSWGAEGQRPGFGYGGRASDYKSAHKGFKGVDAQGTLSKIFKLGGRDSRSGSPMARR

[0269] (SEQ ID NO: 22)

[0270] Human myelin-associated glycoprotein (MAG):

[0271] >NP_002352.1 Myelin-associated glycoprotein isoform a precursor [Homo sapiens]

[0272] MIFLTALPLFWIMISASRGGHWGAWMPSSISAFEGTCVSIPCRFDFPDELRPAVVHGVWYFNSPYPKNYPPVVFKSRTQVVHESFQGRSRLLGDLGLRNCTLLLSNVSPELGGKYYFRGDLGGYNQYTFSEHSVLDIVNTPNIVVPPEVVAGTEVEVSCMVPDNCPELRPELSWLGHEGLGEPAVLGRLREDEGTWVQVSLLHFVPTREANGHRLGCQASFPNTTLQFEGYASMDVKYPPVIVEMNSSVEAIEGSHVSLLLCGADSNPPPLLTWMRDGTVLREAVAESLLLELEEVTPAEDGVYACLAENAYGQDNRTVGLSVMYAPWKPTVNGTMVAVEGETVSILCSTQSNPDPILTIFKEKQILSTVIYESELQLELPAVSPEDDGEYWCVAENQYGQRATAFNLSVEFAPVLLLESHCAAARDTVQCLCVVKSNPEPSVAFELPSRNVTVNESEREFVYSERSGLVLTSILTLRGQAQAPPRVICTARNLYGAKSLELPFQGAHRLMWAKIGPVGAVVAFAILIAIVCYITQTRRKKNVTESPSFSAGDNPPVLFSSDFRISGAPKYESERRLGSERRLLGLRGEPPELDLSYSHSDLGKRPTKDSYTLTEELAEYAEIRVK

[0273] (SEQ ID NO: 23)

[0274] Human myelin proteolipid protein (PLP1):

[0275] >NP_000524.1 Myelin proteolipid protein isoform 1 [Homo sapiens]

[0276] MGLLECCARCLVGAPFASLVATGLCFFGVALFCGCGHEALTGTEKLIETYFSKNYQDYEYLINVIHAFQYVIYGTASFFFLYGALLLAEGFYTTGAVRQIFGDYKTTICGKGLSATVTGGQKGRGSRGQHQAHSLERV CHCLGKWLGHPDKFVGITYALTVVWLLVFACSAVPVYIYFNTWTTCQSIAFPSKTSASIGSLCADARMYGVLPWNAFPGKVCGSNLLSICKTAEFQMTFHLFIAAFVGAAATLVSLLTFMIAATYNFAVLKLMGRGTKF

[0277] (SEQ ID NO: 24)

[0278] The invention is further illustrated by the following non-limiting embodiments.

[0279] Example

[0280] Example 1:

[0281] The method for producing recombinant peptides of the present invention

[0282] -Expi-293F cells (Thermo Fisher), cultured in Expi-293™ expression medium (Thermo Fisher): Using the Expifectamine™ 293 transfection kit (Thermo Fisher), DNA was combined with Expifectamine using Opti-MEM (Thermo Fisher), and 1 µg of DNA was transfected into 2.5 × 10⁻⁶ cells. 6 In a cell incubation rate of 100 cells / ml, after 18-20 hours, add the enhancer according to the experimental protocol. Collect the supernatant 4-6 days later (37℃, 8% CO2, humidified incubator), 19 mm. 2 Track-mounted shaking table, 125 rpm.

[0283] -Spot-tag protein purification: Spot-Cap resin equilibration: Transfer the required amount of slurry to a suitable centrifuge tube, centrifuge to precipitate the resin beads (4°C, 4 min, 2500 g), remove and discard the supernatant, add 10 column volumes of PBS buffer (cold) to the resin beads, invert to mix, centrifuge to precipitate the resin beads (4°C, 4 min, 2500 g), remove and discard the supernatant, repeat 2 times.

[0284] Add the required volume of magnetic beads to the supernatant and incubate at 4°C on a vortex mixer. Wash the magnetic beads by repeated centrifugation (4°C, 4 minutes, 2500 g) and remove the supernatant.

[0285] - Prepare a spotting peptide solution with a concentration of 500 µM (dissolved in PBS), remove the supernatant, and incubate 1 / 3 of the spotting peptide solution with the sample for 5-10 minutes.

[0286] Centrifugal precipitate beads. Protein concentration was performed using an Amicon Ultra-4 centrifugal filter (15 kDa molecular weight cutoff) and spot peptide removal was performed using a 15 kDa Amicon molecular weight cutoff column.

[0287] The Amicon Ultra-4 centrifuge filter (15 kDa molecular weight cutoff) was rinsed with PBS and then with 0.1 N NaOH (4000 g, 4 °C centrifugation) to remove trace amounts of glycerol.

[0288] ELISPOT:

[0289] 1) Cell Culture

[0290] A) Peripheral blood mononuclear cell isolation (under laminar flow hood)

[0291] To isolate peripheral blood mononuclear cells (PBMCs), density centrifugation was performed using leukocytes from the leukopenia chamber and a density gradient medium (e.g., Ficoll or ROTI Sep 1077). Cells were centrifuged continuously at 1200×g for 20 min, and then the intermediate phase loop was collected and washed with 1× PBS (5 min, 300×g). The PBMCs were frozen until further use.

[0292] B) PBMC pulse stimulation (under laminar flow hood)

[0293] One day before PBMC pulse stimulation (d-1), PBMCs were thawed and incubated overnight at 37°C in 5 ml of X-VIVO 15 medium containing 5% human AB serum in the wells of a 6-well plate.

[0294] On day 2 (d0), cells were counted and suspended in X-VIVO 15 complete medium (5% hAB serum and cytokine mixture: 20 ng / ml hIL-2, 20 ng / ml hGM-CSF, 10 ng / ml hIL-4 and 10 ng / ml hTGF-b1) at a cell density of 3 × 10⁶ cells / ml. 6 Cells / ml.

[0295] In the experiment, 3×106 Cells were seeded into each well of a 12-well plate to a final volume of 1000 µl of X-VIVO complete medium containing a cytokine mixture and 5 µg / ml of AIM Bio molecules or a corresponding control group.

[0296] On day 3, 1 ml of complete culture medium (containing cytokines) was added; on day 6, a second pulse treatment was performed, adding 5 µg / ml of the recombinant polypeptide of this invention or its alternative molecules (collectively referred to as "AIM Bio") (after removing the culture medium). On days 7, 10, and 12, 1 ml of complete culture medium (containing cytokines) was added.

[0297] Material:

[0298] X-VIVO 15 culture medium + 5% human AB serum

[0299] X-VIVO 15 complete culture medium: X-VIVO 15 medium + 5% human AB serum, with the addition of a cytokine mixture: 10 ng / ml TGF-β1, 10 ng / ml IL-4, 20 ng / ml IL-2, 20 ng / ml GM-CSF

[0300] 6-hole and 12-hole plates

[0301] 2) ELISPOT

[0302] On day 13, ELISPOT plates were coated with anti-hIL10 (clone 9D-7, diluted 1:500 with PBS and sterile filtered) and aIL10 (10G8-biotin); on day 14, 200,000 cells were seeded into each well of an ELISPOT plate, with two replicates per well, including a negative control (cells plus PBS) and a positive control (e.g., LPS).

[0303] Activate the PFDF membrane with 50 µl / well EtOH (35% v / v) for 1 min, then wash 5 times with 200 µl of distilled sterile water. Coat the membrane with 100 µl / well of antibody solution and incubate overnight at 4°C. The next day, remove unbound coated antibody, wash 5 times with 200 µl of PBS, add 200 µl of blocking buffer (X-VIVO 15 5% hAB serum), and incubate the plate at room temperature for 30 min–2 h.

[0304] Prepare the corresponding antigenic peptide (e.g., MOG157) in DMSO or use DMSO as a control, and add a final volume of 5 μg peptide / ml to a final volume of 100 μl / well. Seed 150,000 cells per well in X-VIVO 15 medium containing 5% human AB serum. Carefully remove the blocking buffer (X-VIVO 15 medium + 5% hAB serum), use medium containing PBS as a negative control, add the stimulant (5 μg / ml total volume per well) to the other wells, and incubate overnight at 37°C.

