MHC Ib-mediated aquaporin 4 (AQP4)-specific immunosuppression as a novel treatment for NMO

Abstract

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

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 neuromyelitis optica (NMO). 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 producing such recombinant polypeptides, pharmaceutical compositions comprising such recombinant polypeptides, and their use in the treatment of neuromyelitis optica (NMO). Background Technology

[0002] Neuromyelitis optica (NMO) is a demyelinating autoimmune disease of the central nervous system (CNS) with an incidence between 0.05 / 100,000 and 0.4 / 100,000. It is characterized by symptoms similar to those of multiple sclerosis (MS). However, while MS is typically characterized by a relapse-remission process, patients with NMO rarely experience remission. Furthermore, some treatments beneficial to MS patients are ineffective or may even worsen NMO. Therefore, NMO is more difficult to treat than MS and usually progresses more rapidly. Current treatment strategies for NMO primarily focus on preventing relapses or treating acute relapses. Acute relapses are typically treated with immunosuppressive therapies, such as corticosteroids or plasma exchange, to clear autoreactive antibodies. In recent years, therapeutic antibodies that inhibit the complement system (eculizumab) or block inflammatory cytokines (satralizumab) have also been approved.

[0003] However, not only is the autoreactive immune function suppressed, but the protective immune function is also suppressed. These measures are crucial for protecting patients from viruses, bacteria, or tumors. Therefore, it is often possible to target only a portion of the immune system. Because autoimmune diseases typically worsen after repeated relapses, considerable effort has been devoted to preventing relapses. However, the overall progression of these autoimmune diseases is slow, and can only be slowed, not stopped. To overcome this predicament, attempts have been made for some time to induce immune tolerance to antigens that attack autoreactive immune cells (antigen-specific immunosuppression, ASI).

[0004] 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.

[0005] WO2018 / 215340 relates to combinations of immunomodulatory MHC class Ib molecules and peptides for targeted therapy.

[0006] In conclusion, there is still a need to improve medications for treating neuromyelitis optica (NMO). Summary of the Invention

[0007] 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.

[0008] 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... bThe constructs of the α1 and α2 domains can present the ovalbumin-derived peptide SIINFEKL (SEQ ID NO 81) to OT-1 T cells. (OT-1 T cells express a transgenic T cell receptor that specifically recognizes this antigen) (WO2018 / 215340).

[0009] Surprisingly, the inventors discovered that by using the recombinant peptides of the present invention, they could suppress the immune response against neuroinflammatory autoantigens and induce cells resistant to human aquaporin 4 (AQP4). Furthermore, using established neuroinflammatory disease models, the inventors demonstrated that alternative molecules of these peptides (suitable for mice) could be used to treat neuroinflammatory diseases. Therefore, according to the present invention, neuromyelitis optica (NMO) can be treated with the recombinant peptides of the present invention.

[0010] 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.

[0011] Furthermore, according to the present invention, the recombinant peptide not only regulates T cell responses but also inhibits the formation of aquaporin 4 (AQP4)-specific autoantibodies. This advantage is expected to translate into clinical improvement in patients with neuromyelitis optica (NMO), as AQP4-specific autoantibodies are involved in the pathological process of NMO.

[0012] Sequencing of peptide antigens dissociated from MHC molecules revealed MHC allele-specific motifs characterized by a preference for specific amino acids at specific positions. These amino acids anchor the presented peptide to the presentation cleft. Replacing less favorable amino acids in the MHC anchoring residues with more favorable amino acids increases the affinity between the epitope and MHC. Consequently, the peptide-MHC complex becomes more stable. Notably, the anchored amino acids do not contact the homologous T cell receptor at the peptide-MHC / T cell receptor interface. Therefore, anchoring residue optimization can improve T cell stimulation (by stabilizing the peptide-MHC complex) without altering the pool of stimulated T cells. The inventors have discovered that optimizing anchoring residues can effectively improve the peptides of the present invention, for example, by increasing peptide production levels.

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

[0014] 1. A recombinant polypeptide capable of presenting a peptide antigen, the recombinant polypeptide comprising, in the order from N-terminus to C-terminus:

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

[0016] ii) Optionally, connector sequence;

[0017] 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;

[0018] iv) Optionally, connector sequence;

[0019] v) The α1 domain of the MHC molecule;

[0020] vi) The α2 domain of the MHC molecule;

[0021] vii) The α3 domain of an MHC Ib molecule or a derivative thereof, wherein the derivative is capable of binding to ILT2 or ILT4.

[0022] viii) Optionally, protease cleavage sites;

[0023] ix) Optionally, an interval sequence; and

[0024] x) Optional, affinity label.

[0025] 2. The recombinant polypeptide according to item 1, wherein the length of the peptide antigen according to i) is 7 to 11 amino acids, preferably 8 to 10 amino acids.

[0026] 3. The recombinant polypeptide according to claim 1 or 2, wherein the peptide antigen comprises, according to i), an amino acid sequence selected from the amino acid sequence group of SEQ ID NO34, 42-51 and 82.

[0027] 4. The recombinant polypeptide according to any one of items 1-3, wherein the peptide antigen according to item i) consists of the amino acid sequence of SEQ ID NO 34.

[0028] 5. The recombinant polypeptide according to any one of items 1-3, wherein the peptide antigen according to item i) consists of the amino acid sequence of SEQ ID NO 48.

[0029] 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, preferably human HLA-A2 molecules, or derived from human MHC class Ib molecules, preferably human HLA-G molecules.

[0030] 7. 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, preferably the α3 domain of human HLA-G.

[0031] 8. The recombinant polypeptide according to any one of the preceding claims, wherein the α3 domain or a derivative thereof according to (vii) has at least 80%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% amino acid sequence identity with the α3 domain having the amino acid sequence of SEQ ID NO 9, SEQ ID NO 21, or SEQ ID NO 53, or is identical to the α3 domain having the amino acid sequence of SEQ ID NO 9, SEQ ID NO 21, or SEQ ID NO 53.

[0032] 9. The recombinant polypeptide according to any one of items 1-8, 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.

[0033] 10. The recombinant polypeptide according to any one of items 1-8, wherein the α3 domain or derivative according to (vii) is identical to the α3 domain having the amino acid sequence of SEQ ID NO 21.

[0034] 11. The recombinant polypeptide according to any one of items 1-8, wherein the α3 domain or derivative according to (vii) is identical to the α3 domain having the amino acid sequence of SEQ ID NO 53.

[0035] 12. The recombinant polypeptide according to any of the preceding claims, wherein the linker sequence according to claim (ii) and / or the linker sequence according to claim (iv) comprises an amino acid sequence (GGGGS)n, wherein n is an integer greater than or equal to 1, and wherein n is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, preferably an integer selected from 2, 3, 4 and 5.

[0036] 13. 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.

[0037] 14. The recombinant polypeptide according to any one of the preceding claims, wherein the polypeptide is a dimer or a polymer.

[0038] 15. The recombinant polypeptide according to any of the preceding claims, wherein the polypeptide comprises or components i) to vii) are composed of all components i) to vii), wherein the polypeptide does not contain components viii) to x), or wherein the polypeptide comprises or is composed of all components i) to x).

[0039] 16. The recombinant polypeptide according to any of the preceding claims further comprises an N-terminal secretion signal peptide sequence.

[0040] 17. A recombinant polypeptide according to any one of claims 1-15, wherein the recombinant polypeptide consists of an amino acid sequence comprising, in order from N-terminus to C-terminus, the following ((a) and (b)):

[0041] (a) A peptide antigen selected from the amino acid sequence group of SEQ ID NO 34, 42-51 and 82,

[0042] (b) The amino acid sequence of SEQ ID NO 16 or SEQ ID NO 55-60.

[0043] 18. The recombinant polypeptide according to any one of the preceding claims, wherein the recombinant polypeptide is soluble.

[0044] 19. A nucleic acid encoding one or more polypeptides according to any one of the preceding claims, wherein the nucleic acid is preferably a carrier.

[0045] 20. A pharmaceutical composition or kit comprising at least one nucleic acid as described in claim 19.

[0046] 21. A pharmaceutical composition or kit comprising at least one recombinant polypeptide according to any one of claims 1-18.

[0047] 22. The pharmaceutical composition or kit according to claim 21, wherein the pharmaceutical composition or kit comprises at least two different recombinant polypeptides according to any one of claims 1-18, wherein each different polypeptide comprises a different peptide antigen as defined in claim 3.

[0048] 23. A pharmaceutical composition or kit according to any one of claims 20-22 for treating neuromyelitis optica in human patients.

[0049] 24. A pharmaceutical composition or kit for the use described in claim 23, wherein the treatment method is immunotherapy, and the treatment method is preferably achieved by inducing immune tolerance to human aquaporin 4.

[0050] 25. A pharmaceutical composition or kit for use according to any one of items 23-24, wherein the treatment is to reduce the level of autoantibodies against human aquaporin 4 in plasma or cerebrospinal fluid, and wherein the human patient is a patient who has autoantibodies against human aquaporin 4 in plasma or cerebrospinal fluid prior to the start of treatment.

[0051] 26. A pharmaceutical composition or kit for the use described in any one of items 23-25, wherein the treatment is performed by inducing myelin-specific regulatory T cells.

[0052] 27. A recombinant host cell comprising the nucleic acid or vector according to claim 32 and expressing a recombinant polypeptide according to any one of claims 1-18.

[0053] 28. A method for obtaining a pharmaceutical composition comprising a polypeptide according to any one of claims 1-18, the method comprising the steps of: (a) culturing a recombinant host cell of claim 27 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

[0054] Figure 1 : Description of soluble MHC Ib molecules of loaded peptides suitable for achieving therapeutic antigen-specific immunomodulation.

