Novel therapeutic methods of MHC Ib-mediated aquaporin 4 (AQP4)-specific immunosuppression as NMO

Abstract

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

Description

Field of the Invention

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

[0002] background

[0003] Neuromyelitis optica (NMO) is a demyelinating autoimmune disease of the central nervous system (CNS) with an incidence of between 0.05 and 0.4 per 100,000. It is characterized by symptoms similar to those of multiple sclerosis (MS). However, whereas MS is typically characterized by a relapsing-remitting course, patients with NMO rarely experience remissions. In addition, some treatments that are beneficial for patients with MS are ineffective for NMO and may even worsen the disease. As a result, NMO is more difficult to treat than MS and often progresses more rapidly. Current treatment strategies for NMO focus on relapse prevention or treatment of acute disease relapses. Acute relapses are typically treated with immunosuppressive therapy, such as glucocorticoids or plasma exchange to remove autoreactive antibodies. Recently, therapeutic antibodies that inhibit the complement system (eculizumab) or block inflammatory cytokines (satralizumab) have also been approved.

[0004] However, not only the autoreactive immune functions are suppressed, but also the protective immune functions. These functions are essential for protecting the patient from viruses, bacteria or tumors. Therefore, only partial suppression is usually aimed at. Since autoimmune diseases often worsen after repeated attacks, a lot of efforts are required to prevent relapses. However, this overall slow progression of autoimmune diseases can only be slowed down but not stopped. In order to avoid this dilemma, people have been trying for some time to induce immune tolerance to antigens attacked by autoreactive immune cells (antigen-specific immunosuppression, ASI).

[0005] So far, at least in early clinical studies, two strategies have been evaluated. Similar to desensitization strategies in allergies, large amounts of antigens have been administered by different routes to induce antigen-specific tolerance. However, in autoimmune diseases, these strategies have caused serious side effects and have not been successful in the clinic. Attempts to induce tolerance by adoptive transfer of antigen-specific regulatory T cells or antigen-loaded tolerogenic dendritic cells appear more promising. However, these strategies are extremely complex and expensive and require GMP-compliant manufacturing and quality control processes for each patient. Therefore, even if small-scale clinical trials are successful, it remains a big question whether many patients will be able to receive adoptive transfer therapy in the foreseeable future.

[0006] WO2018 / 215340 relates to a combination of MHC class Ib molecules and peptides for targeted therapeutic immunomodulation.

[0007] In conclusion, there remains a need for improved drugs to treat neuromyelitis optica (NMO). Summary of the invention

[0008] The inventors have found that human MHC class Ib molecules, such as HLA-G, have the ability to induce antigen-specific tolerance to presented peptide antigens. Therefore, although the structure and sequence of MHC class Ib molecules are similar to the classical human MHC class Ia molecules that induce antigen peptide-specific immune responses, according to the present invention, MHC class Ib molecules can be advantageously used to suppress immune responses in an antigen-specific manner. In addition, the inventors have found that molecules other than naturally occurring MHC class Ib molecules, particularly polypeptides that only comprise at least one domain of MHC class Ib molecules, preferably at least α3 domains of MHC class Ib molecules, can be used to suppress immune responses as described in the present invention: the α1 and α2 domains of various class Ia molecules can be combined with the [α]3 domains of human MHC class Ib molecules to suppress immune responses to peptides presented by these antigens.

[0009] HLA-G molecules loaded with antigens may be unstable. Therefore, the inventors designed soluble recombinant polypeptides comprising peptide antigens, MHC class Ib molecules (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 MHC class Ib molecules (e.g., HLA-G) are replaced with corresponding domains of other MHC molecules to enhance the flexibility and versatility of these recombinant polypeptides (e.g., see Figure 2 ). These alternative recombinant polypeptides can be designed with antigen-binding domains of other human HLA molecules. bThe construct of the α1 and α2 domains can present the ovalbumin-derived peptide SIINFEKL to OT-1 T cells. (OT-1 T cells express a transgenic T cell receptor that specifically recognizes this antigen) (WO2018 / 215340).

[0010] Surprisingly, the inventors have found that by using the recombinant polypeptides of the present invention, immune responses to neuroinflammatory autoantigens can be suppressed and cells capable of inducing tolerance to human aquaporin 4 (AQP4) can be induced. In addition, using established neuroinflammatory models, the inventors have demonstrated that alternative molecules to these polypeptides (suitable for mice) can be used to treat neuroinflammatory diseases. Therefore, according to the present invention, neuromyelitis optica (NMO) can be treated by the recombinant polypeptides of the present invention.

[0011] The inventors' experimental data indicate that a suitable peptide antigen and the α3 domain of MHC class Ib molecules (e.g., HLA-G) are required to achieve the desired effect. Thus, this approach goes beyond previously described strategies that either use antigenic peptides without co-stimulation (leading to anergic T cells rather than tolerant T cells) or use MHC class Ib molecules in an antigen-nonspecific context.

[0012] Furthermore, according to the present invention, the recombinant polypeptide of the present invention not only modulates T cell responses, but also prevents the formation of AQP4-specific autoantibodies. This advantage is expected to translate into clinical improvements in human patients with neuromyelitis optica (NMO), since AQP4-specific autoantibodies are associated with the pathology of neuromyelitis optica (NMO).

[0013] Therefore, the present invention relates to the following preferred embodiments:

[0014] A recombinant polypeptide capable of presenting a peptide antigen, the recombinant polypeptide comprising, in order from N-terminus to C-terminus:

[0015] i) a peptide antigen presented by the recombinant polypeptide, wherein the peptide antigen is a peptide of human aquaporin 4;

[0016] ii) optionally, a linker sequence;

[0017] iii) optionally, a human polypeptide domain sequence, the human polypeptide domain sequence comprising a human β2 microglobulin sequence, or an amino acid sequence that is at least 90% identical to the human β2 microglobulin amino acid sequence shown in SEQ ID NO: 5;

[0018] iv) optionally, a linker sequence;

[0019] v) optionally, an α1 domain of an MHC molecule;

[0020] vi) optionally, an α2 domain of an MHC molecule;

[0021] vii) an α3 domain of an MHC class Ib molecule or a derivative of the α3 domain of an MHC class Ib molecule, said derivative being capable of binding to ILT2 or ILT4;

[0022] viii) optionally, a protease cleavage site;

[0023] ix) optionally, a spacer sequence; and

[0024] x) Optionally, an affinity tag.

[0025] 1. The recombinant polypeptide according to item 1, wherein the peptide antigen according to i) has a length of 7 to 11 amino acids, preferably a length of 8 to 10 amino acids.

[0026] 2. The recombinant polypeptide according to item 1 or 2, wherein the peptide antigen according to i) consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 2, 22, 23 and 24.

[0027] 3. The recombinant polypeptide according to any one of the above items, wherein the peptide antigen consists of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 22.

[0028] 4. The recombinant polypeptide according to any one of items 1 to 4, wherein the peptide antigen consists of the amino acid sequence of SEQ ID NO: 2.

[0029] 5. The recombinant polypeptide according to any one of items 1 to 3, wherein the peptide antigen consists of the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24.

[0030] 6. The recombinant polypeptide according to any one of the above items, wherein the α1 domain according to (v) and the α2 domain according to (vi) are derived from human MHC I class a molecules or from human MHC class Ib molecules.

[0031] 7. The recombinant polypeptide according to any one of the above items, wherein the α1 domain according to (v) and the α2 domain according to (vi) are from human MHC I class Ia molecules.

[0032] 8. The recombinant polypeptide according to item 8, wherein the α1 domain according to (v) and the α2 domain according to (vi) are from human HLA-A2 molecules.

[0033] 9. The recombinant polypeptide according to item 7, wherein the α1 domain according to (v) and the α2 domain according to (vi) are from human MHC class Ib molecules.

[0034] 10. The recombinant polypeptide according to item 10, wherein the α1 domain according to (v) and the α2 domain according to (vi) are from human HLA-G molecules.

[0035] 11. The recombinant polypeptide according to any one of the above items, wherein the α3 domain of the MHC class Ib molecule according to (vii) is the α3 domain of human HLA-E, human HLA-F or human HLA-G.

[0036] 12. The recombinant polypeptide according to any one of the above items, wherein the α3 domain of the MHC class Ib molecule according to (vii) is the α3 domain of human HLA-G.

[0037] 13. A recombinant polypeptide according to any of the above items, wherein the α3 domain or derivative according to (vii) is identical to the α3 domain having the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:21, or has at least 80% amino acid sequence identity, preferably at least 90% amino acid sequence identity, with the α3 domain having the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:21.

[0038] 14. A recombinant polypeptide according to item 14, wherein the α3 domain or derivative according to (vii) is identical to the α3 domain having the amino acid sequence of SEQ ID No: 9 or SEQ ID No: 21, or has at least 92% amino acid sequence identity with the α3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 21.

[0039] 15. A recombinant polypeptide according to item 14, wherein the α3 domain or derivative according to (vii) is identical to the α3 domain having the amino acid sequence of SEQ ID No: 9 or SEQ ID No: 21, or has at least 94% amino acid sequence identity with the α3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 21.

[0040] 16. A recombinant polypeptide according to item 14, wherein the α3 domain or derivative according to (vii) is identical to the α3 domain having the amino acid sequence of SEQ ID No: 9 or SEQ ID No: 21, or has at least 96% amino acid sequence identity with the α3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 21.

[0041] 17. A recombinant polypeptide according to item 14, wherein the α3 domain or derivative according to (vii) is identical to the α3 domain having the amino acid sequence of SEQ ID No: 9 or SEQ ID No: 21, or has at least 98% amino acid sequence identity with the α3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 21.

[0042] 18. A recombinant polypeptide according to item 14, wherein the α3 domain or derivative according to (vii) is identical to the α3 domain having the amino acid sequence of SEQ ID No: 9 or SEQ ID No: 21, or has at least 99% amino acid sequence identity with the α3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 21.

[0043] 19. The recombinant polypeptide according to item 14, wherein the α3 domain according to (vii) is identical to the α3 domain having the amino acid sequence of SEQ ID No: 9 or SEQ ID No: 21.