[0305] Laminar flow hood outside

[0306] Secondary antibody preparation: 1 µg / ml aIL-10-biotinylated antibody was dissolved in 0.5% BSA / 1× PBS (1:1000 dilution) and horseradish peroxidase-conjugated streptavidin (0.5% BSA / PBS, 1:750 dilution). Tetramethylbenzidine solution was filtered through a 0.45 µm filter and stored at 4 °C until use.

[0307] Remove the cell supernatant and wash five times with 100 µl PBS. Finally, remove excess buffer with paper.

[0308] Add 25 µl of diluted HRP-streptavidin (1:750) to each well, incubate at room temperature in the dark for 1 h, and then wash 5 times with sterile 1×PBS.

[0309] Add 100 µl of filtered TMB substrate to each well and let stand for 15–25 minutes until blue spots appear. Rinse the wells thoroughly with tap water to stop the reaction.

[0310] Remove the plastic drain pipe from the board and rinse the bottom and sides of the board with tap water and let it air dry.

[0311] The data from the board was read using an ImmunoSpot S6 Ultra-V analyzer (Cellular Technology Limited), analyzed in Excel, and then plotted / statistical data in Graphad Prism.

[0312] Required: Capture antibody: anti-hIL10 (clone: ​​9D-7, Mabtech #3430-3-250; 1:500 dilution), anti-hIL10 biotinylated (Mabtech, #3430-6-250), 1×PBS (sterile), 35% EtOH (v / v); Blocking buffer: X-vivo 5% hAB serum (sterile) [blocked in the same medium as cell culture]; Dilution buffer: 0.5% BSA in PBS; Wash buffer: 1×PBS; Culture medium: for T cells, X-VIVO 15 medium (Lonza); Filter syringe: Millex GV, ELISPOT PVDF plate (#MSIP4510, Millipore), TMB substrate

[0313] Example 2 (Reference): The alternative molecule of the recombinant polypeptide of the present invention induces Tregs to secrete IL10 in mice.

[0314] Six wild-type black mice were injected with 100 µg of a recombinant polypeptide (also known as "AIMBio"), the sequence of which is as follows.

[0315] Ova_KbG

[0316] SIINFEKLGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQP KIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGC YNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDPPKTHVTHHPVFDY EATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC (SEQ ID NO: 46)

[0317] and

[0318] Gp34_KbG

[0319] AVYNFATMGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQP KIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGC YNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDPPKTHVTHHPVFDY EATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC (SEQ ID NO: 47)

[0320] Tolerance to either the OVA peptide or the viral Gp34 peptide was induced, respectively. Gp34 is a well-characterized T-cell epitope derived from a lymphocytic choriomeningovirus (LCMV) glycoprotein. Although this epitope was traditionally named Gp33, it was later discovered that H2-K... b The epitopes presented only contain amino acids 34-41. (Conversely, epitopes starting from amino acid 33 are presented on H2-K.) d (Above.) Therefore, we will use H2-K b The epitope is named Gp34, which aligns with the latest recommendations. However, the use of the nomenclature Gp33 and Gp34 in the literature remains ambiguous. The first eight amino acids of SEQ ID NO: 47 show the corresponding peptide sequence. Two weeks later, mice were sacrificed and splenocytes were challenged again with matching or non-matching peptides. IL-10 secretion cells were quantified using ELIspot. Results are as follows... Figure 3 As shown.

[0321] Example 3 (Reference): The alternative molecule of the recombinant polypeptide of the present invention can prevent EAE induced by CD8+ T cells in mice.

[0322] As described in (Na et al, Brain. 2008 Sep;131(Pt 9):2353-65.), H2-K will be identified.b CD8 of ovalbumin epitopes in the background + Adoptive transfer of OT-I T cells to mice expressing ovalbumin in oligodendrocytes leads to experimental autoimmune encephalomyelitis (EAE), which re-emerges with many symptoms of MS and MOGAD. In this animal model, a single injection of 500 µg of the recombinant peptide substitute molecule (also known as "AIM Bio") induces tolerance to the target ovalbumin epitope and almost completely prevents EAE symptoms, while the substitute molecule presenting the control peptide has no significant protective effect. Figure 4 The sequence of the recombinant polypeptide substitution molecule is shown in Example 2.

[0323] In the same model, the inventors further demonstrated that effective bystander immunosuppression could be generated using antigenic peptides presented in target tissues or cells. Here, each mouse was injected with 250 µg of the recombinant peptide substitute molecule (also known as “AIMBio”) (Figure 5). The sequence of the recombinant peptide substitute molecule is as follows:

[0324] Mog44_DbG

[0325] FSRVVHLYRNGGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTL SQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSMRYFETAVSRPGLEEPRYISVGYVDNKEFVRFDSDAENPRYEPRAPWMEQEGPEYWERETQKAKGQEQWFRVSLRNLL GCYNQSAGGSHTLQQMSGCDLGSDWRLLRGYLQFAYEGRDYIALNEDLKTWTAADMAAQITRRKWEQSGAAEHYKAYLEGECVEWLHRYLKNGNATLLRTDPPKTHVTHHPVFD YEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC (SEQ ID NO: 48)

[0326] Mog37_DbGVGWYRSPFSRGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACR VNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSMRYFETAVSRPGLEEPRYISVGYVDNKEFVRFDSDAENPRYEPRAPWMEQEGPEYWERETQKAKGQEQWFRVSL RNLLGCYNQSAGGSHTLQQMSGCDLGSDWRLLRGYLQFAYEGRDYIALNEDLKTWTAADMAAQITRRKWEQSGAAEHYKAYLEGECVEWLHRYLKNGNATLLRTDPPKTHVTHHPV FDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC (SEQ ID NO: 49)

[0327] Example 4 (Reference): Some recombinant peptide alternatives of the present invention can selectively prevent CD4 in mice. + EAE caused by T cells

[0328] On day 0, 33 µg or 100 µg of the recombinant polypeptide (“AIM Bio”), the alternative molecule of this invention, was administered intraperitoneally. 100 μl of MOG35-55 peptide / CFA (complete Freund's adjuvant; final concentrations of Mycobacterium tuberculosis H37RA and peptide were each 1 mg / ml) emulsion was administered subcutaneously to the left and right flanks. 250 ng of pertussis toxin (in 200 μl PBS) was administered intraperitoneally. A second pertussis toxin injection was given 3 days later. In this animal model, a single injection of the AIM Bio alternative molecule (inducing tolerance to the Mog epitope (Mog44_DbG)) significantly reduced EAE symptoms, while alternative molecules presenting the control peptide (Gp34) or the non-functional Mog peptide (Mog37) did not show significant protective effects. Figure 6 In this model, Mog44 AIM Bio also prevented spinal cord inflammation and CD8 T cell infiltration. Figure 7 The sequence of the recombinant polypeptide substitution molecule is shown in Example 3.

[0329] In this model, ELISA analysis showed that Mog44 AIM Bio completely prevented the formation of MOG-specific autoantibodies in serum. Figure 8 and Figure 11 This strongly suggests that the recombinant peptides of the present invention are effective therapeutic agents for MS and MOGAD, both diseases typically characterized by antibody responses against MOG. Therefore, the patient population is defined by common autoimmune-associated antigens.

[0330] Mog-reactive antibodies in the serum of mice treated with AIM Bio (33 or 100 µg) were detected using a standard ELISA protocol, with three washes between each step. In brief, the ELISA plate was coated with 10 µg / ml Mog35-55 peptide, blocked with PBS 1% BSA, and then mouse serum diluted 1:25 in PBS 1% BSA was added and incubated for 1 h. Detection was performed using anti-mouse IgG-HRP or anti-mouse heavy and light chain-HRP antibodies diluted 1:5000.

[0331] Example 5: Human recombinant peptide candidates for MS and MOGAD of the present invention

[0332] The recombinant polypeptide of this invention is a newly developed protein complex derived from the pregnancy-associated immunosuppressive MHC molecule HLA-G. HLA-G may enable the embryo to influence the maternal immune system, making it tolerant to embryonic antigens, but further antagonizing pathogen antigens. The recombinant polypeptide containing the variable peptide of this invention can selectively eliminate peptide-specific cytotoxic effector T cells and induce peptide-specific regulatory T cells in vitro.

[0333] Figure 9 A list of human MS and MOGAD recombinant peptide candidates is shown.