[0055] 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., Proc NatlAcad Sci US A. 2005 Mar 1;102(9):3360-5 and Hansen et al., Trends Immunol. 2010 Oct;31(10):363-9.

[0056] Figure 2Examples of vector-based constructs encoding single-chain MHC Ib molecules suitable for therapeutic peptide-specific immunomodulation.

[0057] 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 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 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 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.

[0058] Figure 3 The alternative molecules of the recombinant polypeptide of the present invention induce Treg secretion of IL10 in mice.

[0059] 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 Composed of α1 and α2 domains, as well as the human HLA-G α3 domain and β-2-microglobulin, the cells were injected intraperitoneally (ip) into 12-week-old C57BL / 6 mice. Fourteen days later, the mice were sacrificed, and splenocytes were restimulated with 5 µg / ml Gp34 or Ova peptide in a standard mouse IL-10 ELIspot assay at 48 hours (Mabtech Mouse IL-10 HRP ELISpot Kit, cells cultured in RPMI 10% FCS 10 ng / ml IL2).

[0060] A significant increase in regulatory T cells was detected, which secrete IL-10 only upon induction of tolerance by alternative molecular injection and in response to re-stimulation by peptides targeting them.

[0061] (A) Experimental design; (B) Results https: / / en.wikipedia.org / wiki / C57BL / 6

[0062] Figure 4 The recombinant polypeptide of this invention can prevent EAE induced by CD8+ T cells in mice.

[0063] In this EAE mouse model, oligodendrocytes express the model antigen ovalbumin (OVA) under the control of the myelin basic protein (MBP) promoter (ODC-OVA). This leads to 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, 500 µg 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 Vis Exp. 2014 Apr 15;(86):51275.

[0064] Only alternative molecules that induce ovalbumin tolerance can almost completely prevent EAE symptoms. (A) Experimental design; (B) Results

[0065] Figure 5 The present invention provides alternative molecules for some recombinant peptides that can selectively prevent CD4 in mice. + EAE caused by T cells.

[0066] In this model, a strong myelin-specific autoimmune response was induced by administration of MOG 35-55 peptides 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 a 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.

[0067] Mog44 peptide, containing a substitute molecule, significantly alleviated EAE symptoms and weight loss.

[0068] (A) Experimental design; (B) EAE score; (C) Body weight

[0069] Figure 6 The Mog44 substitution molecule of the recombinant polypeptide of the present invention can prevent spinal cord inflammation and CD8 T cell infiltration.

[0070] 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.

[0071] 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.

[0072] (A) Toluidine; (B) CD8-DAB

[0073] Figure 7 Detection of anti-MOG35-55 antibody (“AIM Bio”) in Mog-EAE mice treated with the recombinant peptide of this invention.

[0074] Mice were euthanized, and serum was collected via cardiac puncture. The mice were coated overnight with 10 µg / ml Mog35-55, wells were blocked with 1% BSA, and anti-Mog35-55 antibodies were detected using the specified HRP-conjugated secondary antibody.

[0075] Mog35-55-induced EAE is associated with high levels of Mog35-55-specific IgG autoantibodies, which are undetectable in animals treated with 100 µg of MOG44_Db_G as a replacement molecule.

[0076] Figure 8 List of examples of human NMO recombinant peptide candidates.

[0077] The exemplary peptide antigen sequences of the NMO recombinant polypeptide candidates shown in the figure are as follows:

[0078] Construct peptide antigen SEQ ID NO

[0079] hAQP4 42-50_HLAG Myc / His label FLAMLIFVLSEQ ID NO 31

[0080] hAQP4 45-53_HLAG Myc / His label MLIFVLLSLSEQ ID NO 32

[0081] hAQP4 65-72_HLAG Myc / His label PLPVDMVLSEQ ID NO 33

[0082] hAQP4 71-79_HLAG Myc / His label VLISLCFGLSEQ ID NO 22

[0083] hAQP4 126-135_HLAG Myc / His label AIIGAGILYLSEQ ID NO 34

[0084] hAQP4 127-135_HLAG Myc / His label IIGAGILYLSEQ ID NO 23

[0085] hAQP4 45-53_A2G Myc / His tag MLIFVLLSLSEQ ID NO 32

[0086] hAQP4 65-72_A2G Myc / His tag PLPVDMVLSEQ ID NO 33

[0087] hAQP4 71-79_A2G Myc / His tag VLISLCFGLSEQ ID NO 22

[0088] hAQP4 126-135_A2G Myc / His tag AIIGAGILYLSEQ ID NO 34

[0089] hAQP4 127-135_A2G Myc / His tag IIGAGILYLSEQ ID NO 23

[0090] hAQP4 156-164_A2G Myc / His tag AGHGLLVELSEQ ID NO 35

[0091] hAQP4 238-247_A2G Myc / His tag IIGAVLAGGLSEQ ID NO 24

[0092] hAQP4 45-HLAG SPOT tag MLIFVLLSLSEQ ID NO 32

[0093] hAQP4 36-43_HLAG SPOT tag KAVTAEFL SEQ ID NO 36

[0094] hAQP4 71-79_HLAG SPOT tag VLISLCFGLSEQ ID NO 22

[0095] hAQP4 64-72_HLAG SPOT tag KPLPVDMVL SEQ ID NO 2

[0096] hAQP4 71-79_A2G SPOT tag VLISLCFGLSEQ ID NO 22

[0097] hAQP4 127-A2G SPOT tag IIGAGILYLSEQ ID NO 23

[0098] hAQP4 238-A2G SPOT tag IIGAVLAGGLSEQ ID NO 24

[0099] hAQP4 137-145_HLAG SPOT tag VTPPSVVGGLSEQ ID NO 37

[0100] Figure 8Further analysis revealed which combinations of the AQP4 peptide and antigen-presenting MHC class I α1 and α2 domains (HLAg = HLA-G, A2G = HLA-A2 presenting domain + HLA-Gα3 domain) yielded successful (high), moderate (average, av.), or (low) results in production, quality control, and priority of healthy blood donors (e.g.). Figure 9 (as described).

[0101] Figure 9 The recombinant polypeptide containing KPLPVDMVL antigen (“AQP_64”) of this invention can upregulate CD8 Treg cells in healthy blood donors.

[0102] In vitro Treg induction was performed using the peptide-HLA-G construct (AIM Biologicals). The specific steps were as follows: 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 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 PBS, followed by the addition of 200 µl blocking buffer (X-VIVO 15 5% hAB serum), and incubated at room temperature for 30 min–2 h. On day 14, 200,000 cells were seeded into each well of the ELISpot plate in duplicate, including a negative control (cells plus PBS) and a positive control (e.g., LPS) for 48 h. 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. The cell supernatant was removed and the cells were washed 5 times with 100 µl PBS. Excess buffer was removed with paper. 25 µl of diluted HRP-streptavidin (1:750) was added to each well and incubated at room temperature in the dark for 1 h, followed by washing 5 times with sterile 1×PBS. 100 µl of filtered TMB substrate was added to each well and allowed to stand for 15–25 min until blue spots appeared. The wells were thoroughly rinsed with tap water to stop the reaction. The plastic drain tube of the plate was removed, and the bottom and sides of the plate were rinsed with tap water and allowed to air dry.

[0107] AQP4_64_G_Spt induced at least a 30% increase in Tregs secreting IL-10 in 65% of healthy blood donors.

[0108] Figure 9 (continued) The percentage increase in IL10 spots in PBMCs is shown as HLA-A2. + and HLA-A2 - Donor (response to AQP4_64_G_Spt treatment).

[0109] Figure 10 : Figure 7 The control experiment of the sample shown indicates that MOG47_Db_G substitution molecule treatment does not reduce total IgG content. Total IgG was quantified using the Easy-Titer™ Human IgG (γ-chain) Detection Kit (Thermo Fisher) according to the manufacturer's instructions. This experiment is consistent with... Figure 7 This combination suggests that single-chain MHC Ib molecules can be used to inhibit selective antibody responses.

[0110] Figure 11 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.

[0111] Figure 12 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).

[0112] Figure 13 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, as well as human β-2-microglobulin and the specified peptide, were constructed or carried in PBS. Ova is a homologous peptide of OT-I TCR in these mice, and Gp34 is 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.

[0113] Figure 14Single-chain MHC Ib molecules inhibit T cell lysis in a dose-dependent manner. 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 dose-escalating Ova_KbG). Cells were then scraped from the culture dish and CD8+ cells were purified using magnetic beads. Sterile 96-well white culture plates were used. Panc02 target cells expressing luciferase were incubated with 20 µg / ml Ova peptide (SIINFEKL) at 37°C. ℃ Loading was performed by shaking at 500 rpm for 60 minutes. CD8+ effector T cells and luciferin were added at a ratio of 50:1. The luminescence intensity was measured at 24 and 48 hours, respectively.

[0114] Figure 15 Serum 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 eight capture microbeads with different fluorescence intensities, each coated with a specific capture antibody against each cytokine. Magnetic beads coated with ten specific capture antibodies were mixed. Subsequently, 25 μL of the mixed capture beads, 25 μL of unknown serum sample or standard dilution, and 25 μL of phycoerythrin (PE) detection reagent were 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).

[0115] Figure 16 Example: Molecular composition diagram of mouse-fit AIM Bio.

[0116] The corresponding exemplary amino acid sequence is as follows:

[0117] MSRSVALAVLALLSLSGLEAVTTVHGNLGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKD EYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRV DLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDPPKTHVTHHPVF DYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC (SEQ ID NO 38)

[0118] Figure 17 Coomassie brilliant blue gel electrophoresis was performed on purified, stability-tested NMO-substituted single-chain MHC Ib molecules. After purification, the stability of the single-chain MHC Ib molecules was analyzed after one and three freeze-thaw cycles, storage at room temperature for 5 days, and heating at 50°C for 30 minutes. Methods are described in [link to method]. Figure 11 .