[0044] 20. The recombinant polypeptide according to any of the above items, wherein the linker sequence according to (ii) and / or the linker sequence according to (iv) comprises the amino acid sequence (GGGGS)n, wherein n is an integer equal to or greater than 1.

[0045] 21. The recombinant polypeptide according to item 21, wherein the linker sequence according to (ii) comprises the amino acid sequence (GGGGS)n, wherein n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, and is preferably selected from the group consisting of 2, 3, 4 and 5.

[0046] 22. A recombinant polypeptide according to item 21 or 22, wherein the linker sequence according to (iv) comprises the amino acid sequence (GGGGS)n, wherein n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, and is preferably selected from the group consisting of 2, 3, 4 and 5.

[0047] 23. A recombinant polypeptide according to any of the above items, wherein the sequence of the human polypeptide domain according to (iii) is at least 95% identical to the amino acid sequence of SEQ ID NO:5, preferably at least 98% identical to the amino acid sequence of SEQ ID NO:5, and more preferably identical to the amino acid sequence of SEQ ID NO:5.

[0048] 24. The recombinant polypeptide according to any one of the above items, wherein the polypeptide is a dimer or a multimer.

[0049] 25. The recombinant polypeptide according to any one of the above items, wherein the polypeptide comprises or consists of all components i) to vii).

[0050] 26. The recombinant polypeptide according to any one of the above items, wherein the polypeptide does not comprise components viii) to x).

[0051] 27. The recombinant polypeptide according to any one of items 1 to 26, wherein the polypeptide comprises or consists of all components i) to x).

[0052] 28. The recombinant polypeptide according to any one of the above items, further comprising an N-terminal secretion signal peptide sequence.

[0053] 29. The recombinant polypeptide according to any one of items 1 to 28, which consists of the following amino acid sequence, which consists of the following ((a) and (b)) in order from N-terminus to C-terminus:

[0054] (a) a peptide antigen selected from the group consisting of the amino acid sequences of SEQ ID NOs: 2, 22, 23 and 24, and

[0055] (b) The amino acid sequence of SEQ ID No:16.

[0056] 30. The recombinant polypeptide according to any one of the above items, wherein the recombinant polypeptide is soluble.

[0057] 31. A nucleic acid encoding one or more polypeptides according to any one of the above.

[0058] 32. The nucleic acid according to item 32, wherein the nucleic acid is a vector.

[0059] 33. A pharmaceutical composition or kit comprising at least one nucleic acid according to item 32 or 33.

[0060] 34. A pharmaceutical composition or kit comprising at least one recombinant polypeptide according to any one of items 1 to 31.

[0061] 35. The pharmaceutical composition or kit according to item 35, wherein the pharmaceutical composition or kit comprises at least two different recombinant polypeptides according to any one of items 1-31, wherein each different polypeptide comprises a different peptide antigen as defined in any one of items 3 to 6.

[0062] 36. The pharmaceutical composition or kit according to any one of items 34 to 36 for use in the treatment of neuromyelitis optica in a human patient.

[0063] 37. The pharmaceutical composition or kit for use according to item 37, wherein the treatment is treatment by immunotherapy.

[0064] 38. The pharmaceutical composition or kit for use according to any one of items 37-38, wherein the treatment is by inducing immune tolerance against human aquaporin-4.

[0065] 39. The pharmaceutical composition or kit for use according to any one of items 37-39, wherein the treatment is used to reduce the level of autoantibodies against human aquaporin 4 in plasma or cerebrospinal fluid.

[0066] 40. The pharmaceutical composition or kit for use according to any one of items 37 to 40, wherein the human patient is a patient who has autoantibodies against human aquaporin 4 in plasma or cerebrospinal fluid before the start of treatment.

[0067] 41. A pharmaceutical composition or kit for use according to any one of items 37-41, wherein the treatment is by inducing myelin-specific regulatory T cells.

[0068] 42. A recombinant host cell comprising the nucleic acid or vector of item 32 or 33, and expressing the recombinant polypeptide of any one of items 1 to 31.

[0069] 43. A method for obtaining a pharmaceutical composition comprising a polypeptide according to any one of items 1 to 31, the method comprising the steps of: (a) culturing the recombinant host cell of item 43 under conditions allowing expression of the recombinant polypeptide from the nucleic acid molecule, (b) recovering the recombinant polypeptide, (c) purifying the recombinant polypeptide, and (d) formulating the recombinant polypeptide into a pharmaceutical composition.

[0070] BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 : Description of peptide-loaded soluble MHC Ib molecules suitable for achieving therapeutic antigen-specific immunomodulation.

[0072] The presented peptide antigen is represented by a dotted sphere, the HLA-Gα1-3 domain is represented by a light gray, and the β2 microglobulin domain is represented by a dark gray. The optional linker connecting the antigenic peptide and the β2 microglobulin molecule is represented by a gray rod pattern, and the optional disulfide bond capture is represented by a black sphere. This figure was generated using Pymol and modified from the structures published in Clements et al., Proc Natl Acad Sci US A. 2005 Mar 1; 102(9): 3360-5 and Hansen et al., Trends Immunol. 2010 Oct; 31(10): 363-9.

[0073] Figure 2: Examples of vector-based constructs encoding single-chain MHC Ib molecules suitable for therapeutic peptide-specific immunomodulation.

[0074] HLA-G1 and HLA-G5 each consist of three α domains (shown in black here), a non-covalently bound β2-microglobulin subunit (shown in dark gray here) and an antigenic peptide presented on HLA-G (short black arrow). HLA-G1 also contains a transmembrane domain and a short intracellular chain (not shown here). As shown, the α3 domain is able to bind to the receptors ILT2 (see Shiroishi et al., Proc Natl Acad Sci US A. 2003 July 22; 100 (15): 8856-8861) and ILT4 (see Shiroishi et al., Proc Natl Acad Sci US A. 2006 Oct 31; 103 (44): 16412-7) on immune cells. Physiologically, these sequences form non-covalently linked MHC class 1 complexes. To simplify the purification of the composite MHC Ib molecules, one or more protein tags (e.g., SpotTag, myc-tag, and / or His(6x)-tag) can be introduced. These tags are introduced in such a way that they can be selectively removed later by cleavage using the optional Factor Xa cleavage site. In addition, the antigenic peptide, β2-microglobulin, and MHC Ib α chain can be linked sequentially to improve stability. The vector map was generated using Snapgene Viewer software.

[0075] Figure 3 :The alternative molecule of the recombinant polypeptide of the present invention can induce Treg cells secreting IL10 in mice

[0076] In this experiment, 100 μg of virus (Gp34) or ovalbumin (Ova) model peptide antigen, mouse H2-K b The surrogate molecules consisting of the α1 and 2 domains and the human HLA-G α3 domain and β2 microglobulin were injected intraperitoneally into 12-week-old C57BL / 6 mice. After 14 days, the mice were sacrificed and splenocytes were restimulated with 5 μg / ml Gp34 or Ova peptide in a 48-hour standard mouse IL-10 ELISpot assay (Mabtech Mouse IL-10 HRPELISpot Kit, cells cultured in RPMI 10% FCS 10 ng / ml IL2).

[0077] A significant increase in IL-10-secreting regulatory T cells was detected only when restimulated with a peptide that induced tolerance to them by injection of a replacement molecule. (A) Experimental design; (B) Results

[0078] Figure 4 : The surrogate molecules of the recombinant polypeptide of the present invention can prevent CD8+T cell-driven EAE in mice.

[0079] In this EAE 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 on the H-2Kb MHC molecules on oligodendrocytes. OT-I mice express a T cell receptor (OT-I) on their CD8+ T cells that accurately recognizes this peptide-MHC combination. When CD8+ T cells from these mice are transferred into 10-day-old ODC-OVA mice, these mice develop experimental autoimmune encephalomyelitis (EAE), whose pathogenesis and symptoms are similar to MS in many aspects (Na et al., Brain, Volume 131, Issue 9, September 2008, Pages 2353–2365). In this experiment, 500 μg of virus (Gp34) or ovalbumin (Ova) model peptide antigen, mouse H2-K b The α1 and 2 domains and a surrogate molecule consisting of the human HLA-G α3 domain and β2 microglobulin, or PBS alone were injected. EAE scoring was performed according to Bittner et al., J Vis Exp. 2014 Apr 15;(86):51275.

[0080] Only the alternative molecule that induced ovalbumin tolerance almost completely prevented EAE symptoms. (A) Experimental design; (B) Results

[0081] Figure 5 :Some surrogate molecules of the recombinant polypeptide of the present invention selectively prevent CD4 + T cell-driven EAE.

[0082] In this model, a strong myelin-specific autoimmune response was induced by administering MOG 35-55 peptide in combination with complete Freund's adjuvant (which activates CD4+ Th17 cells) and pertussis toxin (which makes the blood-brain barrier more permeable) (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, each mouse was injected with 100 μg of virus (Gp34) or two Mog peptide antigens (Mog37 or Mog44), mouse H2-D on the first day. b The α1 and 2 domains, as well as alternative molecules of human HLA-G α3 domain and β2 microglobulin or PBS alone were injected.

[0083] Mog44 peptide containing the replacement molecule significantly alleviated EAE symptoms and weight loss.

[0084] (A) Experimental design; (B) EAE score; (C) Body weight

[0085] Figure 6 : The Mog44 replacement molecule of the recombinant polypeptide of the present invention can prevent inflammation and CD8 T cell infiltration in the spinal cord.

[0086] 10 μm fresh frozen sections were stained with commercially available toluidine 1× staining reagent for 1 hour at room temperature. Extensive immune cell infiltration was detected in EAE but was blocked by Mog44_Db_G.

[0087] 10 μm fresh frozen sections were briefly dried at room temperature, fixed with acetone, blocked with 5% BSA 10% normal goat serum in PBS, and stained with 1:100 anti-CD8 antibody and secondary antibody coupled 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 + The cells infiltrated the spinal cord en masse, and the Mog44_Db_G replacement molecule prevented this process.