[0334] The inventors' research indicates that single-chain proteins containing myelin peptide antigen and HLA-G α3 domain can induce tolerant T cells in healthy donors. Therefore, proteins containing VLLAVLPVL antigen (also known as "Mog157_A2G"; see [link to related research]) can induce tolerance T cells. Figure 10 The recombinant polypeptide of the present invention upregulated CD8 Treg by at least 30% in 75% of all healthy blood donors.

[0335] Example 6 (Reference): Further proof of concept regarding the stability and efficacy of the recombinant polypeptide of the present invention.

[0336] Furthermore, the inventors set out to obtain and test recombinant peptides having the general structure of the recombinant peptides of the present invention but containing various different peptide antigens, in order to obtain further proof of concept and demonstrate that the recombinant peptides of the present invention and their alternative molecules are stable and effective. Figure 12 and Figure 13 As shown, the tested recombinant peptides remained stable during freeze-thaw cycles and storage, and exhibited thermal stability. Furthermore, they induced Treg (Treg) in a dose-dependent manner. Figure 14 ) and inhibits T cell lysis in a dose-dependent manner ( Figure 15 The effects of recombinant peptides on serum cytokine profiles in EAE-ODC Ova mice are as follows: Figure 16 As shown, IL-10 was induced, and IL-4 was potentially induced as well; both are known immunosuppressive cytokines that can downregulate immune responses in an inflammatory environment. This requires the HLA-G α3 domain plus a homologous peptide. IL-2 appears to be induced upon presentation of a homologous peptide unrelated to the α3 domain to cells. IL-2 is essential for T cell activation and survival.

[0337] Example 7 (Reference): The MOG tolerance-inducing recombinant peptide of the present invention can prevent spinal cord cell apoptosis (cleaved caspase 3) and CD3. + Immune cell infiltration and myelin damage. Mouse models such as... Figure 6 As shown. Immunofluorescence analysis of the spinal cord in a MOG-induced EAE model.

[0338] Material:

[0339] Primary resistance:

[0340] α-CD3i clone CD3-12 (1:100); rat monoclonal (rat anti-human, cross-reactive with mouse, Bio-rad); α-MBP (1:300, rabbit monoclonal, MBL); α-cleaved caspase 3 (1:400, rabbit monoclonal, CellSignaling)

[0341] DAPI (1:500, Sigma# D8417)

[0342] Secondary antibody

[0343] α-Rabbit-Cy3 (red, 1:300, Dianova #111-165-144); α-Rat-AF488 (green, 1:300, Invitrogen); or α-Rabbit-Cy3 (red, 1:300, Dianova #112-165-167).

[0344] plan

[0345] The frozen sections were heated at room temperature for 5 minutes, the stained areas were marked with Pap-Pen, fixed in acetone, and placed at -20°C for 10 minutes.

[0346] Washing: Wash 3 times with 1×PBS

[0347] Blocking for 1 hour: 1× PBS containing 5% BSA + 0.2% Triton-X100 + 5% NGS

[0348] Primary antibody: 1% BSA + 1% NGS + 0.2% Triton-X100; 4 o C, overnight

[0349] Washing: Wash 3 times with 1×PBS

[0350] Secondary antibody: 1% BSA + 1% NGS + 0.2% Triton-X100; RT, 1h

[0351] Washing: Wash 3 times with 1×PBS

[0352] DAPI: 1:300 in 1× PBS, 10 min, RT

[0353] Washing: Wash twice with 1×PBS

[0354] Use Aquapolymount to cover

[0355] Detailed results:

[0356] Figure 17 A and B: Caspase 3 activation (apoptosis) can be blocked in MOG-induced EAE spinal cord. A: Image; B: Quantitative analysis.

[0357] Figure 17 C and D: MOG-induced EAE spinal white matter lesions can be blocked. C: Image; D: Quantitative analysis.

[0358] Figure 17 E, F: CD3 infiltration of the spinal cord in MOG-induced EAE can be blocked. E: Image; F: Quantitative analysis.

[0359] Example 8: Increased IL10 spots after MOG157_A2G treatment

[0360] Figure 18 The increased IL10 spots observed after treatment with MOG157_A2G indicate that tolerant Tregs have been induced. This is Figure 10 Another description of it.

[0361] Example 9:

[0362] Expression and testing of polypeptides having the polypeptide structure defined in claim 1 and containing the peptide antigen or alternative antigen of the present invention (“AIM Bio(s)”).

[0363] Sandwich ELISA scheme

[0364] Nunc 96-well microtiter plates (Thermo Fisher, 442404) were coated overnight with 50 µl of 1 µg / ml anti-HLA-G (2A12) capture antibody (MAI-10358, Thermo Fisher). The plates were washed three times with 200 µl of PBS / 0.05% Tween 20 (PBS / T), then blocked for 2 hours at room temperature with 150 µl of 1% BSA PBS (blocking buffer), followed by three washes with 200 µl of PBS / T. The Expi293F expression culture was diluted 1:10 with blocking buffer and tested in duplicate. Biotin-labeled anti-β2 microglobulin antibody (MA1-19506, Thermo Fisher) was diluted 1:1000 with blocking buffer, and 100 µl was added to each well and incubated for 1 hour. After washing three times with 200 µl PBS / T, 100 µl of horseradish peroxidase (HRP) and streptavidin conjugate (405210, Biolegend; diluted 100-fold with blocking buffer) were added and incubated for 20 minutes. Then, the mixture was washed three times with 200 µl PBS / T. Finally, 100 µl of TMB substrate solution (BioLegend) was added and incubated for 10–15 minutes to observe the binding of the biotinylated anti-β2 microglobulin detection antibody. The reaction was terminated with 100 μl of 1N H2SO4. The absorbance was measured at 450 nm using a TecanSunrise™ ELISA plate reader and calibrated at 620 nm. The results were evaluated using Magellan™ software.

[0365] Transfection protocol

[0366] Expi-293F cells (Thermo Fisher) were cultured in Expi-293™ expression medium (Thermo Fisher) and transfected using the Expifectamine™ 293 transfection kit (Thermo Fisher). Transfection was performed in 50 ml round-bottom tubes (TPP, 91056) using the filter caps of 50 ml bioreactor tubes (TPP, 87050). Expi293F cells were counted at 4.5–5.7 × 10⁻⁶ cells / year. 6 Transfection was performed at a density of 1 live cells / mL (viability >99%).

[0367] To obtain a final culture volume of approximately 12 ml, 3 × 10⁻⁶ ppm was used.7 One live cell was diluted in 10 ml of Expi293™ expression medium containing GlutaMAX (gibco, A14351-01). A total transfection volume of 10 µg DNA / ml was diluted in 600 µl of Opti-MEM (Thermo Fisher, 31985-047). 30 µl of Expifectamine™ 293 reagent was diluted in 570 µl of Opti-MEM or Opti-Plex complex buffer (Thermo Fisher). After 3–5 minutes, DNA was added to the Expifectamine-293™ / Opti-MEM™ transfection mixture and incubated at room temperature for 15–20 minutes to form the DNA-Expifectamine-293F™ reagent complex. Then, 1200 µl of the DNA-Expifectamine-293™ reagent complex was added dropwise to each 50 ml bioreactor containing 30 million cells and 10 ml of medium. The cells were placed in a humidified incubator and cultured on a track shaker at 37°C and 8% CO2. The shaker diameter was 19 mm² and the rotation speed was 225 rpm.

[0368] After 18-20 hours of incubation, while shaking the bioreactor, 60 µl of Enhancer I and 600 µl of Enhancer II (ExpiFectamine™ 293 Transfection Enhancer 1 + 2, catalog number A14524, part of the ExpiFectamine™ 293 Transfection Kit) were added dropwise to enhance the cells. On day 5, the cells were centrifuged at 300 g for 5 minutes at room temperature, and then centrifuged at 4000 g for 20 minutes at 4°C, and the supernatant was collected.