[0119] Figure 18 Western blot analysis (2A12HLA-G5 antibody 1:5000) was performed on purified and stability-tested NMO-substituted single-chain MHC Ib molecules. Both experiments showed that these molecules were very stable, but AQP203_H2KbG was more prone to polymerization.

[0120] Figure 19 Thermal displacement analysis (TSA). The results obtained in the previous figure are also confirmed in the TSA, where the polymer exhibits high autofluorescence at low temperatures.

[0121] Figure 20The AQP147_KbG score completely protected 2D2 mice from EAE. Mice were injected with either the treatment or control AIM Bios on days 0, 15, and 30. They were assessed daily using the 0-10 EAE scoring system described in Table 1. Untreated mice developed severe EAE symptoms during the experiment. One untreated mouse reached a score of 6 on day 14 and was euthanized. Its score was assumed to be 6 for the remainder of the assessment period. Control treatment with gp34_KbG did not provide the expected protection. (*p<0.05, **p<0.01, ***p<0.001)

[0122] Figure 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. Figure A (optic nerve) and Figure B (spinal cord) show CD3 levels obtained from each treatment group. + and CD8 + Fluorescence signal. DAPI images show the total number of cell nuclei present in each slice. CD3 in the optic nerve (Fig. C) and spinal cord (Fig. D) of control and untreated mice. + and CD8 + The number of T cells was significantly higher. AQP203_KbG was more effective than the control and untreated groups in inhibiting immune infiltration. Data are expressed as mean ± standard error. Scale bar: 25 micrometers. One-tailed t-test.

[0123] Figure 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. Fluorescence signals obtained from staining for cleaved caspase-3 (an apoptosis marker) in the optic nerve (Fig. A, B), spinal cord (Fig. C, D), and retina (Fig. E, F) are shown. Untreated and control mice had the highest levels of cleaved-casp-3, and therefore the highest levels of apoptosis. Data are expressed as mean ± standard error. Scale bar: 25 μm. One-tailed t-test.

[0124] Figure 23 CD8 in the optic nerve + CD122 + and CD4 + FoxP3 + Regulatory T cells. The figure shows that although the number of CD8 T cells in mice treated with AIM was low, the number of regulatory T cells in the optic nerve, though low, was significantly higher (using a one-tailed T test).

[0125] Figure 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 of AQP147_KbG mice was preserved, while the inner retinal thickness of untreated and control mice was thinner. Data are expressed as mean ± standard error. Scale bar 25 μm. Two-tailed t-test.

[0126] Figure 25 A, B, D: Simple Western blotting results; C, E: Supernatant concentrations measured in ELISA.

[0127] The constructs used in this figure are described in the "Sequence" section below and in Example 8. Simple Western blotting was performed as described in Example 8; sandwich ELISA was performed as described in Example 8.

[0128] Figure 26 ELISpot assay was performed using peripheral blood mononuclear cells from the patient. A: Images of each well during the assay. The image shows the number of spots counted. B: Quantitative analysis.

[0129] ELISpot was performed as described in Example 8.

[0130] Compared to untreated PBMCs, those treated with AIM Bio AQP65 showed better results. P65K Of the 5 NMO patients treated with _G, 4 had increased IL-10 spots in their PBMCs.

[0131] Figure 27 Residue optimization of anchor hAQPA_HLA-G AIM Bios.

[0132] A: HLA-G peptide binding motif

[0133] B: Di Marco et al. described HLA-G binding peptide motifs (Moreno Di Marco, Heiko Schuster, Linus Backert, Michael Ghosh, Hans-Georg Rammensee, Stefan Stevanović; Revealing HLA-C and HLA-G peptide motifs from natively presented peptides and generating binding prediction matrices. J Immunol 2017 Oct 15; 199(8): 2639–2651. https: / / doi.org / 10.4049 / jimmunol.1700938). Their analysis showed that certain amino acids are located at specific positions that facilitate anchoring the peptide chain in the grooves formed by the α1 and α2 domains of native HLA-G. The inventors believe that these findings provide a basis for optimizing the recombinant peptides of the present invention. The inventors found that anchoring residue optimization of the peptides of the present invention (also referred to herein as “AIM Bios”) is advantageous and can produce more stable molecules and improve production levels.

[0134] Figure 28 Purified AQP4 126-135 Size exclusion chromatography analysis of A2G

[0135] Figure 29 Thermal displacement analysis: AIM Bio AQP 126-135 Melting curve analysis of _A2G.

[0136] Figure 30 According to AQP 65-72_P65K _G and AQP 65-72_P65R Optimization of _G anchor residues using Jess SimpleWestern manipulation in AIM Bio.

[0137] Figure 31 ILT4-Fc combined with ELISA Invention Details

[0139] Definitions and General Techniques

[0140] 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. References may be cited by way of their full reference numbers in the text.

[0141] 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.

[0142] 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).

[0143] 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).

[0144] Similarly, it should be understood that the recombinant polypeptides of the present invention are intended to optionally include the corresponding propeptides.

[0145] 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.

[0146] 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.

[0147] According to the present invention, the MHC molecule is preferably a human MHC molecule.

[0148] The recombinant polypeptide of the present invention is preferably an isolated recombinant polypeptide.

[0149] 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.

[0150] According to the present invention, the recombinant peptides, pharmaceutical compositions and kits of the present invention are preferably suitable for human patients.

[0151] According to the present invention, the recombinant peptides, pharmaceutical compositions and kits of the present invention are preferably suitable for treating neuromyelitis optica in human patients.

[0152] According to the present invention, the recombinant peptides, pharmaceutical compositions and kits of the present invention are preferably suitable for inducing immune tolerance to human aquaporin 4, for example in human patients.

[0153] 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 for the treatment of neuromyelitis optica (NMO) in HLA-A2 positive human patients. This use is expected to have a more favorable (lower immunogenicity) immunogenicity profile compared to HLA-A2 negative patients.

[0154] 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.

[0155] It should be understood that, according to the present invention, the recombinant peptides, pharmaceutical compositions and kits of the present invention are stable.

[0156] 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).

[0157] According to the present invention, each occurrence of the term "comprising" can be selectively replaced with the term "consisting of".

[0158] Methods and Techniques

[0159] 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.

[0160] 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.

[0161] 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 performed at 25°C. Suitable 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.

[0162] The sequence alignment according to the present invention is performed using the BLAST algorithm (see Altschul et al. (1990) "Basic local alignment search tool." Journal of Molecular Biology 215. pp. 403-410.; Altschul et al. (1997) Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acid Research 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 extension; conditional combination score matrix adjustment. Therefore, when using sequence-related terms such as "identity" or "same," it preferably refers to the identity value obtained using the BLAST algorithm.

[0163] Preparation of the pharmaceutical composition of the present invention

[0164] The pharmaceutical compositions according to the present invention are prepared according to known standards for the preparation of pharmaceutical compositions.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] peptide antigens according to the present invention

[0169] 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.

[0170] Peptides that can be presented on MHC molecules can be generated using methods known in the art (e.g., see Rammensee, Bachmann, Emmerich, Bachor, Stevanović. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics, Nov 1999; 50(3-4):213-9; Pearson et al., MHC class I-associated peptides derive from selective regions of the human genome. Journal of Clinical Research, Nov 1216; 126(12):4690-4701; and Rock, Reits, Neefjes, Presentation of yourself via MHC class I and MHC class II molecules, Trends in Immunology, Nov 2016; 37(11):724-737).

[0171] 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.

[0172] 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:

[0173] Rammensee, Bachmann, Emmerich, Bachor, Stevanović. SYFPEITHI:database for MHC ligands and peptide motifs.Immunogenetics.1999 Nov;50(3-4):213-9;

[0174] 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

[0175] Rock, Reits, Neefjes.Present Yourself! By MHC Class I and MHC ClassII Molecules.Trends Immunol.2016 Nov;37(11):724-737.

[0176] These methods include experimental methods and methods for predicting peptide-antigen binding.

[0177] 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.

[0178] 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.

[0179] Preferably, such predictions are made according to the descriptions in any of the following publications:

[0180] - Rammensee et al., SYFPEITHI: MHC ligand and peptide motif database. Immunogenetics (1999) 50: 213-219

[0181] - Nielsen et al, Protein Sci (2003) 12:1007-1017

[0182] - Neefjes et al. Nat Rev Immunol. 2011 Nov 11;11(12):823-36

[0183] - Diehl et al. Curr Biol. March 1, 1996; 6(3):305-14,

[0184] - Lee et al. Immunity. November 1995; November 1995; 3(5):591-600.

[0185] - Desai & Kulkarni-Kale, T-cell epitope prediction methods: anoverview. Methods Mol Biol. 2014;1184:333-64.

[0186] - Jumper et al. Highly accurate protein structure prediction withAlphaFold. Nature 2021; 596: 583–589

[0187] In this invention, the peptide antigen is derived from human aquaporin 4.

[0188] It should be understood that the non-anchored amino acid residues of the peptide antigen of the present invention may or may not contain conserved substitutions relative to the corresponding amino acid sequence of the peptide antigen of human aquaporin 4, preferably no more than two conserved substitutions, and more preferably only one conserved substitution.

[0189] 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 antigen of the present invention comprises a peptide mimic of the indicated peptide antigen amino acid sequence of human aquaporin 4.

[0190] Methods for synthesizing peptide antigens, including methods for synthesizing peptide antigens according to the present invention, are well known in the art.

[0191] Therapeutic applications of the present invention

[0192] The recombinant polypeptide of this invention can be used to treat neuromyelitis optica.

[0193] 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.