[0088] (A) Toluidine; (B) CD8-DAB

[0089] Figure 7 : Detection of anti-MOG35-55 antibodies in Mog-EAE mice treated with a surrogate molecule of the recombinant polypeptide of the present invention ("AIM Bio"

[0090] After mice were sacrificed, mouse serum was collected by cardiac puncture. Plates were coated overnight with 10 μg / ml Mog35-55, blocked with 1% BSA, and then detected with the indicated HRP-conjugated secondary antibodies against MOG35-55.

[0091] Mog35-55-induced EAE was associated with high levels of MOG35-55-specific IgG autoantibodies, whereas such antibodies were undetectable in animals treated with 100 μg of the Mog44_Db_G replacement molecule.

[0092] Figure 8 : List of human NMO recombinant polypeptide candidates.

[0093] The peptide antigen sequences of the NMO recombinant polypeptide candidates shown in the figure are as follows:

[0094] Construct peptide antigen SEQ ID NO:

[0095] hAQP4 42-50_HLAG Myc / His tag FLAMLIFVL SEQ ID No: 31

[0096] hAQP4 45-53_HLAG Myc / His Tag MLIFVLLSL SEQ ID No: 32

[0097] hAQP4 65-72_HLAG Myc / His tag PLPVDMVL SEQ ID No: 33

[0098] hAQP4 71-79_HLAG Myc / His Tag VLISLCFGL SEQ ID NO:22

[0099] hAQP4 126-135_HLAG Myc / His tag AIIGAGILYL SEQ ID No:34

[0100] hAQP4 127-135_HLAG Myc / His tag IIGAGILYL SEQ ID NO:23

[0101] hAQP4 45-53_A2G Myc / His Tag MLIFVLLSL SEQ ID No: 32

[0102] hAQP4 65-72_A2G Myc / His tag PLPVDMVL SEQ ID No: 33

[0103] hAQP4 71-79_A2G Myc / His tag VLISLCFGL SEQ ID NO:22

[0104] hAQP4 126-135_A2G Myc / His tag AIIGAGILYL SEQID No: 34

[0105] hAQP4 127-135_A2G Myc / His tag IIGAGILYL SEQ ID NO:23

[0106] hAQP4 156-164_A2G Myc / His tag AGHGLLVEL SEQ ID No: 35

[0107] hAQP4 238-247_A2G Myc / His tag IIGAVLAGGL SEQ ID NO:24

[0108] hAQP4 45-HLAG SPOTtag MLIFVLLSL SEQ IDNo:32

[0109] hAQP4 36-43_HLAG SPOTtag KAVTAEFL SEQ ID No:36

[0110] hAQP4 71-79_HLAG SPOTtag VLISLCFGL SEQ IDNO:22

[0111] hAQP4 64-72_HLAG SPOTtag KPLPVDMVL SEQ IDNO:2

[0112] hAQP4 71-79_A2G SPOTtag VLISLCFGL SEQ IDNO:22

[0113] hAQP4 127-A2G SPOTtag IIGAGILYL SEQ ID NO:23

[0114] hAQP4 238-A2G SPOTtag IIGAVLAGGL SEQ ID NO:24

[0115] hAQP4 137-145_HLAG SPOTtag VTPPSVVGGL SEQ ID No:37

[0116] Figure 8It is further shown which combinations of AQP4 peptides and antigen presenting MHC class I α1 and 2 domains (HLAg = HLA-G, A2G = HLA-A2 presenting domain + HLA-G α3 domain) can lead to successful (high), intermediate (average, av.) or poor (low) results in terms of production, quality control and prioritization in healthy blood donors (e.g. Fig. 9 (shown)

[0117] Fig. 9 :The recombinant polypeptide of the present invention containing KPLPVDMVL antigen ("AQP_64") upregulates CD8 Treg in healthy blood donors

[0118] In vitro Treg induction mediated by peptide-HLA-G-containing constructs (AIM Biologicals) was performed as follows: PBMCs from healthy donors were purified by density centrifugation of leukocytes from the leukopenia chamber using Ficoll. Cells were centrifuged at 1200×g for 20 minutes without interruption, and then the mesophase rings were collected and washed with 1×PBS (5 minutes, 300×g). PBMCs were frozen for future use.

[0119] PBMCs were thawed 1 day before PBMC pulse (d-1) and stored in 5 ml X-VIVO 15 medium containing 5% human AB serum in 6-well plates at 37°C overnight.

[0120] On the next day (d0), the cells were counted and suspended in X-VIVO 15 complete medium (5% hAB serum and cytokine mixture: 20 ng / ml hIL-2, 20 ng / ml hGM-CSF, 10 ng / ml hIL-4 and 10 ng / ml hTGF-b1) at a cell density of 3 × 10 6 cells / ml. 6 Each cell was seeded into the corresponding wells of a 12-well plate in a final volume of 1000 μl X-VIVO complete medium containing a cytokine cocktail and 5 μg / ml AIM Bio molecules or corresponding controls.

[0121] On day 3, add 1 ml of complete medium (with cytokines), and on day 6, add 5 μg / ml AIM Bio molecules for the second pulse (after removing the medium). On days 7, 10, and 12, add 1 ml of complete medium (with cytokines).

[0122] On day 13, the ELISpot plate PVDF membrane was activated with 50 μl / well EtOH (35% v / v) for 1 minute, and then washed 5 times with 200 μl distilled sterile water. The plate was coated with 100 μl / well anti-hIL10 (clone 9D-7, diluted 1:500 in PBS, sterile filtered) at 4°C overnight. The next day, the unbound coating antibody was removed, washed 5 times with 200 μl of PBS, and 200 μl of blocking buffer (X-VIVO 15 5% hAB serum) was added, and the plate was incubated at room temperature for 30 minutes-2 hours. On day 14, 200,000 cells were seeded per well on the ELISpot plate, in duplicate, including negative controls (cells plus PBS) and positive controls (e.g., LPS), and incubated for 48 hours. Prepare secondary antibodies: 1 μg / ml aIL-10-biotinylated antibody (1:1000 dilution) and horseradish peroxidase-conjugated streptavidin (1:750 dilution in 0.5% BSA / 1×PBS), tetramethylbenzidine solution filtered using a 0.45 μm filter and stored at 4°C until use. Remove cell supernatant and wash 5 times with 100 μl of PBS. Use paper towels to remove the final excess buffer. Add 25 μl of diluted HRP-streptavidin (1:750) to each well, incubate in the dark at room temperature for 1 hour, and then wash 5 times with sterile 1× PBS. Add 100 μl of filtered TMB substrate to each well and react for 15-25 minutes until blue spots appear. Wash the wells thoroughly with water to stop the reaction. Remove the plastic drain of the plate, and rinse the bottom and sides of the plate with tap water and let it dry.

[0123] AQP4_64_G_Spt induced at least 30% increase in IL-10 secreting Tregs in 65% of healthy blood donors.

[0124] Fig. 9 Continued: In HLA-A2 + and HLA-A2 - Percent increase in IL10 spots in PBMCs of the indicated donors in response to AQP4_64_G_Spt treatment.

[0125] Fig.10 : Figure 7 Control experiments with samples shown in showed that treatment with the MOG47_Db_G surrogate molecule did not reduce total IgG. TM Total IgG was quantified using the Human IgG (γ chain) Detection Kit (Thermo Fisher). Figure 7 The binding demonstrated that selective antibody responses could be inhibited using single-chain MHC Ib molecules.

[0126] Fig.11 : Stability of purified single-chain MHC Ib molecules. After purification, the stability of single-chain MHC Ib molecules was analyzed after 1 and 3 freeze-thaw cycles, storage at room temperature for 5 days, and heating to 50°C for 30 minutes. For this purpose, aHLA-G Western Blot was performed under non-reducing conditions using A) 2 μg of AIM Bio in 12% polyacrylamide gels with Coomassie staining and B) 1 μg of protein with 2A12 aHLA-G antibody (1:1000). Both monomers and dimers were detected.

[0127] Fig.12 :Single-chain MHC Ib molecules are thermally stable. For thermal shift assay (TSA), 3 μg of the corresponding single-chain MHC Ib molecule or Motavizumab as a control molecule was diluted to a volume of 25 μl with PBS and 5×SYPRO Orange dye (stock solution 5000×, final concentration: 5×). The melting curve program was set up on the StepOnePlus instrument using StepOnePlus software 2.3. The starting temperature was 25°C for one minute, and then the temperature was increased to a final temperature of 95°C at a rate of 1°C per minute for 2 minutes, thereby measuring autofluorescence in arbitrary units. The data was exported and graphed in Prism V7.04. To determine the melting temperature (Tm), the Boltzman sigmoid function was used.

[0128] Fig.13 :Single-chain MHC Ib molecules induce Treg in a dose-dependent manner. OT-I mice were intraperitoneally injected with the indicated amount of single-chain H2_K b α1+2, HLA-Gα3 domain, human β2 microglobulin and the constructs of the indicated peptides, or vehicle (PBS). In these mice, Ova is the homologous peptide of the OT-I TCR and Gp34 is an unrelated virus-derived control peptide. After 14 days, the mice were sacrificed and IL10-secreting cells in splenocytes were detected in the memory immune mouse IL-10 ELISpot (200,000 cells per well, MabTech mouse IL-10 ELISpot kit, supplemented with 5 μg / ml of the indicated peptide or PBS alone, 48 hours). When 50 and 500 μg of mouse-adapted Ova_KbG were injected, a clear induction of IL-10-secreting cells reactive to the Ova peptide was observed.

[0129] Fig.14:Single-chain MHC Ib molecules inhibit T cell lysis in a dose-dependent manner. OT1 / BL6 mice were sacrificed, spleen cells were collected and washed once in RPMI 5% FCS. Red blood cells were removed with 2ml of 1× sterile RBC lysis buffer for 3 minutes. Cells were cultured at high density (10mio cells / ml) for 72 hours in RPMI 10% FCS medium containing GMCSF 20ng / mL, IL-2 20ng / ml and IL-4 10ng / ml and increasing doses of Ova_KbG. The cells were then scraped from the culture dish and CD8+ cells were purified by magnetic beads. A sterile 96-well white plate was used. Panc02 target cells expressing luciferase were loaded with 20μg / ml Ova peptide (SIINFEKL) and shaken at 500rpm for 60 minutes at 37°C. CD8+ effector T cells were added at a ratio of 50:1, as well as luciferin. Luminescence was measured after 24 hours and 48 hours.