[0369] Simple Western Solution

[0370] Jess Simple Western blot experiments were performed under non-reducing conditions using EZ Standard Pack 1 reagents (bio-techne, catalog number PS-ST01E7). A 12–230 kDa column (bio-techne, catalog number SM W004-1), 2A12 αHLA-G antibody (Thermo Fisher, MAI-10358, diluted 1:50 in bio-techne antibody dilution 2 as part of the anti-mouse assay kit), and an anti-mouse assay kit (bio-techne, catalog number DM-002) were used. The supernatant transfected with Expi293F cells was pre-diluted 10-fold with 1×PBS, then further diluted 10-fold with 0.1×sample buffer. After a 1:5 dilution with 5-fold fluorescent stock solution, 3 µl of the diluted solution was added to each well of a single cartridge along with the molecular weight marker. Molecular weight was determined using 5 µl of protein molecular weight standard (EZ Standard Pack 1) in one well of the first row. In the next row, add 10 µl of antibody dilution 2 (from bio-techne, part of the anti-mouse assay kit) to each well as a blocking agent; in the third row, add 10 µl of 1:50 diluted primary antibody 2A12αHLA-G to each well; in the fourth row, add 10 µl of the mouse secondary antibody from the anti-mouse assay kit to each well; in the fifth row, add 15 µl of luminol-peroxide mixture to each well; and in the last three rows, add 500 µl of wash buffer to each well. Centrifuge the plate at 1000 g for 5 minutes at room temperature to remove air bubbles, then remove any remaining capping foil. Then, load the kit and plate into the Jess system (bio-techne) using Compass 6.2.0 software and start the machine.

[0371] The separation time was 375 V for 25 minutes, followed by blocking with antibody dilution buffer (bio-techne) for 5 minutes, incubation with primary antibody for 30 minutes, incubation with secondary antibody for 30 minutes, and then detection.

[0372] The ELISpot protocol for patients with neuromyelitis optica spectrum disorder (NMOSD)

[0373] ELISpot analysis was performed according to the following protocol:

[0374] Phase 1: PBMC: AIM Bio Culture

[0375] reagents

[0376] ● Human peripheral blood mononuclear cells from patients with neuromyelitis optica spectrum disorder (NMOSD)

[0377] ●AIM Bio: AQP64_G, AQP65 P65K _G

[0378] ●Complete culture medium: X-vivo 15 (Lonza, Biozym 881026) supplemented with 5% human AB serum and human cytokines

[0379] -rhIL-2 (20ng / ml); Immunotools, 11340027

[0380] -rhGM-CSF (20ng / ml); Immunotools, 11343128

[0381] -rhIL-4 (10ng / ml); Immunotools, 11340047

[0382] -rhTGFβ (10ng / ml); Immunotools, 11343160

[0383] ●24-well culture plate

[0384] ●PBS (sterile)

[0385] Thawing peripheral blood mononuclear cells (Day -1)

[0386] ● Thaw frozen PBMCs by rotating them in a water bath for about 20-30 seconds.

[0387] ● Immediately transfer the cells to a 15ml centrifuge tube containing PBS and centrifuge at 300g for 5 minutes at room temperature.

[0388] ● Wash the cells twice more with PBS buffer.

[0389] ● Resuspend the cells in 5 ml of X-vivo 15 medium supplemented with 5% hAB serum. Place the cells in 6-well plates and incubate at 37°C. ℃ Let it stand overnight.

[0390] Treg induction: PBMC culture for 14 days using AIM Bios (aseptic conditions).

[0391] ●Test conditions:

[0392] ○ Negative control (not treated with AIM Bio / untreated)

[0393] ○AQP64_G processing (Sequence AQP64_G: MSRSVALAVLALLSLSGLEA KPLPVDMVLGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRA DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRY TCHVQHEGLPEPLMLRW SKEGDGGIMSVRESGSLSEDLGS PDRVRAVSHWSSC* (SEQ ID NO: 64; Note: The asterisk indicates a stop codon ））

[0394] ○AQP65 P65K _G Treatment (Sequence AQP65 P65K _G: MSRSVALAVLALLSLSGLEA KLPVDMVLGGGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGAYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRA DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAA VVVPSGEEQRYTCHVQHEGLPEPLMLRW SKEGDGGIMSVRESGSLSEDLGS PDRVRAVSHWSSC* (SEQ ID NO:65; Note: The asterisk indicates a stop codon))

[0395] ● PBMC quantity: 1.2 × 10 6 Each cell was dissolved in 400 μL of complete culture medium.

[0396] ● Count the cells left to stand overnight and calculate the number of cells required for the test.

[0397] ●Wash the cells and resuspend them in complete culture medium to a final concentration of 3 × 10⁻⁶. 6 Cells / mL

[0398] ● Add 400 µl (1.2 × 10⁻⁶) to each well of the 24-well plate. 6 Cell suspension (cells).

[0399] ●Use 5 µg / ml AIM Bios AQP64_G and AQP65 P65K _G pulse treatment of cells (day 0)

[0400] ● Add 400 µl of complete culture medium to the cells every 2 days.

[0401] ●On day 6, use 5 µg / ml AIM Bios AQP64_G and AQP65 P65K _G performs pulse processing

[0402] ● On day 14, cells were seeded onto ELISpot plates.

[0403] Phase Two: ELISpot Testing

[0404] (EliSpot testing was performed according to the guidelines provided with the Human IL-10 ELISpot (Basic) Kit (Mabtech; 3430-2H))

[0405] reagents

[0406] ●ELISPOT PVDF plate (Millipore)

[0407] ● Human IL-10 EliSpot Basic Reagent Kit (Product Code: 3430-2H)

[0408] ●35% Ethanol

[0409] ●Blocking culture medium: X-vivo 5% hAB serum

[0410] ●Dilution buffer: 0.5% BSA PBS solution or 0.5% FCS PBS solution

[0411] ● EliSpot medium: X-VIVO 15 (Lonza, Biozym 881026) supplemented with 5% human AB serum and hIL-2 (20 ng / ml)

[0412] ●Streptavidin-HRP used for detection (Biolegend #405210; 1:750 dilution)

[0413] ●ELISpot substrate: TMB for HRP (Mabtech code: 3651–10).

[0414] ●PBS (sterile)

[0415] Cell coating (under sterile conditions)

[0416] ● Activate the PVDF pores with 25 µl of ethanol (35% v / v) for up to 1 minute.

[0417] ● Wash the culture plate 5 times with sterile water (200 µl / well).

[0418] ● Dilute the coated antibody anti-hIL-10 (9D7) with sterile PBS to a final concentration of 2 µg / ml. Add 50 µl of the diluted antibody to each well (Guideline: 15 µg / ml; 100 µl / well).

[0419] ●Incubate overnight at 4-8℃

[0420] Culture cells in a petri dish (under sterile conditions).

[0421] ●Prepare a layout diagram. All samples should be tested in duplicate.

[0422] ●Sample:

[0423] ○ Negative control: Untreated cells (no stimulation)

[0424] ○ Positive control: Untreated cells + 1µg / ml α-CD3 / CD28 stimulation mixture

[0425] ○Test samples: AQP64_G treated cells and AQP65 P65K _G Processed cells

[0426] ● Count PBMCs in 14D cultures. Calculate the required number of cells for each condition.

[0427] ● Minimum cell viability > 50%. Otherwise, discard the sample.

[0428] ●In a fume hood, remove excess coating antibody and wash the microplate five times with 200 µl of sterile PBS.

[0429] ● Add 50 µl of blocking solution to each well. Incubate at room temperature for 2 hours.

[0430] ● Peripheral blood mononuclear cells (PBMCs) under each test condition were resuspended in Elispot medium to a final concentration of 1.5 × 10⁻⁶. 6 / ml.

[0431] ● Resuspend the positive control cells to a higher concentration and make up the volume with stimulating solution.

[0432] ●Under the lid, carefully remove any excess sealing cushioning.

[0433] ● Take 100 µl of PBMC (1.5 × 10⁻⁶) under each condition. 6 The cell suspensions were added to the corresponding wells.

[0434] ●Incubate at 37℃ for 12-48 hours.

[0435] Spot detection

[0436] ● Dilute the biotin-labeled detection antibody (12G8-Biotin) with dilution buffer to a concentration of 1 µg / ml (1:1000).

[0437] ● Remove the cell supernatant and wash the culture plate five times with 200µl PBS. Press with a paper towel to remove any remaining excess buffer.

[0438] ● Add 50 µl of detection antibody to each well. Incubate overnight at 4°C.

[0439] ● Wash 5 times with 200 µl PBS. Press with a paper towel to absorb excess liquid.

[0440] ●Preparation of horseradish peroxidase-labeled streptavidin (diluted to 1:750 with dilution buffer).