[0194] 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. See, for example:

[0195] Junfeng Liu, Dacan Chen, Golay D. Nie, and Zhenhua Dai, A Newly EmergingRegulator with Central Memory Cell Phenotypes, Front. Immunol. doi: 10.3389 / fimmu.2015.00494; and Niederlova, V., Tsyklauri, O., Chadimova, T., and Stepanek, O. (2021), CD8 + Tregs revisited: A heterogeneous population with different phenotypes and properties, Eur. J. Immunol., 51: 512-530. https: / / doi.org / 10.1002 / eji.202048614

[0196] 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:

[0197] 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).

[0198] The treatment method described in this invention can be used to reduce the level of autoantibodies against human aquaporin 4 in plasma or cerebrospinal fluid (CSF). Human patients can be those who have autoantibodies against human aquaporin 4 in their plasma or CSF prior to the start of treatment.

[0199] 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 (e.g., aquaporin 4) of the autoantibody 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 flow cytometry (CBA-FACS) or by visual scoring of immunofluorescence under a microscope (CBA-IF), typically requiring titration. Other methods, such as enzyme-linked immunosorbent assay (ELISA) or Western blotting, are also feasible, but are generally less sensitive because conformationally sensitive antibodies may not be detectable by these methods. Suitable methods have been described in the following aspects:

[0200] Waters, P., Pettingill, P., and Lang, B, Detection methods for neuralautoantibodies. Handb. Clin. Neurol. 133, 147–163 (2016).

[0201] A method for detecting Aqp4-specific autoantibodies is described in Lennon, VA, Kryzer, TJ, Pittock, SJ, Verkman, AS & Hinson, SR IgG marker of optic-spinal multiple sclerosis binds to the aquaporin-4 water channel. J. Exp. Med. 202, 473–477 (2005).

[0202] sequence

[0203] 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.

[0204] Exemplary sequences of the recombinant polypeptides of this invention:

[0205] Optional lead peptides (not present in the recombinant peptide due to processing during cell expression): for example, MSRSVALAVLALLSLSGLEA (SEQ ID NO 1), encoded by the DNA sequence: ATGAGCAGATCTGTGGCCCTGGCTGTTCTGGCTCTGCTGTCTCTGTCTGGCCTGGAAGCC (SEQ ID NO 41)

[0206] Optional alternative lead peptides:

[0207] For example, mouse immunoglobulin heavy chain peptide: MGWSCIILFLVATATGVHS (SEQ ID NO 88)

[0208] For example, human OSM peptide: MGVLLTQRTLLSLVLALLFPSMASM (SEQ ID NO 89)

[0209] For example, VSV-G peptide: MKCLLYLAFLFIGVNC (SEQ ID NO 90)

[0210] For example, mouse Ig Kappa peptide: METDTLLLWVLLLWVPGSTGD (SEQ ID NO 91)

[0211] For example, BM40 peptide: MRAWIFFLLCLAGRALA (SEQ ID NO 92)

[0212] For example, the Secreton peptide: MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO 93)

[0213] For example, the human IgKVIII peptide: MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO 94)

[0214] For example, the CD33 peptide: MPLLLLLPLLWAGALA (SEQ ID NO 95)

[0215] For example, tPA peptide: MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO 96)

[0216] For example, human chymotrypsinogen peptide: MAFLWLLSCWALLGTTFG (SEQ ID NO 97)

[0217] For example: Human trypsinogen-2 peptide: MNLLLILTFVAAAVA (SEQ ID NO 98)

[0218] For example, human IL-2 peptide: MYRMQLLSCIALSLALVTNS (SEQ ID NO 99)

[0219] For example, gaussin peptide: MGVKVLFALICIAVAEA (SEQ ID NO 100)

[0220] For example, albumin (HSA) peptide: MKWVTFISLLFSSAYS (SEQ ID NO 101)

[0221] For example, influenza hemagglutinin peptide: MKTIIALSYIFCLVLG (SEQ ID NO 102)

[0222] For example: Human insulin peptide: MALWMRLLPLLALLALWGPDPAAA (SEQ ID NO 103)

[0223] For example, silk fibroin LC peptide: MKPIFLVLLVVTSAYA (SEQ ID NO 104)

[0224] Peptide antigen: Any MHC class I peptide corresponding to the α1&2 domain of MHC class I, such as KPLPVDMVL (SEQ ID NO 2), or any peptide antigen according to the present invention (all derived from human AQP4):

[0225] hAQP4 126-135_A2G Myc / His tag AIIGAGILYL SEQ ID NO 34

[0226] AQP4_111-119_A2G_fs-_Spt SIAKSVFYISEQ ID NO42

[0227] AQP4_242-251_A2G_fs-_Spt VLAGGLYEYV SEQ ID NO 43

[0228] AQP4_292-301_A2G_fs-_Spt LILKPGVVHV SEQ ID NO 44

[0229] AQP4_293-301_A2G_fs-_Spt ILKPGVVHV SEQ ID NO 45

[0230] AQP4_210-218_A2G_fs-_Spt ASMNPARSF SEQ ID NO 46

[0231] AQP134-142_A2G_fs-_Spt YLVTPPSVV SEQ ID NO 82

[0232] AQP4_293-301_G_fs-_Spt ILKPGVVHV SEQ ID NO 47

[0233] AQP4_65-72_P65K_G_fs-_Spt KLPVDMVL SEQ ID NO 48

[0234] AQP4_65-72_P65R_G_fs-_Spt RLPVDMVL SEQ ID NO 49

[0235] AQP4_126-134_A126R_G_fs-_Spt RIIGAGILY SEQ ID NO 50

[0236] AQP4_164-172_L164R_F172L_G_fs-_Spt RIITFQLVL SEQ ID NO 51

[0237] First connector: for example, GGGGSGGGGSGGGGS (SEQ ID NO 3) or GCGASGGGSGGGGS (SEQ ID NO 4) or GCGASGGGSGGGGS (SEQ ID NO 52)

[0238] β2 microglobulin, for example:

[0239] IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (SEQ ID NO 5, human β2 microglobulin)

[0240] Second connector, for example:

[0241] GGGGSGGGGSGGGGSGGGGS (SEQ ID NO 6)

[0242] 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 can be C or A, for example, the α1 and α2 domains derived from human HLA-G.

[0243] For example

[0244] GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRA (SEQ ID NO 7)

[0245] Alternatively: derived from human HLA-A2 α1 and α2 domains: for example

[0246] GSHSMRYFFTSVSRPGRGEPRIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRT (SEQ ID NO 8)

[0247] 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; human HLA-G α3 sequence, wherein the C-terminal amino acid sequence of human HLA-G intron 4 contains a furin cleavage site), or

[0248] DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESGSLSEDL (SEQ ID NO 53; 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).

[0249] Note: The following underlined amino acids in this sequence are associated with interactions with ILT2 or ILT4 receptors:

[0250] DPPKTHVTHH PVFDYE ATLRCWALGFYPAEIILTWQRDGEDQTQD V ELVETRPAGDGTFQKWAAV V VPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDL, or

[0251] DPPKTHVTHH PVFDYE ATLRCWALGFYPAEIILTWQRDGEDQTQD V ELVETRPAGDGTFQKWAAV V VPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESGSLSEDL

[0252] Alternatively, a shorter human HLA-G [alpha]3 domain can be used, which lacks an optional C-terminal amino acid sequence from intron 4 (SKEGDGGIMSVRESRSLSEDL; SEQ ID NO 20, or SKEGDGGIMSVRESGSLSEDL; SEQ ID NO 54), i.e.:

[0253] DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO 21).

[0254] 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 SEQ ID NO 53 sequence, 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.

[0255] Xa factor restriction site: IEGRTGTKLGP (SEQ ID NO 10)

[0256] Spot label: PDRVRAVSHWSSC (SEQ ID NO 11)

[0257] Myc tag: EQKLISEEDL (SEQ ID NO 12)

[0258] His tag: HHHHHH* (SEQ ID NO 13)

[0259] Spacer sequences: such as NSAPD (SEQ ID NO: 14) or GS

[0260] The following are exemplary reference peptide antigens that can serve as components of recombinant polypeptides:

[0261] VLISLCFGL (SEQ ID NO 22), preferably in a recombinant polypeptide containing HLA-G α1 and α2 domains.

[0262] IIGAGILYL (SEQ ID NO 23), preferably in recombinant peptides containing HLA-A2 α1 and α2 domains.

[0263] IIGAVLAGGL (SEQ ID NO 24), preferably in a recombinant polypeptide containing HLA-A2 α1 and α2 domains.

[0264] Exemplary peptide antigens that can be used as part of the recombinant polypeptides of the present invention are as follows:

[0265] AIIGAGILYL (SEQ ID NO 34) is preferably found in recombinant peptides containing HLA-A2 α1 and α2 domains and HLA-G α3 domain.

[0266] SIAKSVFYI (SEQ ID NO 42) is preferably found in recombinant peptides containing HLA-A2 α1 and α2 domains and HLA-G α3 domain.

[0267] VLAGGLYEYV (SEQ ID NO 43) is preferably found in recombinant peptides containing HLA-A2 α1 and α2 domains and HLA-G α3 domain.

[0268] LILKPGVVHV (SEQ ID NO 44) is preferably found in recombinant peptides containing HLA-A2 α1 and α2 domains and HLA-G α3 domain.

[0269] ILKPGVVHV (SEQ ID NO 45) is preferably found in recombinant peptides containing HLA-A2 α1 and α2 domains and HLA-G α3 domain.

[0270] ASMNPARSF (SEQ ID NO 46) is preferably found in recombinant peptides containing HLA-A2 α1 and α2 domains and HLA-G α3 domain.

[0271] YLVTPPSVV (SEQ ID NO 82) is preferably found in recombinant peptides containing HLA-A2 α1 and α2 domains and HLA-G α3 domain.

[0272] ILKPGVVHV (SEQ ID NO 47) is preferably found in recombinant peptides containing α1 and α2 domains and HLA-G α3 domain derived from human HLA-G.