[0130] Fig.15 :Serum cytokines from EAE-ODC Ova mice. EAE-ODC Ova mouse serum cytokines were measured using the Th1 / Th210plex Flowcytomix kit (eBioscience) according to the manufacturer's instructions. The 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), t interleukin-17 (IL-17) and tumor necrosis factor (TNF) in a single sample. The array kit provides eight capture bead mixtures with different fluorescence intensities, which have been coated with specific capture antibodies for each cytokine. Beads coated with ten specific capture antibodies are mixed together. Subsequently, 25 μL of mixed capture beads, 25 μL of unknown serum sample or standard dilution, and 25 μL of phycoerythrin (PE) detection reagent were added to each well of the 96V bottom plate in sequence and incubated at room temperature in the dark for 2 hours. The samples were washed with 1 mL of wash buffer for 5 minutes and centrifuged. After discarding the supernatant, the bead pellet was resuspended in 200 μL of buffer. TM The cells were measured on an NxT flow cytometer and samples were analyzed using Attune flow cytometer software (Thermo Fisher Scientific).

[0131] Fig.16 : Diagram of the molecular composition of the most preferred mouse-adapted AIM Bio.

[0132] The corresponding exemplary amino acid sequence is:

[0133] MSRSVALAVLALLSLSGLEAVTTVHGNLGCGASGGGGSGGGGSIQRTPKIQVYSRHPAEN

[0134] GKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEY

[0135] ACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGL

[0136] GEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFR

[0137] VDLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWT

[0138] AADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDDPPKTHVTHH

[0139] PVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPS

[0140] GEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC(SEQ IDNo:38)

[0141] Fig.17 : Coomassie gel of purified, stability-tested NMO substituted single-chain MHC Ib molecules. After purification, the single-chain MHC Ib molecules were analyzed for stability after 1 and 3 freeze-thaw cycles, storage at room temperature for 5 days, and heating to 50°C for 30 minutes. Methods refer to Fig.11 .

[0142] Fig.18 : Western blot of purified and stability tested NMO substituted single chain MHC Ib molecules (2A12HLA-G5 antibody 1:5000). Both experiments show that these molecules are very stable, but AQP203_H2KbG prefers to multimerize.

[0143] Fig.19: Thermal shift analysis (TSA). The results obtained in the above figure were also confirmed in TSA, where polymers produce high autofluorescence at low temperatures.

[0144] Fig. 20 : EAE scoring. AQP147_KbG provided complete protection against EAE in treated 2D2 mice. Mice were injected with treatment or control AIM Bios at D0, D15 and D30. Mice were evaluated daily according to the EAE scoring system of 0-10 described in Table 1. Untreated mice developed severe EAE symptoms during the experiment. One untreated mouse reached a score of 6 on D14 and was sacrificed. Its score was assumed to be 6 for the remaining days during the evaluation period. Control treatment with gp34_KbG did not provide satisfactory protection. (*p<0.05, **p<0.01, ***p<0.001)

[0145] Fig.21 : Immune cell infiltration in the optic nerve of wild-type and 2D2 mice. AQP147_KbG treatment inhibited immune cell infiltration in the optic nerve. Panel A (optic nerve) and panel B (spinal cord) show the CD3 + and CD8 + Fluorescent signal, DAPI images depict the total number of nuclei present in each section. CD in the optic nerve (panel C) and spinal cord (panel D) of control and untreated mice 3+ and CD8 + The number of T cells was significantly higher. The effect of AQP203_KbG in suppressing immune infiltration was better than that of the control group and the untreated group. Data are expressed as mean + / - SEM. Scale bar 25 μm. One-tailed T test.

[0146] Fig. 22 : Quantitative analysis of cleaved caspase 3 in the optic nerve, spinal cord and retina of wild-type and 2D2 mice. AQP147_KbG completely inhibited apoptosis in the optic nerve, spinal cord and retina. The figure shows the fluorescent signal obtained by staining cleaved caspase 3 (apoptosis marker) in the optic nerve (sub-figures A, B), spinal cord (sub-figures C, D) and retina (sub-figures E, F). The highest levels of cleaved caspase 3 and therefore apoptosis were found in untreated and control-treated mice. Data are expressed as mean + / - SEM. Scale bar 25 μm. One-tailed T test

[0147] Fig.23 : CD8 in the optic nerve + CD122 + and CD4 + FoxP3 +Regulatory T cells. This figure shows that while there were fewer CD8 T cells in mice treated with AIM, there were a small but significantly more regulatory T cells in the optic nerve (using a one-tailed T test).

[0148] Fig.24 : Quantification of inner retinal thickness in wild-type and 2D2 mice. AQP147_KbG completely rescued IRL cells from degradation. Hematoxylin-eosin staining results showed that the inner retinal thickness was preserved in AQP147_KbG, while the IRL was thinner in untreated and control-treated mice. Data are expressed as mean + / - SEM. Scale bar 25 μm. Two-tailed T-test. DETAILED DESCRIPTION OF THE INVENTION

[0150] Definition and general techniques

[0151] Unless otherwise defined below, the terms used in the present invention should be understood according to the ordinary meaning known to those skilled in the art. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. Publications cited herein may be cited by specifying complete references in the text.

[0152] All proteins of the present invention, including the recombinant polypeptides of the present invention, can be obtained by methods known in the art. Such methods include methods for producing recombinant polypeptides. The recombinant polypeptides of the present invention can be expressed in the recombinant host cells of the present invention. The recombinant host cells of the present invention are preferably mammalian cells, such as CHO and HEK cells.

[0153] It should be understood that the recombinant polypeptide of the present invention is intended to optionally include a secretion signal peptide sequence. Similarly, the recombinant polypeptide of the present invention is intended to also optionally include an affinity tag, such as for the convenience of purification, and an optional protease cleavage site between the tag and the polypeptide, such as for the convenience of removing the tag by protease cleavage.

[0154] It should also be understood that any amino acid sequence mentioned herein is intended to cover not only the unmodified amino acid sequence, but also typical post-translational modifications of these amino acid sequences that occur in cell expression systems known in the art (e.g., glycosylation or deamidation of amino acids, cleavage of specific amino acids or other post-translational modifications), including mammalian cells, such as CHO and HEK cells.

[0155] Likewise, it is to be understood that the recombinant polypeptides of the present invention are intended to optionally include the corresponding propeptides.

[0156] It should also be understood that the recombinant polypeptides of the present invention may be in their soluble form or membrane-bound form. Whether a recombinant polypeptide is "soluble" under these conditions can be determined by methods known in the art, such as by measuring the turbidity of the recombinant polypeptide under the above-mentioned reference conditions. As used herein, soluble means that at least 95% of the recombinant polypeptide is determined to be soluble under these reference conditions.

[0157] Single-chain MHC molecules can be stored, for example, at -80°C in PBS (with or without 0.1% human albumin as a carrier, depending on the protein concentration), or at -20°C in 50% glycerol.

[0158] According to the present invention, the MHC molecule is preferably a human MHC molecule.

[0159] The recombinant polypeptide of the present invention is preferably an isolated recombinant polypeptide.

[0160] It should be understood how to prepare recombinant polypeptides according to the present invention that can bind and present peptide antigens. For example, peptide antigen binding domains such as α1 and α2 domains are well known, and these domains can be modified. The ability of peptide antigens to bind to polypeptides of the present invention and MHC molecules can be determined by techniques known in the art, including but not limited to exploratory methods such as mass spectrometry and computer bioinformatics predictions after MHC peptide elution, and confirmatory methods such as MHC peptide multimer binding methods and stimulation tests.

[0161] According to the present invention, the recombinant polypeptide, pharmaceutical composition and kit of the present invention are preferably suitable for use in human patients.

[0162] According to the present invention, the recombinant polypeptide, pharmaceutical composition and kit of the present invention are preferably suitable for treating neuromyelitis optica in human patients.

[0163] According to the present invention, the recombinant polypeptide, pharmaceutical composition and kit of the present invention are preferably suitable for inducing immune tolerance against human aquaporin 4, for example, in human patients.

[0164] It should be understood that according to the present invention, the recombinant polypeptides, pharmaceutical compositions and kits of the present invention are stable.

[0165] It should be understood that for peptide antigens used according to the present invention, any length of these peptide antigens mentioned herein (e.g. "length of 7 to 11 amino acids") refers to the length of the peptide antigen itself. Therefore, the length of the peptide antigen mentioned herein does not include the length conferred by additional amino acids that do not belong to the peptide antigen, such as additional amino acids from possible linker sequences, etc.

[0166] According to the present invention, each occurrence of the term "comprising" may optionally be replaced by the term "consisting of".

[0167] Methods and techniques

[0168] Generally, unless otherwise defined herein, the methods used in the present invention (e.g., cloning methods or methods related to antibodies) are performed according to procedures known in the art, such as those described in Sambrook et al. ("Molecular Cloning: A Laboratory Manual.", 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1989), Ausubel et al. ("Current Protocols in Molecular Biology." Greene Publishing Associates and Wiley Interscience; New York 1992), and Harlow and Lane ("Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1988), all of which are incorporated herein by reference.

[0169] Protein-protein binding, e.g. binding of an antibody to its respective target protein, can be assessed by methods known in the art. Protein-protein binding is preferably assessed by surface plasmon resonance spectroscopy measurements.

[0170] For example, the binding of the MHC class Ib molecule or recombinant polypeptide according to the present invention to its receptor, including ILT2 and ILT4, is preferably assessed by surface plasmon resonance spectroscopy. More preferably, the binding of the MHC class Ib molecule or recombinant polypeptide according to the present invention to its receptor is assessed by surface plasmon resonance measurement at 25°C. Shiroishietal., Proc Natl Acad Sci US A. 2003July 22; 100(15): 8856-8861 describes suitable conditions for such surface plasmon resonance measurement.