[0441] ● Add 50 µl of streptavidin-HRP conjugate to each well. Incubate at room temperature for 1 hour.

[0442] ● Wash 5 times with 200 µl PBS. Press with a paper towel to absorb excess liquid.

[0443] ● Add 50µl to each well After filtration Incubate the TMB substrate in the dark for 10-20 minutes until blue spots appear.

[0444] ● Rinse the hole with water (tap water is fine) to stop the reaction.

[0445] ● Remove the plastic drain tube from the bottom of the plate and thoroughly wash the bottom and sides of the plate with tap water.

[0446] Analysis was performed using an S6 Universal M2 reader (Immunospot, Cleveland, Ohio).

[0447] result:

[0448] Figure 19 A shows that the AIM Bios of the present invention are expressed in Expi293F cells, and the monomers (~50 kDa) of all compounds are detectable; B: Expression in the supernatant was detected by ELISA and quantitatively analyzed. This indicates that these molecules can be successfully produced.

[0449] Figure 20 The results show that, compared to untreated PBMCs, the AIM Bios of the present invention, after being treated with AIM Bio AQP65... P65K Regulatory IL-10-secreting T cells were induced in PBMCs of 4 out of 5 NMO patients treated with _G. This suggests that these molecules can be used to successfully induce tolerance in patients with autoimmune diseases.

[0450] Furthermore, by using alternative constructs that conform to the claims but contain substituted peptides, the inventors have found that those constructs capable of being expressed at good expression levels are functional, i.e., capable of inducing antigen-specific immune tolerance. Given the good expression levels of the constructs of the present invention, it is anticipated that the constructs of the present invention are functional and can be used to treat the medical indications defined in the claims.

[0451] Example 10:

[0452] 2D2 animal model

[0453] Soluble MOG-specific AIM Bio showed therapeutic efficacy in a spontaneous optic neuritis model.

[0454] The amino acid sequence of the recombinant peptide used in this experimental example (the mouse alternative construct of the recombinant peptide of the present invention) is as follows:

[0455] mAIM_MOG 37-44 _K b G:

[0456] peptide sequence MOG 37-44 :VGWYRSPF (SEQ ID NO: 51)

[0457] Protein sequence MOG 37-44 _H2-K b G:

[0458] VGWYRSPF GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRT (SEQ ID NO: 52), followed by the sequence of human HLA-G α3 domain (i.e., with C from intron 4) of SEQ ID NO: 54. The human HLA-G α3 domain of the terminal amino acid sequence (where the furin cleavage site has been removed) and a tag.

[0459] mAIM_MOG 37-47 _K b G

[0460] peptide sequence MOG 37-47 :VGWYRSPFSRV (SEQ ID NO: 60)

[0461] Protein sequence MOG 37-47 _H2-K b G:

[0462] VGWYRSPFSRVGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRT (SEQ ID NO: 61), followed by the sequence of human HLA-G α3 domain (i.e., with C from intron 4) of SEQ ID NO: 54. The human HLA-G α3 domain of the terminal amino acid sequence (where the furin cleavage site has been removed) and a tag.

[0463] mAIM_gp 34-41 _K b G

[0464] peptide sequence gp 34-41 :AVYNFATM(SEQ ID NO: 62)

[0465] protein sequence gp 34-41 _H2-K b G:

[0466] AVYNFATMGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRT (SEQ ID NO: 63), followed by the sequence of the human HLA-Gα3 domain of SEQ ID NO: 54 (i.e., human HLA-Gα3 domain with the C-terminal amino acid sequence from intron 4). (The α3 domain, in which the furin cleavage site has been removed) and a tag.

[0467] The 2D2 transgenic mice express only one TCR that targets the MOG peptide (AA 35-55). They develop spontaneous optic neuritis and ocular inflammation (Bettelli et al., 2003; Bell et al., 2013; Gupta et al., 2012). This model allows for therapeutic administration of the test item and can represent MOGAD symptoms with high face validity. After observing optic neuritis symptoms, the presence of MOG antibodies was confirmed, and the mice were treated three times at 15-day intervals with 3 µg / g mAIM_MOG per treatment. 37-44 _K b G, mAIM_MOG 37-47 _K b G or the corresponding negative control (mAIM_gp) 34-41 _K b G)( Figure 21 a).

[0468] Use mAIM_MOG 37-44 _K b After G treatment, the prognosis of mice was significantly better than that of the control group: no weight loss was observed (data not shown), ocular inflammation was reduced, and the development of EAE was completely prevented. Figure 21 b, c). Based on immunohistochemical staining for lysed caspase 3, apoptosis of target cells in the spinal cord and retina was prevented. Figure 21df). Apoptosis of optic nerve cells was completely prevented, and T cells (CD3) + and CD8 + Infiltration became undetectable (data not displayed). mAIM_MOG 37-47 _K b G treatment showed some protective effect, but its effect was not as good as mAIM_MOG. 37-44 _K b G is effective ( Figure 21 bf). Given mAIM_MOG 37-47 _K b The first 8 amino acids of the peptide presented by G are similar to mAIM_MOG. 37-44 _K b The G is the same, but slightly longer. The inventors believe that the presented peptide may affect the folding, antigen presentation, and efficacy of AIM Bio molecules.

[0469] In summary, the inventors have demonstrated the efficacy of MOG-specific AIM Bios in spontaneous optic neuritis models, even when administered in difficult-to-treat settings (e.g., in the 2D2 model where all T cells express homologous TCRs). Consistent with the reduction in ocular inflammation and EAE development, apoptosis was inhibited, and T cell (CD3+) activity was reduced. + and CD8 + Reduced infiltration.

[0470] Example 11: Characterization of the human recombinant polypeptide of the present invention (“AIM Bios”)

[0471] Size exclusion chromatography

[0472] To characterize the purified proteins, size exclusion chromatography was performed at room temperature using an Äkta FPLC (Amersham Biosciences) and a Superdex 200 increase 10 / 300 column with Unicorn 5.31 software (GE Healthcare). Before the run, the buffer was replaced with 2 CV H₂O from 20% EtOH (stock solution). During analysis, the column was washed with 2 CV PBS (Sigma, D8537-500 ml) at a flow rate of 0.5 ml / min. Subsequently, 10–20 µg of AIM Bio (500 µl) was loaded onto the Superdex 200 increase 10 / 300 column (Cytiva), and the column was run for one column volume at a flow rate of 0.5 ml / min to collect the protein fraction.

[0473] For AIM Bio MOG 157-165 _A2G, observed monomeric (approximately 50 kDa) and small dimer (approximately 100 kDa) components ( Figure 22 The "MOG" used in this experimental example. 157-165 The “A2G” construct contains the amino acid sequence of SEQ ID NO: 58, followed by the sequence of the human HLA-G α3 domain of SEQ ID NO: 54 (i.e., the human HLA-G α3 domain having the C-terminal amino acid sequence from intron 4, wherein the furin cleavage site has been removed) and an optional tag (described below).

[0474] To analyze the protein stability of purified AIM Bios, thermal shift analysis (TSA) was performed: 3 µg of AIM Bio was dissolved in 25 µl PBS (Sigma, D8537-500 ml) containing SYPRO Orange dye (Merck, S5592, final dilution 1:2000, dissolved in PBS). Melting curves were recorded using a StepOnePlus instrument (ThermoFisher), starting at 25 °C, increasing to 95 °C at a rate of 1 °C per minute, and holding for 2 minutes. The melting curves were analyzed using StepOnePlus 2.3 software. To determine Tm, a Boltzmann sigmoid curve was fitted, and Tm was calculated accordingly.

[0475] MOG 157-165 The TSA values ​​of _A2G were in the same temperature range (56.52℃ and 57.34℃), independent of the production cell lines (Expi293F™ and ExpiCHO™) (Figure 23).

[0476] MOG discovered 157-165 The _A2G peptide is more stable than other recombinant peptides mentioned in this article, and has higher yield and purification efficiency.