[0273] KLPVDMVL (SEQ ID NO 48) is preferably found in recombinant peptides containing α1 and α2 domains and HLA-G α3 domain derived from human HLA-G.

[0274] RLPVDMVL (SEQ ID NO 49) is preferably found in recombinant peptides containing α1 and α2 domains and HLA-G α3 domain derived from human HLA-G.

[0275] RIIGAGILY (SEQ ID NO 50), preferably in a recombinant polypeptide containing the α1 and α2 domains and the HLA-G α3 domain derived from human HLA-G.

[0276] RIITFQLVL (SEQ ID NO 51), preferably in a recombinant polypeptide containing the α1 and α2 domains and the HLA-G α3 domain derived from human HLA-G.

[0277] Refer to the example of a recombinant peptide (including an optional leader peptide):

[0278] MSRSVALAVLALLSLSGLEAKPLPVDMVLGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTE KDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTD RMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHH PVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC* (SEQ ID NO: 15; note, asterisk indicates stop codon)

[0279] Please note that the peptide antigen (here: KPLPVDMVL) sequence of the full-length recombinant polypeptide described above can be replaced by any peptide antigen sequence according to the present invention, i.e., 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 consist of the following sequence: this sequence contains a peptide antigen, which is a peptide of human aquaporin 4 (e.g., any one of SEQ ID NO 2, 22, 23 and 24, or preferably any one of SEQ ID NO 34, 42-51 and 82 according to the present invention), followed by the following sequence:

[0280] GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWD RDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGCYNQS EASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC* (SEQ ID NO 16; note that the asterisk indicates a stop codon), or followed by the following sequence

[0281] CGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSE AGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESGSLSEDLGSPDRVRAVSHWSSC* (SEQID NO 55; note that the asterisk indicates a stop codon), or followed by the following sequence

[0282] GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQS EAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESGSLSEDLGSPDRVRAVSHWSSC* (SEQ ID NO 56; note that the asterisk indicates the stop codon), or followed by the following sequence

[0283] GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGGSPDRVRAVSHWSSC* (SEQ ID NO 57; note that the asterisk indicates the stop codon),

[0284] 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:

[0285] CGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAH SQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO 58), or followed by the following sequence

[0286] GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTL SQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAH SQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO 59), or followed by

[0287] GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAH AQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO 60)

[0288] Examples of preferred recombinant peptides of the present invention (optional leader peptide, optional C-terminal amino acid sequence from intron 4, and optional tag not shown in the figure):

[0289] HLA-A2G construct:

[0290] AQP 126-135 _A2G; Optimal Selection

[0291] Protein sequence:

[0292] AIIGAGILYLGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTL SQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAH SQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO 87)

[0293] Encoded by DNA sequences (note that the DNA sequences encoding the N-terminal leader peptide are not shown below):

[0294] GCCATCATCGGAGCCGGCATTCTGTATCTT

[0295] AQP4_111-119_A2G_fs-_Spt

[0296] Protein sequence:

[0297] SIAKSVFYI GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLS QPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQ THRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO 61)

[0298] Encoded by DNA sequences (note that the DNA sequences encoding the N-terminal leader peptide are not shown below):

[0299] AGCATCGCCAAGAGCGTGTTCTACATC

[0300] AQP4_242-251_A2G_fs-_Spt

[0301] Protein sequence:

[0302] VLAGGLYEYV GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTL SQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAH SQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO 63)

[0303] Encoded by DNA sequences (note that the DNA sequences encoding the N-terminal leader peptide are not shown below):

[0304] GTGCTGGCCGGCGGCCTGTACGAGTACGTG

[0305] AQP4_292-301_A2G_fs-_Spt

[0306] Protein sequence:

[0307] LILKPGVVHV GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTL SQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAH SQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO 65)

[0308] Encoded by DNA sequences (note that the DNA sequences encoding the N-terminal leader peptide are not shown below):

[0309] CTGATCCTGAAGCCCGGCGTGGTGCACGTG

[0310] AQP4_293-301_A2G_fs-_Spt

[0311] Protein sequence:

[0312] ILKPGVVHV GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTL SQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAH SQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO 67)

[0313] Encoded by DNA sequences (note that the DNA sequences encoding the N-terminal leader peptide are not shown below):

[0314] ATCCTGAAGCCCGGCGTGGTGCACGTG

[0315] AQP4_210-218_A2G_fs-_Spt - Single Component

[0316] Protein sequence:

[0317] ASMNPARSFGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETR KVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLR RYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO 69)

[0318] Encoded by DNA sequences (note that the DNA sequences encoding the N-terminal leader peptide are not shown below):

[0319] GCCAGCATGAACCCCGCCAGGAGCTTC

[0320] AQP134 - 142_A2G_fs-_Spt

[0321] Protein sequence

[0322] YLVTPPSVVGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO 83)

[0323] DNA sequence

[0324]

[0325] HLA-G construct:

[0326] AQP4_293-301_G_fs-_Spt

[0327] Protein sequence:

[0328] ILKPGVVHV GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAH AQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO 71)

[0329] Encoded by DNA sequences (note that the DNA sequences encoding the N-terminal leader peptide are not shown below):

[0330] ATCCTGAAGCCCGGCGTGGTGCACGTG

[0331] Anchoring residue optimization

[0332] AQP4_65-72_P65K_G_fs-_Spt; Optimal Selection

[0333] Protein sequence:

[0334] KLPVDMVL GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAH AQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO 73)

[0335] Encoded by DNA sequences (note that the DNA sequences encoding the N-terminal leader peptide are not shown below):

[0336] AAACTGCCTGTGGATATGGTGCTCGGATGCGGAGCTAGCGGAGGAGGGAGGTTCTGGAGGAGGGGGTAGTATCCAGCGAACCCCAAAGATTCAGGTGTACTCACGTCACCCCGCTGAGAACGGCAAATCCAACTTCCTGAATTGTTATGTCTCAGGATTTCATCCCTCCGACATCGAAGTGGATCTGCTGAAGAATGGCGAAAGAATTGAGAAAGTGGAACACAGCGACCTGTCTTTCAGTAAGGATTGGTCTTTTTACCTGCTGTACTATACTGAATTCACCCCTACAGAGAAAGACGAATATGCTTGCAGGGTCAACCATGTGACCCTG AGCCAGCCAAAGATCGTCAAATGGGACCGGGATATGGGCGGAGGAGGCTCTGGCGGAGGAGGTTCTGGCGGAGGAGGTAGTGGAGGAGGAGGTTCAGGATCCCACAGCATGAGGTACTTCTCAGCCGCCGTGTCCAGGCCAGGACGAGGAGAGCCTAGGTTCATCGCCATGGGCTATGTCGACGATACCCAGTTCGTGCGCTTTGACTCTGATAGTGCATGTCCTAGGATGGAGCCTCGAGCACCATGGGTGGAACAGGAGGGCCCAGAATACTGGGAGGAAGAGACTCGGAACACCAAGGCTCAC GCACAGACTGATAGAATGAACCTGCAGACCCTGCGCGGCTGCTATAATCAGAGCGAGGCTTCCAGCCATACACTGCAGTGGATGATCGGATGTGACCTGGGCTCTGATGGACGACTGCTGCGTGGGTACGAGCAGTACGCTTATGACGGCAAGGATTATCTGGCACTGAATGAAGACCTGCGAAGTTGGACCGCAGCCGATACAGCTGCACAGATTTCCAAGCGTAAATGCGAGGCAGCTAACGTCGCAGAACAGAGGCGAGCTTACCTGGAGGGGACATGCGTGGAATGGCTGCACAGGTATCTGGAGAATGGCAAGGAAATGCTGCAGCGGGCAGACCCCCCTAAAACACATGTCACTCACCATCCAGTGTTCGATTACGAGGCAACTCTGAGGTGCTGGGCCCTGGGCTTTTACCCTGCCGAGATCATCCTGACCTGGCAGAGGGACGGCGAGGACCAGACCCAGGATGTGGAACTGGTGGAAACCAGACCTGCCGGCGACGGCACCTTCCAGAAATGGGCTGCTGTGGTGGTGCCCTCCGGCGAGGAACAGCGGTACACCTGTCACGTGCAGCACGAGGGACTGCCCGAGCCCCTGATGCTGCGGTGG (SEQ ID NO 74)

[0337] AQP4_65-72_P65R_G_fs-_Spt

[0338] Protein sequence:

[0339] RLPVDMVL GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO 75)

[0340] Encoded by the DNA sequence (note that the DNA sequence encoding the N-terminal leader peptide is not shown below):

[0341] AGGCTGCCCGTGGACATGGTGCTG

[0342] AQP4_126-134_A126R_G_fs-_Spt

[0343] Protein sequence:

[0344] RIIGAGILY GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAH AQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO 77)

[0345] Encoded by DNA sequences (note that the DNA sequences encoding the N-terminal leader peptide are not shown below):

[0346] AGGATCATCGGAGCCGGCATTCTGTAT

[0347] AQP4_164-172_L164R_F172L_G_fs-_Spt

[0348] Protein sequence:

[0349] RIITFQLVL GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAH AQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (SEQ ID NO 79)

[0350] Encoded by DNA sequences (note that the DNA sequences encoding the N-terminal leader peptide are not shown below):

[0351] AGGATCATCACCTTCCAGCTGGTGCTG

[0352] The recombinant polypeptide of the present invention, as shown above, may further comprise any of the following optional components: a leader peptide, a C-terminal amino acid sequence of intron 4 of an HLA-G α3 domain sequence (e.g., SKEGDGGIMSVRESRSLSEDL; SEQ ID NO 20, or SKEGDGGIMSVRESGSLSEDL; SEQ ID NO 54), an optional protease cleavage site, an optional linker (e.g., GS), and / or an optional tag, as shown in the example above.