[0171] The sequence alignment of the present invention is performed using the BLAST algorithm (see Altschul et al. (1990) "Basic local alignment search tool." Journal of Molecular Biology 215.p.403-410.; Altschul et al.: (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402.). Suitable parameters for sequence alignment of short peptides using the BLAST algorithm, which are suitable for peptide antigens according to the present invention, are known in the art. Most software tools using the BLAST algorithm automatically adjust the parameters for sequence alignment of short input sequences. In one embodiment, the following parameters are used: Max target sequences 10; Word size 3; BLOSUM 62 matrix; gap costs: existence 11, extension 1; conditional compositional score matrix adjustment. Therefore, terms such as "identity" or "identical" when used in connection with a sequence preferably refer to identity values ​​obtained using the BLAST algorithm.

[0172] Preparation of the pharmaceutical composition of the present invention

[0173] The pharmaceutical composition of the present invention is prepared according to known pharmaceutical composition preparation standards.

[0174] For example, the pharmaceutical composition should be prepared in such a way that it can be easily stored and administered.Thus, the pharmaceutical composition of the present invention may comprise pharmaceutically acceptable components, such as carriers, excipients and / or stabilizers.

[0175] Such pharmaceutically acceptable components are non-toxic in the amounts used when the pharmaceutical composition is administered to a human patient. The pharmaceutically acceptable components added to the pharmaceutical composition may depend on the chemical nature of the active ingredients present in the composition, the specific intended use of the pharmaceutical composition, and the route of administration.

[0176] In general, pharmaceutically acceptable components associated with the present invention are used according to existing knowledge in the art, such as from Remington's Pharmaceutical Sciences, Ed. AR Gennaro, 20th edition, 2000, Williams & Wilkins, PA, USA. Pharmaceutical compositions comprising nucleic acids (e.g., RNA) of the present invention can also be formulated according to existing knowledge in the art, such as using liposome formulations targeted to dendritic cells.

[0177] Peptide antigens according to the present invention

[0178] The peptide antigens that can be used according to the present invention include the peptide antigens defined above, and there is no particular limitation except that they can be presented on MHC molecules. It should be understood that the "peptide antigen presented by the recombinant polypeptide" related to the present invention refers to the peptide antigen presented by the recombinant polypeptide to human T cells (if such T cells exist) by binding to the T cell receptor on human T cells.

[0179] As is known in the art, peptides that can be presented on MHC molecules can be produced (see, e.g., Rammensee, Bachmann, Emmerich, Bachor, and Rock,Reits,Neefjes.PresentYourself! By MHC Class I and MHC Class II Molecules.Trends Immunol.2016Nov;37(11):724-737).

[0180] Peptide antigens are well known in the art. Generally, the peptide antigens according to the present invention are capable of binding to MHC class I proteins. It will be appreciated by those skilled in the art that for each MHC class Ib molecule or polypeptide capable of presenting a peptide according to the present invention, a peptide antigen capable of binding to the MHC class Ib molecule or recombinant polypeptide will preferably be used. These peptide antigens can be selected based on methods known in the art.

[0181] The binding of a peptide antigen to an MHC class Ib molecule according to the present invention or a polypeptide capable of binding to a peptide antigen can be assessed by methods known in the art, such as the following method:

[0182] Rammensee,Bachmann,Emmerich,Bachor, SYFPEITHI:database forMHC ligands and peptide motifs.Immunogenetics.1999Nov;50(3-4):213-9;

[0183] Pearson et al.MHC class I-associated peptides derive from selective regions of the human genome.J Clin Invest.2016Dec 1;126(12):4690-4701; and

[0184] Rock,Reits,Neefjes.Present Yourself! By MHC Class I and MHC Class II Molecules.Trends Immunol.2016Nov;37(11):724-737.

[0185] These methods include experimental approaches and methods for predicting peptide antigen binding.

[0186] Anchor residues for anchoring peptide antigens to MHC class I molecules and ensuring binding of peptide antigens to MHC class I molecules are known in the art.

[0187] In a preferred embodiment according to all embodiments of the present invention, the peptide antigen used according to the present invention comprises any anchor or preferred amino acid residues at positions predicted for MHC class I molecules.

[0188] Preferably, such predictions are performed as described in any of the following publications:

[0189] -Rammensee et al, SYFPEITHI: database for MHCligands and peptidemotifs. Immunogenetics (1999) 50:213-219

[0190] -Nielsen et al,Protein Sci(2003)12:1007-1017

[0191] -Neefjes et al. Nat Rev Immunol.2011Nov 11;11(12):823-36

[0192] -Diehl et al.Curr Biol.1996Mar 1;6(3):305-14,

[0193] -Lee et al.Immunity.1995Nov;3(5):591-600.

[0194] -Desai&Kulkarni-Kale,T-cell epitope prediction methods:anoverview.Methods Mol Biol. 2014;1184:333-64.

[0195] -Jumper et al.Highly accurate protein structure prediction withAlphaFold.Nature2021;596:583–589

[0196] In the present invention, the peptide antigen is derived from human aquaporin 4.

[0197] It should be understood that, relative to the corresponding amino acid sequence of the peptide antigen from human aquaporin 4, the non-anchor amino acid residues of the peptide antigen of the present invention may or may not contain conservative substitutions, preferably no more than two conservative substitutions, more preferably one conservative substitution.

[0198] The peptide antigens of the present invention are preferably composed of natural amino acids. However, non-natural amino acids, such as modified amino acids, may also be used. For example, in one embodiment, the peptide antigens of the present invention include a peptidomimetic of the amino acid sequence of the peptide antigen of human aquaporin 4.

[0199] Methods for the synthesis of peptide antigens, including peptide antigens according to the present invention, are well known in the art.

[0200] Therapeutic applications of the invention

[0201] The recombinant polypeptide of the present invention can be used to treat neuromyelitis optica.

[0202] Treatment can be treatment by inducing myelin-specific regulatory T cells. Because such regulatory T cells (e.g., CD8 positive regulatory T cells) are activated in myelin structures, they confer target cell protection to resist cytotoxic T cells that recognize the same or another myelin antigen. Regulatory T cells (e.g., CD8 positive regulatory T cells) are known in the art and can be detected by their secretion of IL-10.

[0203] Although CD8+ regulatory T cells are not as well known as CD4CD25+ regulatory T cells, they are considered even more potent. See, for example:

[0204] and

[0205] Niederlova,V.,Tsyklauri,O.,Chadimova,T.and Stepanek,O.(2021),CD8 + Tregs revisited: A heterogeneous population with different phenotypes andproperties. Eur.J.Immunol.,51:512-530.https: / / doi.org / 10.1002 / eji.202048614

[0206] While these cells are characterized by the expression of CD122 and CD8 in mice, the corresponding cells in humans are described as being positive for CD8 and CXCR3. See, for example:

[0207] Shi Z, Okuno Y, Rifa'i M, Endharti AT, Akane K, Isobe K, et al. Human CD8+CXCR3+Tcells have the same function as murine CD8+CD122+Treg. Eur J Immunol (2009) 39:2106–2119.doi:10.1002 / eji.200939314).

[0208] The treatment according to the present invention may be a treatment to reduce the level of autoantibodies against human aquaporin 4 in plasma or cerebrospinal fluid (CSF). The human patient may be a patient having autoantibodies against human aquaporin 4 in plasma or cerebrospinal fluid (CSF) before the start of treatment.

[0209] According to the present invention, autoantibodies can be detected by various methods known in the art. A preferred method is a cell-based assay (CBA), in which the suspected target antigen of the autoantibody (e.g., aquaporin 4) is overexpressed in HEK293 or CHO cells, and the cells are then incubated with serum or cerebrospinal fluid, usually at room temperature for 1 hour. Simulated transfected sister cells are used as controls. Autoantibodies bound to cells are detected using different fluorescently labeled anti-human specific secondary antibodies, which recognize total human IgG (heavy chain and light chain), IgG-Fc (constant chain) or IgG1. Binding is quantified by visual scoring by flow cytometry (CBA-FACS) or by microscopic assessment of immunofluorescence (CBA-IF), usually titrated. Other methods such as enzyme-linked immunosorbent assay (ELISA) or western blotting can also be used, but are generally less sensitive because these methods may not be able to detect conformation-sensitive antibodies. Suitable methods have been described in:

[0210] Waters,P.,Pettingill,P.&Lang,B.Detection methods for neural autoantibodies.Handb.Clin.Neurol.133,147–163(2016).

[0211] The method for detecting AQP4-specific autoantibodies is described in:

[0212] Lennon, VA, Kryzer, TJ, Pittock, SJ, Verkman, AS & Hinson, SRIgGmarker of optic-spinal multiple sclerosis binds to the aquaporin-4waterchannel. J. Exp. Med. 202, 473–477 (2005).

[0213] sequence

[0214] The preferred amino acid sequences mentioned in the present application can be independently selected from the following sequences. These sequences are represented in the order from N-terminus to C-terminus; and are represented by single-letter amino acid codes.

[0215] Exemplary sequences that are part of a recombinant polypeptide of the invention:

[0216] Optional leader peptide (deleted from the recombinant polypeptide due to processing during cell expression): for example MSRSVALAVLALLSLSGLEA (SEQ ID No: 1)

[0217] Peptide antigen: Any MHC class I peptide corresponding to the MHC class I α1 & 2 domains, such as KPLPVDMVL (SEQ ID No: 2)

[0218] First linker: for example, GGGGSGGGGSGGGGS (SEQ ID No: 3) or GCGASGGGGSGGGGS (SEQ ID No: 4)

[0219] beta-2 microglobulin, such as:

[0220] IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSK DWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO: 5, human β2 microglobulin)

[0221] Second connector, for example:

[0222] GGGGSGGGGSGGGGSGGGGS(SEQ ID No:6)

[0223] α1 and 2 domains, derived from human HLA-G or any other MHC class I α1 & 2 domains suitable for presenting the selected antigenic peptide, Y84 in the DT variant can be C, or, Y84 can be A

[0224] For example, the α1 & 2 domains from human HLA-G: e.g.