[0477] Jess Simple Western Observation

[0478] Jess simple Western blotting was performed under non-reducing conditions using EZ Standard Pack 1 reagents (bio-techne, catalog number PS-ST01E7). A 12–230 kDa column (bio-techne, catalog number SM W004-1), 2A12αHLA-G antibody (Thermo Fisher, MAI-10358, diluted 1:50 in bio-techne antibody dilution 2 as part of the anti-mouse assay kit) as the primary antibody, and an anti-mouse assay kit (bio-techne, catalog number DM-002) were used. The supernatant was pre-diluted 1:10, and the purified protein was pre-diluted 1:100 with 1×PBS, then further diluted 1:10 with 0.1× sample buffer. After a 1:5 dilution with 5x fluorescent stock solution, 3 µl of the diluted protein was added to a single cartridge well along with the molecular weight marker. Molecular weight was determined using 5 µl of protein molecular weight standard (EZ Standard Pack 1) in one well of the first row. Add 10 µl of antibody dilution 2 (from bio-techne, part of the anti-mouse detection kit) to each well in the next row as a blocking agent. Add 10 µl of 2A12αHLA-G primary antibody (diluted 1:50) to the third row. Add 10 µl of mouse secondary antibody from the anti-mouse detection kit to the fourth row. Add 15 µl of luminol-peroxide mixture to the fifth row. Add 500 µl of wash buffer to each compartment of the last three rows. Centrifuge the plate at 1000 g for 5 minutes at room temperature to remove air bubbles, then remove any remaining capping foil. Then, load the kit and plate into the Jess system (bio-techne) using Compass 6.2.0 software and start the machine.

[0479] The separation time was 375 V for 25 minutes, followed by blocking with antibody dilution buffer (bio-techne) for 5 minutes, incubation with primary antibody for 30 minutes, incubation with secondary antibody for 30 minutes, and then detection.

[0480] ELISpot and Dextramer staining cell cultures from MOGAD patients

[0481] For the ELISpot assay, PBMCs from MOGAD patients were thawed and left to stand overnight in a 6-well plate (Greiner, #657160). On the following day (d0), cells were counted and seeded at a density of 3 million cells / mL in 12-well plates (Greiner, 665180) with 1.5 million cells / well. The culture medium was X-VIVO 15 complete medium (X-VIVO 15 medium (Lonza / Biozym#881026), 5% human AB serum (HUABSE-0100, Biosell), 20 ng / ml human IL-2 (Immunotools, #11340027), 20 ng / ml human GM-CSF (Immunotools, #11343128), 10 ng / ml human IL-4 (Immunotools, #11340047) and 10 ng / ml human TGFβ1 (Biolegend, #781804)), and stimulated with 20 µg / ml AIMBio (MOG157_A2G). On day 6, cells were second-stimulated with 20 µg / ml AIM Bio (MOG157_A2G). On days 3, 6, 8, 10, and 13, cells were fed with X-VIVO 15 complete medium, with a 20 mM HEPES / 150 mM NaCl buffer as a solvent control. On day 14, cells were counted and used for ELISpot assays and dextran staining.

[0482] ILT2-Fc combined with ELISA

[0483] For the ILT2 binding assay, 125 ng of AIM Bio (25 µl 5 µg / ml solution) was coated onto a 96-well half-area microplate (Greiner, 675061), covered with sealing foil, and incubated overnight at 4°C. The plate was then washed three times with 120 µl PBS / 0.05% Tween 20 (PBST). PBS without AIM Bio served as a control (AIM Bio was buffered with PBS). Blocking was performed for 1.5 h with 100 µl 1% BSA (albumin grade V, Roth, #0163.2) in PBS, followed by three washes with 120 µl PBS / T on a shaker (also used for further blocking steps). Human ILT2-Fc (stock concentration in PBS: 250 µg / ml) (Sino Biological, #16014-H02H) was diluted with PBS / T and titrated with 25 µl (100 µg / ml, 20 µg / ml, 4 µg / ml, 0.8 µg / ml, 0.16 µg / ml) for 2 hours at room temperature. PBS / T without ILT4-Fc served as a control. The plate was washed three times with 120 µl of PBS / T. For assay, 25 µl of α-hIgGγ antibody (Thermo, #628420) was diluted 1:2000 with PBS / T and added to each well. The plate was incubated for 1 hour and then washed three times with 120 µl of PBS / T. Then, 25 µl of TMB substrate (Biolegend, #421101) was added and reacted for 3 minutes until the color turned blue. The reaction was terminated with 25 µl of 1 NH2SO4. The plate was then analyzed at 450 nm using a Sunrise microplate reader (Tecan) and Magellan 7.5 software (Tecan), with a reference wavelength of 620 nm. Control wells were calibrated. Data were calculated in Excel 365 (Microsoft), and graphs were plotted in Prism 10.2 (GraphPad).

[0484] In ELISA experiments, MOG was found to... 157-165 _A2G_His is combined with ILT2-Fc (Figure 24).

[0485] ILT4-Fc combined with ELISA

[0486] For the ILT4 binding assay, 25 µl of 5 µg / ml AIM Bio was coated onto a 96-well half-area microplate (Greiner, 675061), covered with sealing foil, and incubated overnight at 4°C. The plate was then washed three times with 120 µl of PBS / 0.05% Tween20 (PBST). PBS without AIM Bio served as a control (AIM Bio was buffered with PBS). Blocking was performed for 1.5 hours with 100 µl of 1% BSA (albumin V grade, Roth, #0163.2) in PBS, followed by three washes with 120 µl of PBS / T on a shaker (subsequent steps were also performed similarly). Human ILT4-Fc (PBS stock concentration 250 µg / ml) (SinoBiological, #14132-H02H) was diluted with PBS / T and titrated with 25 µl (10 µg / ml, 2 µg / ml, 0.4 µg / ml, 0.08 µg / ml, 0.016 µg / ml) at room temperature for 2 hours. PBS / T without ILT4-Fc served as a control. The plate was washed three times with 120 µl of PBS / T. For assay, 25 µl of α-hIgGγ antibody (Thermo, #628420) was diluted 1:2000 with PBS / T and added to each well. The plate was incubated for 1 hour and then washed three times with 120 µl of PBS / T. Then, 25 µl of TMB substrate (Biolegend, #421101) was added, and the reaction was allowed to proceed for 3 minutes until the color turned blue. The reaction was terminated with 25 µl of NH2SO4. The plate was then analyzed at 450 nm using a Sunrise Tecan microplate reader and Magellan 7.5 software (Tecan), with a reference wavelength of 620 nm. The control wells were calibrated. Data were calculated in Excel 365 (Microsoft), and graphs were plotted in Prism 10.2 (GraphPad).

[0487] ILT-4-Fc was found to be compatible with AIM Bio (MOG) 157-165 The binding of _A2G is very similar and is unrelated to the tags used in the molecule. Figure 25 Note that "tag-" means "no tag".

[0488] ELISpot testing

[0489] Activate ELISpot plates (Millipore, MSIPS 4510) with 50 µl of 35% EtOH per well for 1 minute, then wash 5 times with 200 µl of PBSS. Next, coat cells overnight at 4°C with a 2 µg / ml anti-human IL-10 coating antibody (9D7, Mabtech, 3430-3-250) (antibody diluted 1:500 with PBS). The next day, remove excess antibody, wash 5 times with 200 µl of PBS per well, and block with 50–100 µl of X-VIVO 15 medium containing 5% hAB serum for 2 hours. Then remove the medium and seed the cell suspension (50 µl for control group, 100 µl for AIM Bio construct containing 150,000 cells) into X-VIVO 15 medium containing 5% hAB serum and 20 ng / ml h-IL2. For control wells, add 50 µl of solution containing 5 µl / 100 µl CD3 / CD28 (Immunocult, #10971, Stemcell Technologies) as a positive control, or 5 µl / 100 µl HEPES as a negative control. Incubate the culture plates in a humidified incubator containing 5% CO2 for 48 hours, and then detect IL-10 spots.

[0490] For this purpose, cells and culture medium were removed, and the culture plate was washed five times with 200 µl PBS. The α-IL10 detection antibody (12G8-Biotin, Mabtech, 3430-6-250) was diluted 1:1000 in PBS containing 0.5% BSA, resulting in a concentration of 1 µg / ml. 50 µl was added to each well, and the plate was incubated at room temperature for 2 hours or overnight at 4°C. The plate was then washed five times with 200 µl PBS, and 50 µl of TMB substrate solution (Mabtech, 3651-10) was added to each well for color development until clear spots appeared. The color development reaction was stopped after thorough washing with deionized water. The drain tube was removed, and the bottom of the membrane was rinsed. The plate was dried, examined, and the spots were counted using an ELISpot reader (Immunospot CTL machine and Immunospot SC Suite, CTL). Data analysis was performed using Excel 365 (Microsoft) software, and chart creation was performed using Prism 10.2 (Graphpad) software.