[0353] 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:

[0354] ILT2:

[0355] MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEETLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (SEQ ID NO 17)

[0356] ILT4:

[0357] MTPIVTVLICLGLSLGPRTHVQTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGAYPKPTLSAQPSPVVT SGGRVTLQCESQVAFGGFILKEGEEEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPGVSKKPSLSVQPGPVVAPGESTLTLQCVSDVGYDRFVLYKEGERDLRQLPGR QPQAGLSQANFTLGPVSRSYGGQYRCYGAHNLSSECSAPSDPLDILITGQIRGTPFISVQPGPTVASGENVTLLCQSWRQFHTFLLTKAGAADAPLRLRSIHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSDPY LLSHPSEPLELVVSGPSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVVLLLLLLLLLLFLILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKDTQ PEDGVEMDTRAAASEAPQDVTYAQLHSLTLRRKATEPPPSQEREPPAEPSIYATLAIH (SEQ ID NO 18)

[0358] The sequence of human aquaporin 4 is known in the art. The preferred amino acid sequence of human aquaporin 4 is as follows:

[0359] >sp|P55087-2|AQP4_HUMAN Aquaporin 4 isoform 1 OS=Homo sapiens OX=9606 GN=AQP4

[0360] MVAFKGVWTQAFWKAVTAEFLAMLIFVLLSLGSTINWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV

[0361] (SEQ ID NO 19)

[0362] >sp|P55087|AQP4_HUMAN Aquaporin-4 isoform 2 OS=Homo sapiens OX=9606 GN=AQP4 PE=1 SV=2

[0363] MSDRPTARRWGKCGPLCTRENIMVAFKGVWTQAFWKAVTAEFLAMLIFVLLSLGSTINWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSV

[0364] (SEQ ID NO 29)

[0365] >NP_001304313.1 Aquaporin-4 isoform M1x [Homo sapiens]

[0366] MSDRPTARRWGKCGPLCTRENIMVAFKGVWTQAFWKAVTAEFLAMLIFVLLSLGSTINWGGTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFA SCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSVXLEDRTESRQDSLELSSDFLPPIKETDLL

[0367] (SEQ ID NO 30; where X can be tryptophan, cysteine, arginine, or serine, or a stop codon)

[0368] The invention is further illustrated by the following non-limiting embodiments. Example

[0369] The method for producing recombinant peptides of the present invention

[0370] Expi-293F cells (Thermo Fisher) were 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 Cells / ml. After 18-20 hours, add the enhancer according to the experimental protocol. Collect the supernatant after 4-6 days (37°C, 8% CO2, humidified incubator) on a 19 mm² track shaker at 125 rpm.

[0371] -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), discard the supernatant, repeat 2 times.

[0372] 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) to remove and discard the supernatant.

[0373] - 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.

[0374] Centrifugation 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.

[0375] 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.

[0376] ELISPOT:

[0377] 1) Cell Culture

[0378] A) Peripheral blood mononuclear cell isolation (under laminar flow hood)

[0379] 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.

[0380] B) PBMC pulse stimulation (performed under a laminar flow hood)

[0381] 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.

[0382] 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.

[0383] In the experiment, 3×10 6 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.

[0384] 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.

[0385] Required:

[0386] X-VIVO 15 culture medium + 5% human AB serum

[0387] 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

[0388] 2) ELISPOT

[0389] Laminar flow hood

[0390] 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).

[0391] 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.

[0392] Prepare the corresponding antigenic peptide 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.

[0393] Laminar flow hood outside

[0394] 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.

[0395] Remove the cell supernatant and wash five times with 100 µl PBS. Finally, remove excess buffer with paper.

[0396] 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.

[0397] 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.

[0398] Remove the plastic drain pipe from the board and rinse the bottom and sides of the board with tap water and let it dry.

[0399] 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.

[0400] Required:

[0401] Capture antibodies: anti-hIL10 (clone: ​​9D-7, Mabtech #3430-3-250; 1:500 dilution), anti-hIL10-biotinylated (Mabtech, #3430-6-250).

[0402] 1× PBS (sterile)

[0403] 35% ethanol (v / v)

[0404] Blocking buffer: X-vivo 5% hAB serum (sterile) [Blocking is performed in the same culture medium as cell culture].

[0405] Dilution buffer: PBS solution with 0.5% BSA

[0406] Washing buffer: 1× PBS

[0407] Culture medium: T cell culture medium, X-VIVO 15 (Lonza)

[0408] Filter injector: Millex GV

[0409] ELISPOT PVDF plate (#MSIP4510, Millipore)

[0410] TMB substrate

[0411] Example 2 (Reference) The alternative molecules of the recombinant polypeptide of the present invention induce Tregs to secrete IL10 in mice.

[0412] Six wild-type black mice were injected with 100 µg of a recombinant polypeptide (also known as "AIM Bio"), the sequence of which is as follows.

[0413] Ova_KbG

[0414] SIINFEKLGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQP KIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGC YNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDPPKTHVTHHPVFDY EATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC (SEQ ID NO 25),

[0415] and

[0416] Gp34_KbG

[0417] AVYNFATMGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQP KIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGC YNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDPPKTHVTHHPVFDY EATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC (SEQ ID NO 26),

[0418] Tolerance to either the OVA peptide or the viral Gp34 peptide was induced, respectively. Gp34 peptide is a well-characterized T-cell epitope derived from a lymphocytic choriomeningitis virus (LCMV) glycoprotein. Although this antigen 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 8 amino acid sequences of SEQ ID NO 26 are correct. 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.

[0419] Example 3 (Reference) The alternative molecules of the recombinant peptides of this invention can selectively prevent EAE induced by CD8+ T cells in mice.

[0420] 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 eventually NMO. In this animal model, a single injection of 500 µg of the recombinant peptide substitute molecule (also known as “AIM Bio”) induced tolerance to the target ovalbumin epitope and almost completely prevented EAE symptoms, while the substitute molecule presenting the control peptide had no significant protective effect. Figure 4 The sequences of the recombinant polypeptide substitute molecules are listed in Example 2.

[0421] Example 4 (Reference) The present invention provides alternative molecules for some recombinant peptides that can selectively prevent CD4 in mice. + EAE caused by T cells

[0422] On the same day as intraperitoneal injection of 33 µg or 100 µg of the recombinant polypeptide (“AIM Bio”), the surrogate left and right flanks were subcutaneously injected with 100 µl of MOG35-55 peptide / CFA (complete Freund's adjuvant; final concentrations of Mycobacterium tuberculosis H37RA and peptide were 1 mg / ml each) emulsion, followed by intraperitoneal injection of 250 ng of pertussis toxin (in 200 µl PBS). A second pertussis toxin injection was administered 3 days later. In this animal model, a single injection of the AIM Bio surrogate molecule (inducing tolerance to the Mog epitope (Mog44_Kb_G)) significantly reduced EAE symptoms, while surrogate molecules presenting the control peptide (Gp34) or the non-functional Mog peptide (Mog37) did not show significant protective effects. Figure 5 In this model, Mog44 AIM Bio also prevented spinal cord inflammation and CD8 T cell infiltration. Figure 6 The sequence of the recombinant polypeptide substitution molecule is shown in Example 2, or as shown below:

[0423] Mog44_DbG

[0424] FSRVVHLYRNGGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSMRYFETAVSRPGLEEPRYISVGYVDNKEFVRFDSDAENPRYEPRAPWMEQEGPEYWERETQKAKGQEQWFRVSLRNLLGCYNQSAGGSHTLQQMSGCDLGSDWRLLRGYLQFAYEGRDYIALNEDLKTWTAADMAAQITRRKWEQSGAAEHYKAYLEGECVEWLHRYLKNGNATLLRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC (SEQ ID NO 27)

[0425] Mog37_DbGVGWYRSPFSRGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSMRYFETAVSRPGLEEPRYISVGYVDNKEFVRFDSDAENPRYEPRAPWMEQEGPEYWERETQKAKGQEQWFRVSLRNLLGCYNQSAGGSHTLQQMSGCDLGSDWRLLRGYLQFAYEGRDYIALNEDLKTWTAADMAAQITRRKWEQSGAAEHYKAYLEGECVEWLHRYLKNGNATLLRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC (SEQ ID NO 28)

[0426] In this model, ELISA testing confirmed that Mog44 AIM Bio completely prevented the formation of MOG-specific autoantibodies in serum. Figure 7 See also Figure 10 (Confirmation). This strongly suggests that the recombinant peptide of the present invention is an effective therapy for NMO, which is typically characterized by an antibody response against human aquaporin 4. Therefore, the patient population is defined by common autoimmune-associated antigens. Certain MHC molecules are also associated with NMO.

[0427] 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.

[0428] Example 5 This invention relates to human recombinant peptide candidates for the treatment of NMO.

[0429] 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.

[0430] Figure 8 A list of human MS and MOGAD recombinant peptide candidates is shown.

[0431] The inventors' research indicates that single-chain proteins containing the AQP4 peptide antigen and the HLA-G α3 domain can induce tolerance T cells in healthy donors. Therefore, in 65% of healthy blood donors, CD8 Tregs were upregulated by at least 30%. Figure 9 ).

[0432] Example 6 (Reference) This provides further proof of concept regarding the stability and efficacy of the recombinant peptides of the present invention.

[0433] 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 11 and Figure 12As 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 13 ) and inhibits T cell lysis in a dose-dependent manner ( Figure 14 The effects of recombinant peptides on serum cytokine profiles in EAE-ODC Ova mice are as follows: Figure 15 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.

[0434] Example 7 (Reference) The therapeutic effects of recombinant peptides (containing mouse aquaporin antigen peptides) in 2D2 TCR transgenic mice.