[0225] GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRA(SEQ ID No:7)

[0226] Or: Human HLA-A2 α1&2 domains: e.g.

[0227] GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRT(SEQ ID No:8)

[0228] Human HLA-G α3 domain (or any MHC Ib α3 domain such as HLA-F, which also interacts with ILT2 and ILT4 receptors), for example:

[0229] DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDL (SEQ ID No: 9; HLA-Gα3 sequence).

[0230] Note that the following underlined amino acids in this sequence are involved in ILT2 or ILT4 receptor interaction:

[0231] DPPKTHVTHH PVFDYE ATLRCWALGFYPAEIILTWQRDGEDQTQD V ELVETRPAGDGTFQKWAAV V VPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDL

[0232] In addition, a shorter form of the human HLA-Gα3 domain may be used that lacks the alternative C-terminal amino acid sequence from intron 4 (SKEGDGGIMSVRESRSLSEDL; SEQ ID No: 20), namely:

[0233] DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW(SEQ ID No:21),

[0234] Factor Xa restriction site: IEGRTGTKLGP (SEQ ID No: 10)

[0235] SpotTag: PDRVRAVSHWSSC(SEQ ID No:11)

[0236] Myc tag: EQKLISEEDL (SEQ ID No: 12)

[0237] His tag: HHHHHH* (SEQ ID No: 13)

[0238] Spacer sequence: for example NSAVD (SEQ ID No: 14) or GS

[0239] Other (alternative) exemplary peptide antigens that may be part of the recombinant polypeptide of the present invention are as follows:

[0240] VLISLCFGL (SEQ ID NO: 22), preferably used for recombinant polypeptides containing HLA-Gα1 and 2 domains;

[0241] IIGAGILYL (SEQ ID NO: 23), preferably used for recombinant polypeptides containing HLA-A2 α1 and 2 domains;

[0242] IIGAGILYL (SEQ ID NO: 24), preferably used for recombinant polypeptides containing HLA-A2 α1 and 2 domains;

[0243] Examples of recombinant polypeptides of the present invention (with optional leader peptides):

[0244] MSRSVALAVLALLSLSGLEAKPLPVDMVLGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEK DEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRM NLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVF DYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC*(SEQ ID NO:15; note that the asterisk indicates the stop codon)

[0245] Note that the peptide antigen (here: KPLPVDMVL) sequence of the above-mentioned full-length recombinant polypeptide can be replaced by any peptide antigen sequence according to the present invention, that is, by any peptide antigen presented by the recombinant polypeptide, wherein the peptide antigen is a peptide of human aquaporin 4. That is, the recombinant polypeptide of the present invention can be composed of the following sequence, which consists of a peptide antigen, which is a peptide of human aquaporin 4 (for example, any one of the peptide antigens of SEQ ID No: 2, 22, 23 and 24), followed by the following sequence:

[0246] GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDR DMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGCYNQSEAS SHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCW ALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC*(SEQID NO:16; note that the asterisk indicates the stop codon)

[0247] The recombinant polypeptides of the present invention may also contain an optional leader peptide as exemplified above.

[0248] The receptors ILT2 (also known as LILRB1) and ILT4 (also known as LILRB2) are known in the art. According to the present invention, the preferred sequences of these receptors are as follows:

[0249] ILT2:

[0250] MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEETLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH(SEQ ID NO:17)

[0251] ILT4:

[0252] MTPIVTVLICLGLSLGPRTHVQTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGAYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEEEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPGVSKKPSLSVQPGPVVAPGESLTLQCVSDVGYDRFVLYKEGERDLRQLPGRQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSECSAPSDPLDILITGQIRGTPFISVQPGPTVASGENVTLLCQSWRQFHTFLLTKAGAADAPLRLRSIHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSDPYLLSHPSEPLELVVSGPSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVVLLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKDTQPEDGVEMDTRAAASEAPQDVTYAQLHSLTLRRKATEPPPSQEREPPAEPSIYATLAIH(SEQ ID No:18)

[0253] The sequence of human aquaporin 4 is known in the art. The preferred amino acid sequence of human aquaporin 4 is as follows:

[0254] >sp|P55087-2|AQP4_HUMAN Isoform 1of Aquaporin-4OS=Homo sapiens OX=9606GN=AQP4

[0255] MVAFKGVWTQAFWKAVTAEFLAMLIFVLLSLGSTINWGGTEKPLPVDMVLISLCFGLSIA

[0256] TMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVV

[0257] GGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDSKRTDVTGSIALAIGFSVAIGHL

[0258] FAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRF

[0259] KEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSS

[0260] V

[0261] (SEQ ID NO:19)

[0262] >sp|P55087|AQP4_HUMAN Aquaporin-4Isoform 2OS=Homo sapiens OX=9606GN=AQP4 PE=1SV=2

[0263] MSDRPTARRWGKCGPLCTRENIMVAFKGVWTQAFWKAVTAEFLAMLIFVLLSLG-STINWG

[0264] GTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIA

[0265] AQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDS

[0266] KRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIG

[0267] AVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVH

[0268] VIDVDRGEEKKGKDQSGEVLSSV

[0269] (SEQ ID NO:29)

[0270] >NP_001304313.1aquaporin-4isoform M1x[Homo sapiens]

[0271] MSDRPTARRWGKCGPLCTRENIMVAFKGVWTQAFWKAVTAEFLAMLIFVLLSLGSTINWGGTEKPLPVDM

[0272] VLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPS

[0273] VVGGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGA

[0274] SMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEV

[0275] EDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSVXLEDRTESRQDSLELSSDFLPPIKETD

[0276] LL

[0277] (SEQ ID No: 30; wherein X can be tryptophan, cysteine, arginine or serine or a stop codon) The present invention is further illustrated by the following non-limiting examples: Example

[0278] Embodiment 1:

[0279] Method for producing the recombinant polypeptide of the present invention

[0280] Expi-293F cells (Thermo Fisher), TM Growth in expression medium (Thermo Fisher): using Expifectamine TM 293 transfection kit (Thermo Fisher), DNA was complexed with Expifectamine using Opti-MEM (Thermo Fisher), and 1 μg of DNA was transfected into 2.5 × 10 6After 18-20 hours, enhancers were added according to the protocol, and supernatants were harvested 4-6 days later (37°C, 8% CO2, humidified incubator) and cultured in 19 mm 2 The culture was shaken at 125 rpm on an orbital shaker.

[0281] -Spot-tag protein purification: Equilibration of Spot-Cap resin: Transfer the required amount of slurry to an appropriate tube, pellet the beads by centrifugation (4°C, 4 min, 2500 g), remove and discard the supernatant, add 10 bed volumes of PBS (cold) to the beads, invert to mix, pellet the beads by centrifugation (4°C, 4 min, 2500 g), remove and discard the supernatant, and repeat twice.

[0282] - Add the required volume of beads to the supernatant, incubate at 4°C on a rotator, wash the beads by repeated centrifugation (4°C, 4 min, 2500 g), and remove the supernatant.

[0283] - Prepare 500 μM Spot peptide solution in PBS, remove supernatant and incubate with 1 / 3rd Spot peptide solution for 5-10 min.

[0284] Beads were pelleted by centrifugation. Protein concentration was performed using an Amicon Ultra-4 centrifugal filter (15 kDa cutoff) and Spot peptides were removed using a 15 kDa Amicon cutoff column.

[0285] Amicon Ultra-4 centrifugal filters (15 kDa cut-off) were rinsed with PBS and then with 0.1 N NaOH (centrifugation at 4000 g, 4°C) to remove traces of glycerol.

[0286] ELISPOT method:

[0287] 1) Cell culture

[0288] A) PBMC isolation (under laminar flow hood)

[0289] To isolate peripheral blood mononuclear cells (PBMCs), density centrifugation was performed using leukocytes from a leukocyte reduction chamber and a density gradient medium (e.g., Ficoll or ROTI Sep 1077). Cells were centrifuged at 1200 × g for 20 min without stopping, and then the interphase rings were collected and washed with 1 × PBS (5 min, 300 × g). PBMCs were frozen until further use.

[0290] B) PBMC pulse (under laminar flow hood)

[0291] PBMCs were thawed 1 day before PBMC pulse (d-1) and stored in 5 ml X-VIVO 15 medium containing 5% human AB serum in wells of 6-well plates at 37°C overnight.

[0292] The next day (d0), the cells were counted and suspended in X-VIVO 15 complete medium (5% hAB serum and cytokine mixture: 20 ng / ml hIL-2, 20 ng / ml hGM-CSF, 10 ng / ml hIL-4 and 10 ng / ml hTGF-b1) at a cell density of 3 × 10 6 cells / ml.

[0293] In the experiment, 3×10 6 Cells were seeded into the corresponding wells of a 12-well plate in a final volume of 1000 μl X-VIVO complete medium containing a cytokine mixture and

[0294] 5 μg / ml AIM Bio molecules or corresponding controls.

[0295] On day 3, 1 ml of complete medium (containing cytokines) was added, and on day 6, a second pulse (after removal of medium) was performed with 5 μg / ml of the recombinant polypeptide of the present invention or its surrogate molecule (collectively referred to as "AIM Bio" molecules). On days 7, 10, and 12, 1 ml of complete medium (containing cytokines) was added.

[0296] Required:

[0297] X-VIVO 15 medium + 5% human AB serum

[0298] X-VIVO 15 complete medium: X-VIVO 15 medium + 2% human AB serum, supplemented with cytokine mixture: 10ng / ml TGF-b1, 10ng / ml IL-4, 20ng / ml IL-2, 20ng / ml GM-CSF

[0299] 2) ELISPOT

[0300] Laminar flow hood

[0301] On day 13, ELISpot plates were coated with anti-hIL10 (clone 9D-7, diluted 1:500 in PBS, sterile filtered) and aIL10 (10G8-biotin), and on day 14, 200,000 cells were seeded per well on the ELISpot plates in duplicate, including a negative control (cells plus PBS) and a positive control (e.g., LPS).