[0491] Compared with the HEPES (20 mM HEPES, 150 mM NaCl, pH 7.5) control group, AIM Bio MOG157-165 A2G tag- (MOG157) treatment of PBMCs can induce IL-10 production in MOGAD patients (Figure 26). Immunocult (αCD3 / αCD28) serves as a positive control for ILT-10 induction.

[0492] In addition, the upregulation of other potentially dangerous cytokines (such as IFN or TNF) in PBMC supernatant from MOGAD patients was analyzed using the Biolegend LEGENDplex™ HU Th cytokine panel (12-fold) according to the manufacturer's instructions. Only IL-10 showed a significant increase after AIM Bio treatment. Figure 27 ).

[0493] Dextran staining

[0494] Material:

[0495] ●X-VIVO 15 complete culture medium: 5% hAB serum

[0496] ●Antibody:

[0497] ●Human FCR blocking agents

[0498] ●α-CD3 APC Cy7 clone HIT3a, Biolegend (RL3)

[0499] ●α-CD8 BV510 (VL2) clone HIT8, Biolegend

[0500] ●α-CD103 FITC clone Ber-ACT8, Biolegend (BL1)

[0501] ●α-IL-10 BV421, clone JES3 / 9D7, Biolegend (VL1)

[0502] ●α-IFN PE-Cy7 clones B27, Biolegend (YL4)

[0503] ●α-GzmB PE Vio770, cloned Qa16A02, Biolegend (BL3)

[0504] ●α-FoxP3, Miltenyi Biotec (APC) (RL1)

[0505] ●CD103, IL10, GzmB, And the biolegend of FoxP3.

[0506] ●MHC dextran can be loaded without load (1 µL / 1×10⁻⁶) 6 (cells)

[0507] ○HLA-A*2 PE (YL1)

[0508] ○HLA-A*11 PE (YL1)

[0509] ●Repairable dyeing kit for living / dead animals (optional)

[0510] ● Peptide antigens, 5 stock solutions (5 mg / mL) (dissolved in DMSO)

[0511] ●Brefeldin A

[0512] ● Human HLA-A2* / 11* PBMC (fresh or amplified AIMbio culture)

[0513] ●PBS

[0514] ● Recombinant hIL-2 stock solution 200 ug / mL

[0515] ●FACS buffer (2% fetal bovine serum PBS solution)

[0516] ●2% PFA PBS solution

[0517] ●10× Permeabilization Buffer

[0518] Thawing and hibernating cells

[0519] ● Place the frozen vial containing PBMC into a water bath and rotate it until only a small amount of ice remains.

[0520] ● Immediately transfer the cells to 10 ml of X-VIVO 15 medium.

[0521] ● Centrifuge the cells at 300g at room temperature.

[0522] ● Discard the supernatant

[0523] ●Wash the cells again with 10 ml of X-VIVO 15 medium.

[0524] ● Discard the supernatant and resuspend the cells in 10 ml X-VIVO 15 completely In the culture medium.

[0525] ● Place the cells in a cell culture plate (preferably a 6-well plate) and incubate overnight at 37°C.

[0526] ●Culturing as needed: Amplification using AIM Bio or without amplification (14-day protocol)

[0527] Dextran staining and flow cytometry analysis

[0528] 1. Load the peptide antigen onto an empty dextran (according to the IMMUDEX protocol, 48-hour protocol).

[0529] Follow the IMMUDEX protocol. Modification: For reagents such as easymers and dextramer PE, use one-quarter of the IMMUDEX protocol. Easymers preparation should be performed 48 hours before staining.

[0530] 2. Use brefedipine A to block the culture.

[0531] ● On a clean bench, transfer the cultured PBMCs to a 15 mL Falcon container and count them.

[0532] ● Centrifuge the cells at 300g at room temperature.

[0533] ● Remove the supernatant, resuspend the cells with h-IL2 (20 ng / mL) or a mixed antibody (depending on previous culture), add homologous peptides and culture for 24 hours (optional), or immediately treat with 1:1000 Brefeldin A and complete X-VIVO15 in 15 mL Falcon. Cell density is 1 µl. Supplement medium: 10,000 cells (ideally, 100 µl can hold 100 cells). 6 (cells).

[0534] ●Incubate cells in 15 mL Falcon or 96-well U-bottom sterile culture plates at 37°C for 4 hours.

[0535] 3. Incubate cells with dextran

[0536] ● Cleanse cells to remove Brefidobacterium A

[0537] ● Resuspend in 30 µl FACS buffer

[0538] ● For AIM Bio amplification culture, a dextran loaded with peptides is used, the peptides loaded on the dextran having sequences similar to those in AIM Bio. Completely identical .

[0539] ● Add 1 µl of peptide-loaded dextran to each well containing 25 µl of resuspended cells for incubation. Use 1 µL of dextran per staining (ideally for up to 2 × 10⁻⁶ cells).6 (cells).

[0540] ● (Optional) Cells stained with empty dextran or dextran loaded with irrelevant peptides can be used as controls.

[0541] ●Incubate at room temperature in the dark for 15 minutes.

[0542] 4. Surface antibody staining

[0543] ● Add 5 µl of FcR blocking reagent (1 µg / ml) to the FACS buffer, and At 4℃ Incubate for 10 minutes.

[0544] ● Prepare an antibody master mixture containing surface markers anti-CD3, CD103, and CD8 antibodies, 25 µl FACS per well, and live / dead cell markers (optional), added to FACS buffer at predetermined concentrations. For surface markers: 1:50. Vortex the Ab mixture.

[0545] ● Add 20 µl of surface antibody premix to each sample.

[0546] ●Incubate at 4°C for 1 hour in the dark.

[0547] ● Add 50 µl of FACS buffer and centrifuge (5 minutes, 300g, 4℃).

[0548] ● Discard the supernatant and wash once with 100 µl FACS buffer.

[0549] 6. Intracellular staining (IL-10 and IFNg)

[0550] ● Discard the supernatant from centrifuged cells and add 25 µl of 2% PFA PBS as fixation buffer. Incubate at room temperature in the dark for 20 minutes.

[0551] ● Centrifuge the sample at 400×g for 5 minutes at room temperature. Discard the supernatant.

[0552] ● Add 100 μL of 1× permeation buffer (made from 10× buffer and dH2O). Resuspend.

[0553] ● Centrifuge the sample at 400×g for 5 minutes at room temperature. Discard the supernatant.

[0554] ● (Optional) Block with 1% FcR blocking reagent in 50 µl of 1× permeation buffer at room temperature for 15 minutes.

[0555] ● After centrifugation, resuspend the precipitate in 25 µl of 1× permeabilization buffer containing anti-human IL-10, FoxP3, GzmB, and (1:75). Vortex Ab mixture. Incubate at room temperature (in the dark) for at least 20 minutes.

[0556] Prepare 10 samples: 25 µl 1XP buffer × 10 = 250 µl. Add 3.5 µl IL-10 and... .

[0557] ● Centrifuge the sample at 400×g for 5 minutes at room temperature. Discard the supernatant.

[0558] ● Resuspend cells tightly in 140 µl FACS buffer / well (96-well plate) (using 2 × 70 µl multichannel pipettes). Store at 4°C protected from light until analysis. Set both the acquisition and stop volumes in the FACS program to 100 µl to avoid bubble formation.

[0559] ●Combined flow cytometry and single-channel staining in vitro are required for analysis (this is essential!).

[0560] Compared to the HEPES control (20 mM HEPES / 150 mM NaCl, pH 7.5) treatment, AIM Bio MOG 157-165 After processing with _A2G_tag- (abbreviated as "MOG157"), CD3 + CD103 + FoxP3 + and CD3 + Dextramer low FoxP3 + MOGAD cells significantly increased ( Figure 28 ).

[0561] Industrial applicability

[0562] The pharmaceutical compositions, peptides, nucleic acids, cells, and products used in this invention all have industrial application value. For example, they can be used to manufacture or as pharmaceuticals.