[0435] Materials and Methods

[0436] Experimental model

[0437] 2D2 TCR transgenic mice were divided into four groups. Each group contained at least five animals. WT (C57BL / 6) littermates were in group 5. The experimental period began when optic neuritis (EAE) spontaneously appeared in the 2D2 mice. Disease onset was determined by visual abnormalities or elevated serum anti-MOG IgG levels. On the second day after disease onset, mice were injected (iv) with either the treatment mouse-appropriate single-chain MHC Ib molecule AQP147_KbG (antigen peptide: VTTHGNL; SEQ ID NO 39) / AQP203_KbG (antigen peptide: FAINYTGASM; SEQ ID NO 40) or the control AIM Bio Gp34_KbG. Control mice were injected with PBS only. The dose was set at 5 mg / kg body weight, 250 µl. The injections for the control and treatment groups were administered intravenously (D0). Untreated mice received PBS injections. Injections were repeated on days 15 and 30, respectively. Mice were weighed daily, and EAE symptoms were monitored (Table 1). Mice with an EAE score of 6 or higher 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 collected and cryopreserved at -20°C. Cardiac blood was collected and processed for serum analysis.

[0438] EAE scores based on a 10-point rating system (Bittner et al., Journal of visualized experiments: JoVE, (86), 51275).

[0439] Immunohistochemistry

[0440] Eyes were cut into 20 µm thick continuous sections and stained with hematoxylin and eosin. Briefly, frozen sections were dried at room temperature for 10 minutes, then stained in hematoxylin staining solution for 10 minutes. The tissue sections were rinsed under running water for 10 minutes, then stained in eosin staining solution for 30 seconds. The sections were then dehydrated sequentially in 70%, 96%, and 100% ethanol for 30 seconds each. Subsequently, the sections were incubated with xylene for 10 minutes and mounted with an in vitro cloud.

[0441] Immunofluorescence

[0442] Serial, 10µm thick longitudinal sections of the optic nerve were prepared for immunohistochemical staining. The tissue sections were fixed in 4% PFA PBS for 10 minutes; then blocked in 1× PBS buffer containing 5% BSA, 0.2% Triton-X100, and 5% NGS at room temperature for 2 hours. The tissues were then stained overnight at 4°C with a single primary antibody or a suitable combination of primary antibodies in 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-lysed caspase 3 (1:400, Cell signaling). The slides were washed three times with PBS and stained in the dark for 1 hour at room temperature with the corresponding fluorescently labeled secondary antibodies: i) anti-rabbit-Cy3 (1:300, Dianova); ii) anti-rat-AF488 (1:300, Invitrogen). The slides were then washed with PBS and stained with DAPI (1:500, Sigma-Aldrich) at room temperature in the dark for 10 minutes to stain the DNA. Finally, the slides were cleaned and mounted with Aquapolymount (Polysciences). Optical slides were acquired using a Zeiss Axiocam camera at 20x or 40x magnification.

[0443] Quantitative Immunostaining Analysis

[0444] Quantitative analysis of immunofluorescence images was performed using ImageJ-Fiji version 1.53t. Three to four slides were analyzed for each animal. Images were split into individual channels and converted to 8-bit images. The threshold for 8-bit images stained with lysed caspase-3 was set to 5 MFI, and the threshold for DAPI staining was set to 100. The area covered by lysed caspase-3 / area covered by DAPI represents the percentage of lysed caspase-3 in the slide.

[0445] Quality control and stability

[0446] 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) 2 µg of single-chain MHC-Ib molecules were stained with Coomassie brilliant blue on a 12% polyacrylamide gel, and B) αHLA-G protein blotting was performed using 2A12aHLA-G antibody (1:1000) and 1 µg of protein. Both monomers and dimers were detectable.

[0447] result:

[0448] Quality control and stability

[0449] Quality control and stability tests showed that both NMO substitutions for single-chain MHC Ib molecules were very stable. Figure 17-19 However, AQP203_H2KbG_spt appears to be more prone to multiplication, which may explain why it performs poorly in some detections.

[0450] Treatment effect

[0451] It is noteworthy that treatment with AQP147_KbG completely protected the treated mice from EAE (Electroencephalopathy). Figure 20 AIM inhibited immune cell infiltration in the optic nerve (Fig. 21) and completely suppressed apoptosis in the optic nerve, spinal cord, and retina (Fig. 22). Furthermore, although mice treated with AIM had fewer CD8 T cells, the number of regulatory T cells in the optic nerve was low but significantly high (Fig. 23). In addition, AQP147_KbG completely rescued IRL (inner retinal layer) cells from degradation (Fig. 24).

[0452] These data confirm the therapeutic efficacy of the recombinant peptides and their alternative molecules of the present invention. Therefore, according to the present invention, the recombinant peptides of the present invention can be used to treat neuromyelitis optica in human patients.

[0453] Example 8:

[0454] Expression and testing of polypeptides having the polypeptide structure defined in claim 1 and containing the peptide antigen according to the invention (“AIM Bio(s)”).

[0455] Sandwich ELISA scheme

[0456] 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.

[0457] Transfection protocol

[0458] 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%).

[0459] To obtain a final culture volume of approximately 12 ml, 3 × 10⁻⁶ ppm was used. 7One 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.

[0460] 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.

[0461] Simple Western Solution

[0462] 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), 2A12αHLA-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 a single 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. 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.

[0463] 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.

[0464] ELISpot solution

[0465] ELISpot analysis was performed according to the following protocol:

[0466] Phase 1: PBMC: AIM Bio Culture

[0467] reagents

[0468] • Human peripheral blood mononuclear cells from patients with neuromyelitis optica spectrum disorder (NMOSD)

[0469] • AIM Bio: AQP64_G, AQP65 P65K _G

[0470] • Complete culture medium: X-vivo 15 (Lonza, Biozym 881026) supplemented with 5% human AB serum and human cytokines

[0471] -rhIL-2 (20ng / ml); Immunotools, 11340027

[0472] -rhGM-CSF (20ng / ml); Immunotools, 11343128

[0473] -rhIL-4 (10ng / ml); Immunotools, 11340047

[0474] -rhTGFβ (10ng / ml); Immunotools, 11343160

[0475] 24-well culture plate

[0476] ·PBS (sterile)

[0477] Thawing peripheral blood mononuclear cells (Day -1)

[0478] Thaw frozen PBMCs by rotating them in a water bath for about 20-30 seconds.

[0479] Immediately transfer the cells to a 15ml centrifuge tube containing PBS and centrifuge at 300g for 5 minutes at room temperature.

[0480] Wash the cells twice more with PBS buffer.

[0481] • 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.

[0482] Treg induction: PBMC culture for 14 days using AIM Bios (aseptic conditions).

[0483] • Test conditions:

[0484] o Negative control (not treated with AIM Bio / untreated)

[0485] oAQP64_G processing (sequence AQP64_G: MSRSVALAVLALLSLSGLEAKPLPVDMVLGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPCIVKWDRDMGGGGSGGGSGGGGSGGGSGSSHSMRYFSAAVSRPGRGE PRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEETRNTKAHAQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRCKCEAAVNAEQRRAYLEGTCVEWLHRYLENGKEMLQRA DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYT CHVQHEGLPEPLMLRW SKEGDGGIMSVRESGSLSEDLGS PDRVRAVSHWSSC* ; SEQ ID NO.

[0486] oAQP65 P65K _G Treatment P65K _G: MSRSVALAVLALLSLSGLEA KLPVDMVLGGGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPCIVKWDRDMGGGSGGGGSGGGSGGGGSGGGSGSSHSMRYFSAAVSRPGR GEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGAYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRCKCEAAVNAEQRRAYLEGTCVEWLHRYLENGKEMLQRA DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAV VVPSGEEQRYTCHVQHEGLPEPLMLRW SKEGDGGIMSVRESGSLSEDLGS PDRVRAVSHWSSC* ; SEQ ID NO.

[0487] ·PBMC quantity:1.2×10 6Each cell was dissolved in 400 μL of complete culture medium.

[0488] • Count the cells left to stand overnight and calculate the number of cells required for the test.

[0489] • Wash the cells and resuspend them in complete culture medium to a final concentration of 3 × 10⁻⁶. 6 Cells / mL

[0490] • Add 400 µl (1.2 × 10⁻⁶) to each well of the 24-well plate. 6 Cell suspension (cells).

[0491] • Use 5 µg / ml AIM Bios AQP64_G and AQP65 P65K _G pulse treatment of cells (day 0)

[0492] • Add 400 µl of complete culture medium to the cells every 2 days.

[0493] • On day 6, use 5 µg / ml AIM Bios AQP64_G and AQP65 P65K _G performs pulse processing

[0494] • On day 14, cells were seeded onto ELISpot plates.

[0495] Phase Two: ELISpot Testing

[0496] (EliSpot testing was performed according to the guidelines provided with the Human IL-10 ELISpot (Basic) Kit (Mabtech; 3430-2H))

[0497] reagents

[0498] ·ELISPOT PVDF plate (Millipore)

[0499] Human IL-10 EliSpot Basic Reagent Kit (Product Code: 3430-2H)

[0500] 35% ethanol

[0501] • Blocking culture medium: X-vivo 5% hAB serum

[0502] • Dilution buffer: 0.5% BSA PBS solution or 0.5% FCS PBS solution

[0503] • EliSpot medium: X-VIVO 15 (Lonza, Biozym 881026) supplemented with 5% human AB serum and hIL-2 (20 ng / ml)

[0504] • Streptavidin-HRP used for detection (Biolegend #405210; 1:750 dilution)

[0505] • ELISpot substrate: TMB for HRP (Mabtech code: 3651–10).

[0506] ·PBS (sterile)

[0507] Cell coating (under sterile conditions)

[0508] • Activate the PVDF pores with 25 µl of ethanol (35% v / v) for up to 1 minute.