[0302] The PFDF membrane was activated with 50 μl / well EtOH (35% v / v) for 1 minute, and then washed 5 times with 200 μl distilled sterile water. The plate was coated with 100 μl / well antibody solution at 4°C overnight. The next day, unbound coating antibody was removed, washed 5 times with 200 μl PBS, and 200 μl blocking buffer (X-VIVO 15 5% hAB serum) was added, and the plate was incubated at room temperature for 30 minutes to 2 hours.

[0303] Prepare DMSO or DMSO containing the corresponding antigen peptide as a control, the final amount of peptide added is 5 μg peptide / ml, and the final volume is 100 μl / well. 150,000 cells are seeded per well in X-VIVO 15 medium containing 5% human AB serum. Carefully remove the blocking buffer (X VIVO 15 medium + 5% hAB serum), use a medium containing PBS as a negative control, and add the stimulator (total volume per well is 5 μg / ml) to other wells and incubate overnight at 37°C.

[0304] Outside the laminar flow hood

[0305] Prepare secondary antibodies: 1 μg / ml aIL-10-biotinylated antibody and horseradish peroxidase-conjugated streptavidin (1:750 in 0.5% BSA / PBS) in 0.5% BSA / 1×PBS (1:1000 dilution). Filter the tetramethylbenzidine solution using a 0.45 μm filter and store at 4°C until use.

[0306] Remove the cell supernatant and wash five times with 100 μl PBS. Finally, wipe off the excess buffer with a paper towel.

[0307] 25 μl of diluted HRP-streptavidin (1:750) was added to each well and incubated for 1 hour at room temperature in the dark, followed by 5 washes with sterile 1× PBS.

[0308] Add 100 μl of filtered TMB substrate to each well and react for 15-25 minutes until blue spots appear. Rinse the wells thoroughly with tap water to terminate the reaction.

[0309] Remove the board's plastic drain and rinse the bottom and sides of the board with tap water and allow to dry.

[0310] Plate data were read using an ImmunoSpot S6 Ultra-V analyzer (Cellular Technology Limited), analyzed in Excel, and graphs / statistics were plotted in Graphad Prism.

[0311] Required:

[0312] Capture antibodies: anti-hIL10 (clone: ​​9D-7, Mabtech #3430-3-250; 1:500 dilution), anti-hIL10-biotinylated (Mabtech, #3430-6-250)

[0313] 1× PBS (sterile)

[0314] 35% ethanol (v / v)

[0315] Blocking buffer: X-vivo 5% hAB serum (sterile) [Block in the same medium as the cell culture medium]

[0316] Dilution buffer: 0.5% BAS in PBS

[0317] Washing buffer: 1× PBS

[0318] Culture medium: For T cells, X-VIVO 15 medium (Lonza)

[0319] Filter syringe: Millex GV

[0320] ELISPOT PVDF plate (#MSIP4510, Millipore)

[0321] TMB substrate

[0322] Embodiment 2: The surrogate molecules of the recombinant polypeptide of the present invention induce Treg cells secreting IL10 in mice

[0323] Wild-type black 6 mice were injected with 100 μg of a recombinant polypeptide (also called “AIMBio”) having the following sequence, Ova_KbG

[0324] SIINFEKLGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC(SEQ ID NO:25),

[0325] and

[0326] Gp34_KbG

[0327] AVYNFATMGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPK IVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGCYN QSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDPPKTHVTHHPVFDYEAT LRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC(SEQ ID NO:26),

[0328] They were used to induce tolerance to OVA peptide or viral Gp34 peptide, respectively. Gp34 peptide is a well-characterized T cell epitope derived from the lymphocytic choriomeningitis virus (LCMV) glycoprotein. Although this antigen was traditionally referred to as Gp33, it was later discovered that H2-K b The epitope presented on H2-K contains only amino acids 34-41. d The epitope presented starts at amino acid 33). Therefore, we added H2-K b The epitope was named Gp34, which is in line with the latest recommendations. However, the use of the nomenclature of Gp33 and Gp34 in the literature is still unclear. The first 8 amino acids of SEQ ID NO:26 show the correct sequence. After 2 weeks, the mice were sacrificed and the splenocytes were restimulated with matched or mismatched peptides. The IL-10 secreting cells were quantitatively analyzed by ELIspot. The results are shown in Figure 2. Figure 3 shown.

[0329] Embodiment 3: The surrogate molecules of the recombinant polypeptides of the present invention selectively prevent CD8+ T cell-driven EAE in mice

[0330] As described in (Na et al, Brain. 2008 Sep; 131(Pt 9): 2353-65.), the bCD8 recognizes ovalbumin epitope in the context of + Adoptive transfer of OT-IT cells into mice expressing ovalbumin in their oligodendrocytes resulted in experimental autoimmune encephalomyelitis, which recapitulates many of the symptoms of MS and advanced NMO. In this animal model, a single injection of 500 μg of a recombinant peptide replacement molecule that induces tolerance to an ovalbumin epitope (also called “AIMBio”) almost completely prevented EAE symptoms, whereas a replacement molecule presenting a control peptide had no significant protective effect ( Figure 4 ). The sequence of the recombinant polypeptide replacement molecule is listed in Example 2.

[0331] Embodiment 4: Some recombinant polypeptide surrogate molecules of the present invention selectively prevent CD4 + T cell-driven EAE

[0332] On the day of intraperitoneal injection of 33 μg or 100 μg of the recombinant polypeptide replacement molecule of the present invention ("AIM Bio"), 100 μl of MOG35-55 peptide / CFA (complete Freund's adjuvant; the final concentration of Mycobacterium tuberculosis H37RA and peptide was 1 mg / ml) emulsion was injected subcutaneously on the left and right sides, respectively, and 250 ng of pertussis toxin (in 200 μl PBS) was injected intraperitoneally. The second pertussis toxin injection was performed 3 days later. In this animal model, a single injection of the AIM Bio replacement molecule (Mog44_Kb_G) that induces tolerance to the Mog epitope can significantly alleviate EAE symptoms, while replacement molecules presenting a control peptide (Gp34) or a non-functional Mog peptide (Mog37) have no significant protective effect ( Figure 5 In this model, Mog44 AIM Bio also prevented inflammation and CD8 T cell infiltration in the spinal cord ( Figure 6 ). The sequence of the recombinant polypeptide replacement molecule is listed in Example 2 or as follows:

[0333] Mog44_DbG

[0334] FSRVVHLYRNGGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSMRYFETAVSRPGLEEPRYISVGYVDNKEFVRFDSDAENPRYEPRAPWMEQEGPEYWERETQKAKGQEQWFRVSLRNLLGCYNQSAGGSHTLQQMSGCDLGSDWRLLRGYLQFAYEGRDYIALNEDLKTWTAADMAAQITRRKWEQSGAAEHYKAYLEGECVEWLHRYLKNGNATLLRTDPPKTHVTHHPVFDYEATLRCW

[0335] ALGF

[0336] YPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC(SEQ ID No:27)

[0337] Mog37_DbG

[0338] VGWYRSPFSRGCGASGGGGSGGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPS

[0339] DIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVK

[0340] WDRDMGGGGSGGGGSGGGGSGGGGSGPHSMRYFETAVSRPGLEEPRYISVGYVDNKEF

[0341] VRFDSDAENPRYEPRAPWMEQEGPEYWERETQKAKGQEQWFRVSLRNLLG

[0342] CYNQSAGGSHTLQQMSGCDLGSDWRLLRGYLQFAYEGRDYIALNEDLKTWTAADMAA

[0343] QITRRKWEQSGAAEHYKAYLEGECVEWLHRYLKNGNATLLRTDPPKTHVTHHPVFDYE

[0344] ATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRY

[0345] TCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC(SEQ ID No:28)

[0346] In this model, Mog44 AIM Bio also completely prevented the formation of MOG-specific autoantibodies in serum, as tested by ELISA ( Figure 7 ; See also Fig.10 This strongly suggests that the recombinant polypeptides of the invention are an effective treatment for NMO, which is often characterized by an antibody response to human aquaporin 4. Thus, the patient population is defined by common autoimmune-related antigens. Certain MHC molecules are also associated with NMO.

[0347] Mog-reactive antibodies in mouse sera treated with AIM Bio (33 or 100 μg) were detected by standard ELISA protocol, with 3 washes between each step. Briefly, ELISA plates were coated with 10 μg / ml MOG35-55 peptide, blocked with PBS 1% BSA, and then mouse sera diluted 1:25 in PBS 1% BSA were added for 1 hour. Anti-mouse IgG-HRP or anti-mouse heavy and light chain-HRP antibodies diluted 1:5000 were used for detection.

[0348] Embodiment 5: Human recombinant polypeptide candidates for NMO of the present invention

[0349] The recombinant polypeptides of the present invention are newly developed protein complexes derived from the pregnancy-related 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 polypeptides of the present invention containing various peptides can selectively eliminate peptide-specific cytotoxic effector T cells and induce peptide-specific regulatory T cells in vitro.

[0350] Figure 8 A list of human MS & MOGAD recombinant polypeptide candidates is shown.

[0351] The inventors' results showed that a single-chain protein containing an AQP4 peptide antigen and an HLA-Gα3 domain could induce tolerant T cells in healthy blood donors. Thus, CD8 Treg in 65% of healthy blood donors was upregulated by at least 30% ( Fig. 9 ).

[0352] Embodiment 6: Further proof of concept of the stability and efficacy of the recombinant polypeptides of the present invention.