Claims

1. A recombinant polypeptide capable of presenting a peptide antigen, said recombinant polypeptide comprising, in the 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 myelin oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG), or human myelin lipoprotein (PLP1). ii) Optionally, connector sequence; iii) A human polypeptide domain sequence comprising a human β2 microglobulin sequence, or an amino acid sequence that is at least 90% identical to the amino acid sequence of the human β2 microglobulin represented by SEQ ID NO: 5; iv) Optionally, connector sequence; v) The α1 domain of the MHC molecule; vi) The α2 domain of the MHC molecule; vii) The α3 domain of an MHC Ib molecule or a derivative thereof, wherein the derivative is capable of binding to ILT2 or ILT4. viii) Optionally, protease cleavage sites; ix) Optionally, an interval sequence; and x) Optional, affinity label.

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

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

2.

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

53.

5. The recombinant polypeptide according to any one of the preceding claims, wherein, According to i), the peptide antigen is a peptide of human myelin oligodendrocyte glycoprotein (MOG).

6. The recombinant polypeptide according to any one of the preceding claims, wherein the α1 domain according to (v) and the α2 domain according to (vi) are derived from human MHC class Ia molecules or from human MHC class Ib molecules.

7. The recombinant polypeptide according to claim 6, wherein the α1 domain according to (v) and the α2 domain according to (vi) are derived from human MHC class Ia molecules.

8. The recombinant polypeptide according to claim 7, wherein the α1 domain according to (v) and the α2 domain according to (vi) are derived from a human HLA-A2 molecule.

9. The recombinant polypeptide according to claim 6, wherein the α1 domain according to (v) and the α2 domain according to (vi) are derived from human MHC class Ib molecules.

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

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

12. The recombinant polypeptide according to any one of the preceding claims, wherein the α3 domain or derivative according to (vii) is identical to the α3 domain having the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 21, or SEQ ID NO: 54, or has at least 80% amino acid sequence identity, preferably at least 90% amino acid sequence identity, with the α3 domain having the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 21, or SEQ ID NO:

54.

13. The recombinant polypeptide according to claim 12, wherein the α3 domain or derivative according to (vii) is identical to the α3 domain having the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 21, or SEQ ID NO: 54, or has at least 92% amino acid sequence identity with the α3 domain having the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 21, or SEQ ID NO:

54.

14. The recombinant polypeptide according to claim 12, wherein the α3 domain or derivative according to (vii) is identical to, or has at least 94% amino acid sequence identity with, the α3 domain having the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 21, or SEQ ID NO:

54.

15. The recombinant polypeptide according to claim 12, wherein the α3 domain or derivative according to (vii) is identical to the α3 domain having the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 21 or SEQ ID NO: 54, or has at least 96% amino acid sequence identity with the α3 domain having the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 21 or SEQ ID NO:

54.

16. The recombinant polypeptide according to claim 12, wherein the α3 domain or derivative according to (vii) is identical to the α3 domain having the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 21 or SEQ ID NO: 54, or has at least 98% amino acid sequence identity with the α3 domain having the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 21 or SEQ ID NO:

54.

17. The recombinant polypeptide according to claim 12, wherein the α3 domain or derivative according to (vii) is identical to, or has at least 99% amino acid sequence identity with, the α3 domain having the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 21, or SEQ ID NO:

54.

18. The recombinant polypeptide according to claim 12, wherein the α3 domain according to (vii) is identical to the α3 domain having the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 21 or SEQ ID NO:

54.

19. The recombinant polypeptide according to claim 12, wherein the α3 domain according to (vii) is identical to the α3 domain having the amino acid sequence shown in SEQ ID NO:

9.

20. The recombinant polypeptide according to claim 12, wherein the α3 domain according to (vii) is identical to the α3 domain having the amino acid sequence shown in SEQ ID NO:

21.

21. The recombinant polypeptide according to claim 12, wherein the α3 domain according to (vii) is identical to the α3 domain having the amino acid sequence shown in SEQ ID NO:

54.

22. The recombinant polypeptide according to any one of the preceding claims, wherein the adapter sequence according to (ii) and / or the adapter sequence according to (iv) comprises an amino acid sequence (GGGGS)n, wherein n is an integer equal to or greater than 1.

23. The recombinant polypeptide according to claim 22, wherein the linker sequence according to (ii) comprises an amino acid sequence (GGGGS)n, wherein n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, and preferably selected from 2, 3, 4 and 5.

24. The recombinant polypeptide according to claim 22 or 23, wherein the linker sequence according to (iv) comprises an amino acid sequence (GGGGS)n, wherein n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, and preferably selected from 2, 3, 4 and 5.

25. The recombinant polypeptide according to any one of the preceding claims, wherein, The sequence of the human polypeptide domain according to (iii) is at least 95% identical to the amino acid sequence of SEQ ID NO: 5, preferably at least 98% identical to the amino acid sequence of SEQ ID NO: 5, and more preferably identical to the amino acid sequence of SEQ ID NO:

5.

26. The recombinant polypeptide according to any one of the preceding claims, wherein the polypeptide is a dimer or a polymer.

27. The recombinant polypeptide according to any one of the preceding claims, wherein the polypeptide comprises components i) to vii) or consists of all components i) to vii).

28. The recombinant polypeptide according to any one of the preceding claims, wherein the polypeptide does not contain components (viii) to (x).

29. The recombinant polypeptide according to any one of claims 1-27, wherein the polypeptide comprises components i) to x) or consists of all components i) to x).

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

31. The recombinant polypeptide according to any one of claims 1-29, wherein the recombinant polypeptide comprises an amino acid sequence consisting of the following ((a) and (b)) in sequence from N-terminus to C-terminus: (a) A peptide antigen selected from the amino acid sequence group of SEQ ID NO: 2 or 53; (b) The amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 56-57.

32. The recombinant polypeptide according to any one of the preceding claims, wherein the recombinant polypeptide is soluble.

33. A nucleic acid encoding one or more polypeptides according to any one of the preceding claims.

34. The nucleic acid according to claim 33, wherein the nucleic acid is a vector.

35. A pharmaceutical composition comprising at least one nucleic acid according to claim 33 or 34.

36. A pharmaceutical composition or kit comprising at least one recombinant polypeptide according to any one of claims 1-32.

37. The pharmaceutical composition or kit of claim 36, wherein the pharmaceutical composition or kit comprises at least two different recombinant polypeptides according to any one of claims 1-32, wherein each different polypeptide comprises a different peptide antigen as defined in any one of claims 3-5.

38. The pharmaceutical composition or kit according to any one of claims 35-37, for treating multiple sclerosis (MS), MOG antibody disease, or MOG antibody-positive neuromyelitis optica in human patients.

39. The pharmaceutical composition or kit for the use of claim 38, wherein the treatment is for multiple sclerosis (MS).

40. The pharmaceutical composition or kit for the use of claim 38, wherein the treatment is a treatment for MOG antibody disease.

41. The pharmaceutical composition or kit for the use of claim 38, wherein the treatment is for MOG antibody-positive neuromyelitis optica.

42. The pharmaceutical composition or kit for the use according to any one of claims 38-41, wherein the treatment is performed via immunotherapy.

43. The pharmaceutical composition or kit for the use of any one of claims 38-42, wherein the treatment is performed by inducing immune tolerance against human myelin-oligodendrocyte glycoproteins.

44. A pharmaceutical composition or kit for use according to any one of claims 38-43, wherein the treatment is for reducing the level of autoantibodies against human myelin-oligodendrocyte glycoprotein in plasma or cerebrospinal fluid.

45. A pharmaceutical composition or kit for use according to any one of claims 38-44, wherein the human patient is a patient with antimyelin-oligodendrocyte glycoprotein autoantibodies in plasma or cerebrospinal fluid prior to the start of treatment.

46. ​​The pharmaceutical composition or kit for use according to any one of claims 38-45, wherein the treatment is performed by inducing myelin-specific regulatory T cells.

47. A recombinant host cell comprising the nucleic acid or vector according to claim 33 or 34, and expressing the recombinant polypeptide according to any one of claims 1-32.

48. A method for obtaining a pharmaceutical composition comprising the polypeptide according to any one of claims 1-32, the method comprising the following steps: (a) culturing the recombinant host cell of claim 47 under conditions that allow expression of the recombinant polypeptide from nucleic acid molecules, (b) recovering the recombinant polypeptide, (c) purifying the recombinant polypeptide, and (d) formulating the recombinant polypeptide into a pharmaceutical composition.

Images