[0509] Wash the culture plate 5 times with sterile water (200 µl / well).

[0510] • 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).

[0511] Incubate overnight at 4-8°C.

[0512] Culture cells in a petri dish (under sterile conditions).

[0513] • Prepare a layout diagram. All samples should be tested, with two replicates for each.

[0514] ·sample:

[0515] negative control: untreated cells (no stimulation)

[0516] o Positive control: Untreated cells + 1µg / ml α-CD3 / CD28 stimulation mixture

[0517] o Test samples: AQP64_G treated cells and AQP65 P65K _G Processed cells

[0518] • Count PBMCs in 14D cultures. Calculate the required number of cells for each condition.

[0519] • Minimum cell viability > 50%. Otherwise, discard the sample.

[0520] • In a fume hood, remove excess coated antibodies and wash the microplate five times with 200 µl of sterile PBS.

[0521] Add 50 µl of blocking solution to each well. Incubate at room temperature for 2 hours.

[0522] • 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.

[0523] • Resuspend the positive control cells to a higher concentration and make up the volume with a stimulating solution.

[0524] • Under the lid, carefully remove any excess sealing cushioning.

[0525] • Take 100 µl of PBMC (1.5 × 10⁻⁶) under each condition. 6 The cell suspensions were added to the corresponding wells.

[0526] Incubate at 37°C for 12-48 hours.

[0527] Spot detection

[0528] • Dilute the biotin-labeled detection antibody (12G8-Biotin) with dilution buffer to a concentration of 1 µg / ml (1:1000).

[0529] • 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.

[0530] Add 50 µl of detection antibody to each well. Incubate overnight at 4 °C.

[0531] Wash 5 times with 200 µl PBS. Press with a paper towel to absorb excess liquid.

[0532] • Prepare horseradish peroxidase-labeled streptavidin (diluted to 1:750 with dilution buffer).

[0533] Add 50 µl of streptavidin-HRP conjugate to each well. Incubate at room temperature for 1 hour.

[0534] Wash 5 times with 200 µl PBS. Press with a paper towel to absorb excess liquid.

[0535] Add 50µl to each well After filtration Incubate the TMB substrate in the dark for 10-20 minutes until blue spots appear.

[0536] The reaction can be stopped by rinsing the hole with water (tap water is fine).

[0537] Remove the plastic drain tube from the bottom of the plate and thoroughly wash the bottom and sides of the plate with tap water.

[0538] Analysis was performed using an S6 Universal M2 reader (Immunospot, Cleveland, Ohio).

[0539] result:

[0540] Example 9:

[0541] Figure 25 The AIM Bios of the present invention were shown to be expressed in Hek293T cells, and monomers (~50 kDa) of all compounds were detectable; furthermore, expression was confirmed and quantified in the supernatant by ELISA. This indicates that these molecules can be successfully produced. It was also shown that anchoring residue optimized sequences such as AQP65-72_6 (P65K) can result in higher yields.

[0542] Figure 26 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.

[0543] ELISpot was performed as described in Example 9, except that only 1.2 million PBMCs were used to seed the cells in 24-well plates, and the volume of all culture media was reduced accordingly.

[0544] Compared to untreated PBMCs, those treated with AIM Bio AQP65 showed better results. P65K Of the 5 NMO patients treated with _G, 4 had increased IL-10 spots in their PBMCs.

[0545] 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.

[0546] Example 10: Characterization of the inventor's recombinant peptide (“AIM Bios”)

[0547] Size exclusion chromatography

[0548] 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.

[0549] Figure 28 shows the purified AQP4. 126-135 Size exclusion chromatography analysis of _A2G was performed. Advantageously, the study revealed that this protein is a pure monomeric protein. Notably, the expression level of this A2G construct was higher than that of the corresponding pure HLA-G peptide.

[0550] The "AQP4" used in this experimental example 126-135 The “A2G” construct contains the amino acid sequence of SEQ ID NO 87, an optional C-terminal amino acid sequence from intron 4 (with furin cleavage site removed) (SKEGGDGGIMSVRESGSLSEDL; SEQ ID NO 54), and an optional tag (described below).

[0551] Thermal displacement analysis

[0552] Thermal shift analysis (TSA) was performed to analyze the protein stability of purified AIM Bios. For TSA, 3 µg of AIM Bio was dissolved in 25 µl PBS (Sigma, D8537-500 ml) containing SYPROOrange dye (Merck, S5592, final dilution 1:2000, dissolved in PBS). Melting curves were recorded using a StepOnePlus instrument (ThermoFisher) with an initial temperature of 25°C, increased to 95°C at a rate of 1°C per minute, and held for 2 minutes. Melting curves were analyzed using StepOnePlus 2.3 software. To determine Tm, a Boltzmann sigmoid curve was fitted, and Tm was calculated accordingly.

[0553] Figure 29 shows the thermal displacement analysis: AIM Bio AQP 126-135 Melting curve analysis of _A2G.

[0554] Jess Simple West

[0555] 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), 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 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. 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.

[0556] 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.

[0557] Figure 30 shows AQP 65-72_P65K _G and AQP 65-72_P65RJess results for AIM Bios with _G anchored residue optimization.

[0558] As shown in the figure, the tendency to form polymers is reduced due to the optimization of anchoring residues.

[0559] ILT4-Fc combined with ELISA

[0560] For the ILT4 binding assay, 125 ng of AIM Bio (25 µl of 5 µg / ml AIM Bio solution) was coated onto 96-well half-area microplates (Greiner, 675061), covered with sealing foil, and incubated overnight at 4°C. The plates were then washed three times with 120 µl of 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 of 1% BSA (albumin grade V, Roth, #0163.2) in PBS, followed by three washes with 120 µl of PBS / T on a shaker (also used for further blocking steps). Human ILT4-Fc (stock concentration in PBS: 250 µg / ml) (Sino Biological, #14132-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) 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 microplate reader (Tecan) and Magellan 7.5 software (Tecan), with a reference wavelength of 620 nm. Calibration was performed on the control wells. Data were calculated in Excel 365 (Microsoft), and graphs were plotted in Prism 10.2 (GraphPad). Results are as follows... Figure 31 As shown.

[0561] As shown in the figure, similar ICs can be obtained using His-tagged or untagged protein variants.50 Concentration indicates that the (optional) label is irrelevant to the binding properties of AIM Bio.

[0562] Industrial applicability

[0563] 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 aquaporin 4; ii) Optionally, connector sequence; iii) A human polypeptide domain sequence containing 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 the peptide antigen according to i) consists of an amino acid sequence selected from the amino acid sequence group of SEQ ID NO34, 42-51 and 82.

4. The recombinant polypeptide according to any one of claims 1-3, wherein, According to i), the peptide antigen consists of the amino acid sequence of SEQ ID NO 34.

5. The recombinant polypeptide according to any one of claims 1-3, wherein, The peptide antigen described in i) consists of the amino acid sequence of SEQ ID NO 48.

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

7. 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, preferably the α3 domain of human HLA-G.

8. The recombinant polypeptide according to any one of the preceding claims, wherein, According to (vii), the α3 domain or its derivatives have at least 80%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO 9, SEQ ID NO 21, or SEQ ID NO 53, or are identical to the α3 domain having the amino acid sequence of SEQ ID NO 9, SEQ ID NO 21, or SEQ ID NO 53.

9. The recombinant polypeptide according to any one of claims 1-8, wherein the α3 domain or derivative according to (vii) is identical to the α3 domain having the amino acid sequence SEQ ID NO 9.

10. The recombinant polypeptide according to any one of claims 1-8, wherein the α3 domain or derivative according to (vii) is identical to the α3 domain having the amino acid sequence of SEQ ID NO 21.

11. The recombinant polypeptide according to any one of claims 1-8, wherein the α3 domain or derivative according to (vii) is identical to the α3 domain having the amino acid sequence SEQ ID NO 53.

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

13. 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.

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

15. 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), wherein the polypeptide does not comprise components viii) to x), or wherein the polypeptide comprises components i) to x) or consists of all components i) to x).

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

17. The recombinant polypeptide according to any one of claims 1-15, 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 34, 42-51 and 82, (b) An amino acid sequence of SEQ ID NO 16 or SEQ ID NO 55-60.

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

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

20. A pharmaceutical composition or kit comprising at least one nucleic acid according to claim 19.

21. A pharmaceutical composition or kit comprising at least one recombinant polypeptide according to any one of claims 1-18.

22. The pharmaceutical composition or kit of claim 21, wherein the pharmaceutical composition or kit comprises at least two different recombinant polypeptides according to any one of claims 1-18, wherein each different polypeptide comprises a different peptide antigen as defined in claim 3.

23. A pharmaceutical composition or kit according to any one of claims 20-22 for treating neuromyelitis optica in human patients.

24. The pharmaceutical composition or kit for the use of claim 23, wherein the treatment method is immunotherapy, and the treatment method is preferably achieved by inducing immune tolerance to human aquaporin 4.

25. The pharmaceutical composition or kit for the use of any one of claims 23-24, wherein the treatment method is to reduce the level of autoantibodies against human aquaporin 4 in plasma or cerebrospinal fluid, and wherein the human patient is a patient who has autoantibodies against human aquaporin 4 in plasma or cerebrospinal fluid prior to the start of treatment.

26. The pharmaceutical composition or kit for the use of any one of claims 23-25, wherein the treatment is performed by inducing myelin-specific regulatory T cells.

27. A recombinant host cell comprising the nucleic acid or vector according to claim 19 and expressing the recombinant polypeptide according to any one of claims 1-18.

28. A method for obtaining a pharmaceutical composition comprising the polypeptide according to any one of claims 1-18, the method comprising the following steps: (a) culturing the recombinant host cell of claim 27 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.

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