[0353] In addition, the inventors set out to obtain and test recombinant polypeptides having the general structure of the recombinant polypeptides of the present invention but containing various different peptide antigens in order to obtain further proof of concept that the recombinant polypeptides of the present invention and their surrogate molecules are stable and effective. Fig.11 and 12 As shown in Figure 2, the tested recombinant polypeptides were stable during freeze-thaw and storage and were thermostable. In addition, they induced Tregs ( Fig.13 ) and inhibited T cell lysis in a dose-dependent manner ( Fig.14 ). Fig.15 The effects of recombinant peptides on the serum cytokine profile of EAE-ODC Ova mice are shown. IL-10 and possibly IL-4 are induced, both known immunosuppressive cytokines that downregulate immune responses in an inflammatory setting. This requires the HLA-G α3 domain plus the cognate peptide. The induction of IL-2 appears to result from presentation of the cognate peptide to the cells without the α3 domain. IL-2 is required for T cell activation and survival.

[0354] Embodiment 7: Therapeutic effects of recombinant peptides containing mouse aquaporin antigen peptides in 2D2 TCR transgenic mice.

[0355] Materials and methods

[0356] Experimental model

[0357] 2D2 TCR transgenic mice were divided into 4 groups. Each group consisted of at least 5 animals. WT (C57BL / 6) littermates belonged to group 5. The experimental period began when 2D2 mice spontaneously developed optic neuritis. Disease onset was determined by visual abnormalities or increased levels of anti-MOG IgG in serum. On the second day of disease onset, mice were injected (iv) with one of the following treatments: mouse adaptive single-chain MHC Ib molecule AQP147_KbG (antigenic peptide: VTTVHGNL; SEQ ID No: 39) / AQP203_KbG (antigenic peptide: FAINYTGASM; SEQ ID No: 40), or control AIM Bio Gp34_KbG. Control mice received PBS only. The dose was set at 250 μl, 5 mg / kg body weight. Control or treatment injections were administered by intravenous injection (D0). Untreated mice received PBS. The injections were repeated on days 15 and 30. Mice were weighed daily and EAE symptoms were monitored (Table 1). Mice with EAE scores of 6 were sacrificed before the end of the study period. All animals were sacrificed with CO2 on day 42. Optic nerves, eyes, spinal cord, brain, serum and lymph nodes were harvested and frozen at -20°C. Cardiac blood was collected and processed for serum analysis.

[0358] EAE was scored according to a 10-point scoring system (Bittner et al., Journal of visualized experiments: JoVE, (86), 51275)

[0359] Immunohistochemistry

[0360] Serial sections of the eyes were cut at 20 μm thickness and stained with hematoxylin and eosin. Briefly, frozen sections were dried at room temperature for 10 minutes and stained in hematoxylin staining solution for 10 minutes. Tissue sections were rinsed under running water for 10 minutes and stained in eosin staining solution for 30 seconds. The sections were then dehydrated in 70%, 96%, and 100% ethanol for 30 seconds in sequence. Subsequently, the sections were incubated with xylene for 10 minutes and mounted with Vitro Cloud.

[0361] Immunofluorescence

[0362] Serial 10 μm thick longitudinal sections of the optic nerve were cut for immunohistochemical analysis. Tissue sections were fixed with 4% PFA in PBS for 10 minutes; then blocked with a buffer containing 5% BSA, 0.2% Triton-X100 and 5% NGS in 1× PBS for 2 hours at room temperature. Tissues were stained overnight at 4°C with a single or appropriate combination of primary antibodies in a staining buffer containing 1% BSA, 1% NGS and 0.2% Triton-X100. The primary antibodies were: i) rat anti-mouse CD3 (1:200, Invitrogen); ii) rat anti-mouse CD8 (1:200, Biorad); iii) rabbit anti-cleaved caspase 3 (1:400, Cellsignaling). The sections were washed three times with PBS and stained with the corresponding fluorescent-labeled secondary antibodies for 1 hour at room temperature in the dark: i) anti-rabbit Cy3 (1:300, Dianova); ii) anti-rat AF488 (1:300, Invitrogen). The sections were washed with PBS and stained for DNA with DAPI (1:500, Sigma-Aldrich) for 10 min at room temperature in the dark. The sections were finally washed and mounted with aquapolymount (Polysciences). Optical sections were obtained using a Zeiss Axiocam at 20x or 40x magnification.

[0363] Quantification of immunostaining

[0364] Immunofluorescence images were quantified using ImageJ-Fiji Version 1.53t. For each animal, 3-4 sections were analyzed. Images were separated into individual channels and converted to 8-bit images. The threshold of 8-bit images was set to 5 MFI for cleaved caspase-3 staining and 100 for DAPI staining. The area covered by cleaved caspase-3 / area covered by DAPI represents the percentage of cleaved caspase-3 in the section.

[0365] Quality Control and Stability

[0366] After purification of the single-chain MHC Ib molecules, their stability was analyzed after 1 and 3 freeze-thaw cycles, storage at room temperature for 5 days, and heating to 50°C for 30 minutes. For this purpose, A) Coomassie gel staining was performed in 12% polyacrylamide gel using 2 μg of single-chain MHC-Ib molecules, and B) Western Blot of αHLA-G using 1 μg of protein under non-reducing conditions using 2A12aHLA-G antibody (1:1000). Both monomers and dimers were detected.

[0367] result

[0368] Quality Control and Stability

[0369] Quality control and stability testing showed that both NMO surrogate single-chain MHC Ib molecules were very stable ( Figure 17-19 ). However, AQP203_H2KbG_spt appears to multimerize to a greater extent, which could explain why it performs less well in some assays.

[0370] Treatment Effects

[0371] Notably, treatment with AQP147_KbG completely protected treated mice from EAE ( Fig. 20 ), inhibiting immune cell infiltration in the optic nerve ( Fig.21 ) and completely inhibited apoptosis in the optic nerve, spinal cord, and retina ( Fig. 22 In addition, although the mice treated with AIM had fewer CD8 T cells, the number of regulatory T cells in the optic nerve was small but significantly increased ( Fig.23 In addition, AQP147_KbG completely prevented the degeneration of IRL (inner retinal layer) cells ( Fig.24 ).

[0372] These data demonstrate that the recombinant polypeptides of the present invention and their replacement molecules have therapeutic effects. Therefore, according to the present invention, the recombinant polypeptides of the present invention can be used to treat neuromyelitis optica in human patients.

[0373] Industrial Applicability

[0374] The pharmaceutical compositions, polypeptides, nucleic acids, cells and products used in the present invention have industrial application value, for example, they can be used to manufacture medicines or used as medicines.

Claims

1. A recombinant polypeptide capable of presenting a peptide antigen, the recombinant polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen presented by the recombinant polypeptide, wherein the peptide antigen is a peptide of human aquaporin 4; ii) optionally, a linker sequence; iii) optionally, a human polypeptide domain sequence, the human polypeptide domain sequence comprising a human β2 microglobulin sequence, or an amino acid sequence that is at least 90% identical to the human β2 microglobulin amino acid sequence shown in SEQ ID NO: 5; iv) optionally, a linker sequence; v) optionally, an α1 domain of an MHC molecule; vi) optionally, an α2 domain of an MHC molecule; vii) an α3 domain of an MHC class Ib molecule or a derivative of the α3 domain of an MHC class Ib molecule, said derivative being capable of binding to ILT2 or ILT4; viii) optionally, a protease cleavage site; ix) optionally, a spacer sequence; and x) Optionally, an affinity tag.

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

3. The recombinant polypeptide according to claim 1 or 2, wherein the peptide antigen according to i) consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 2, 22, 23 and 24.

4. The recombinant polypeptide according to any one of the preceding claims, wherein the α1 domain according to (v) and the α2 domain according to (vi) are derived from a human MHC class Ia molecule, preferably a human HLA-A2 molecule, or from a human MHC class Ib molecule, preferably a human HLA-G molecule.

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

6. A recombinant polypeptide according to any of the above claims, wherein the α3 domain or derivative according to (vii) is identical to the α3 domain having the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:21 or has at least 80% amino acid sequence identity, at least 90% amino acid sequence identity, at least 92% amino acid sequence identity, at least 94% amino acid sequence identity, at least 96% amino acid sequence identity, at least 98% amino acid sequence identity, or at least 99% amino acid sequence identity with the α3 domain having the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:21, or is identical to the α3 domain having the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:

21.

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

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

5.

9. The recombinant polypeptide according to any one of the above claims, wherein the polypeptide is a dimer or a multimer.

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

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

12. The recombinant polypeptide according to any one of claims 1 to 10, wherein the recombinant polypeptide consists of the following amino acid sequence, which consists of the following ((a) and (b)) in order from N-terminus to C-terminus: (a) a peptide antigen selected from the group consisting of the amino acid sequences of SEQ ID NOs: 2, 22, 23 and 24, and (b) The amino acid sequence of SEQ ID No:

16.

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

14. A nucleic acid encoding one or more polypeptides according to any one of the preceding claims, wherein the nucleic acid is preferably a vector.

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

16. A pharmaceutical composition or kit comprising at least one recombinant polypeptide according to any one of claims 1-13.

17. The pharmaceutical composition or kit according to claim 16, wherein the pharmaceutical composition or kit comprises at least two different recombinant polypeptides according to any one of claims 1 to 13, and wherein each different polypeptide comprises a different peptide antigen as defined in claim 3.

18. The pharmaceutical composition or kit according to any one of claims 15 to 17, for use in treating neuromyelitis optica in a human patient.

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

20. A pharmaceutical composition or kit for treatment for said use according to any one of claims 18-19, wherein said treatment is for reducing the level of autoantibodies against human aquaporin-4 in plasma or cerebrospinal fluid, and wherein said human patient is a patient having autoantibodies against human aquaporin-4 in plasma or cerebrospinal fluid before the start of treatment.

21. The pharmaceutical composition or kit for use according to any one of claims 18 to 20, wherein the treatment is performed by inducing myelin-specific regulatory T cells.

22. A recombinant host cell comprising the nucleic acid or vector according to claim 14, and expressing the recombinant polypeptide according to any one of claims 1-13.

23. A method for obtaining a pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 13, the method comprising the following steps: (a) culturing the recombinant host cell of claim 22 under conditions that allow expression of the recombinant polypeptide from the nucleic acid molecule, (b) recovering the recombinant polypeptide, (c) purifying the recombinant polypeptide, and (d) formulating the recombinant polypeptide into a pharmaceutical composition.

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