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

Abstract

This invention relates to the therapeutic use of non-classical human major histocompatibility complex (MHC) molecules (also referred to as MHC class Ib molecules) in combination with peptide antigens for the treatment of neuromyelitis optica (NMO). 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. This invention also relates to methods for producing such recombinant polypeptides, pharmaceutical compositions comprising them, and their use for 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. This invention also relates to methods for producing such recombinant polypeptides, pharmaceutical compositions comprising them, and their use for treating (managing) neuromyelitis optica (NMO). [Background technology]

[0002] Neuromyelitis optica (NMO) is a demyelinating autoimmune disease of the central nervous system (CNS) with an incidence of between 0.05 and 0.4 per 100,000. NMO is characterized by symptoms similar to multiple sclerosis (MS). However, while multiple sclerosis often follows a relapsing-remitting disease course, patients with NMO rarely experience remission. Furthermore, some treatments beneficial in patients with MS are ineffective or even worsen in NMO. Therefore, NMO is more difficult to treat and usually progresses more rapidly than MS. Current treatment strategies for NMO focus on preventing relapses or treating acute disease relapses. Acute relapses are usually treated with immunosuppressive therapies such as glucocorticoids or plasmapheresis to remove autoreactive antibodies. More recently, therapeutic antibodies that inhibit the complement system (eculizumab) or those that interfere with inflammatory cytokines (satralizumab) have also been approved.

[0003] However, not only autoreactive immune function but also protective immune function is inhibited. These are essential for protecting the patient from viruses, bacteria, or tumors. Therefore, often only partial inhibition can be targeted. Autoimmune diseases usually worsen after relapse, so considerable effort is put into preventing relapse. However, the overall slow progression of this autoimmune disease can be slowed, but not prevented. To circumvent this dilemma, attempts have been made for some time to induce immune tolerance to antigens attacked by autoreactive immune cells (antigen-specific immunosuppression, ASI).

[0004] Two strategies have been evaluated so far, at least in early clinical trials. Similar to desensitization strategies in allergies, large amounts of antigen have been administered via different pathways 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 regulatory T cells or antigen-loaded toxicogenic dendritic cells appear more promising. However, these strategies are extremely complex and expensive, each requiring GMP-compliant production and quality control processes for individual patients. Therefore, even if small-scale clinical trials are successful, it is highly doubtful whether adoptive transfer therapy will be available to many patients in the foreseeable future.

[0005] WO 2018 / 215340 relates to the combination of MHC class Ib molecules with peptides for targeted therapeutic immunomodulation.

[0006] In summary, improvements to medications for the treatment of neuromyelitis optica (NMO) are still needed. [Overview of the project]

[0007] The inventors have found that human MHC class Ib molecules, such as HLA-G, have the ability to induce antigen-specific tolerance to presented peptide antigens. Therefore, despite having a structure and sequence similar to classical human MHC class Ia molecules that induce antigen-peptide-specific immune responses, MHC class Ib molecules can be advantageously used according to the present invention to suppress immune responses in an antigen-specific manner. Furthermore, the inventors have found that molecules other than naturally occurring MHC class Ib molecules, particularly polypeptides comprising at least one domain of an MHC class Ib molecule, preferably only at least the [alpha]3 domain of an MHC class Ib molecule, can be used for suppressing immune responses according to the present invention: the [alpha]1 and [alpha]2 domains of a variable class Ia molecule can be combined with the [alpha]3 domain of a human MHC class Ib molecule to suppress immune responses to peptides presented by these antigens.

[0008] HLA-G molecules loaded with antigens can be unstable. Therefore, the inventors designed soluble recombinant polypeptides containing a peptide antigen, an MHC class Ib molecule such as HLA-G, and β2-microglobulin (b2m), and covalently linked these three components (e.g., via a covalent linker). Alternatively, the antigen-binding α1 and α2 domains of an MHC class Ib molecule such as HLA-G were replaced with the respective domains of other MHC molecules to enhance the flexibility and versatility of these recombinant polypeptides (see, for example, Figure 2). These alternative recombinant polypeptides can be designed using the antigen-binding domains of other human HLA molecules. (Mouse H2-K) b It has been previously found that constructs containing 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) (WO 2018 / 215340).

[0009] Surprisingly, the inventors found that by using the recombinant polypeptides of the present invention, it is possible to suppress the immune response to neuroinflammatory autoantigens and induce cells that induce tolerance to human aquaporin 4 (AQP4). Furthermore, using established models of neuroinflammatory conditions, the inventors demonstrated that neuroinflammatory diseases can be treated using surrogates of these polypeptides (adapted for use in mice). Thus, according to the present invention, neuromyelitis optica (NMO) can be treated with the recombinant polypeptides of the present invention.

[0010] Our experimental data indicate that a suitable peptide antigen and the [α]3 domain of an MHC class Ib molecule (e.g., HLA-G) are necessary to achieve the desired effect. Therefore, this approach goes beyond previously described strategies that use antigenic peptides in the absence of co-stimulation (resulting in anergy T cells rather than tolerogenic T cells) or use MHC class Ib molecules in an antigen-nonspecific state.

[0011] Furthermore, according to the present invention, the recombinant polypeptide not only modulates the T cell response but also prevents the formation of aquaporin 4 (AQP4)-specific autoantibodies. Since aquaporin 4 (AQP4)-specific autoantibodies are involved in the pathology of neuromyelitis optica (NMO), this advantage is expected to lead to clinical improvement in human patients with neuromyelitis optica (NMO).

[0012] Sequencing of peptide antigens eluted from MHC molecules revealed MHC allele-specific motifs characterized by a preference for specific amino acids at specific positions. These amino acids tether the presented peptide to the peptide presentation cleft. Substituting less desirable amino acids in the MHC anchor residue with more desirable amino acids increases the affinity between the epitope and MHC. Consequently, the peptide-MHC complex becomes more stable. Notably, the anchor amino acids do not come into contact with the congeneral T cell receptor at the peptide-MHC / T cell receptor interface. Therefore, optimization of the anchor residue improves T cell stimulation (by stabilizing the peptide-MHC complex) without altering the repertoire of stimulated T cells. Advantageously, we have found that the polypeptide of the present invention can be improved by using anchor residue optimization, for example, by increasing the polypeptide production level.

[0013] Therefore, the present invention relates to the following preferred embodiments: 1. A recombinant polypeptide capable of presenting a peptide antigen, wherein the recombinant polypeptide is arranged in the order from the N-terminus to the C-terminus. i) A peptide antigen presented by the recombinant polypeptide, wherein the peptide antigen is a peptide of human aquaporin 4, ii) Depending on the case, the linker sequence, iii) A sequence of a human polypeptide domain containing the sequence of human β2 microglobulin, or an amino acid sequence that is at least 90% identical to the amino acid sequence of human β2 microglobulin represented by Sequence ID No. 5. iv) Depending on the case, the linker sequence, v) The [α]1 domain of the MHC molecule, vi) [α]2 domain of MHC molecule, vii) The [α]3 domain of an MHC class Ib molecule, or a derivative of the [α]3 domain of an MHC class Ib molecule, wherein the derivative is capable of binding to ILT2 or ILT4, viii) optionally, a protease cleavage site, ix) optionally, a spacer sequence, and x) optionally, an affinity tag The recombinant polypeptide as described above, comprising the same.

[0014] 2. The recombinant polypeptide according to item 1, wherein the peptide antigen described in i) has a length of 7 to 11 amino acids, preferably a length of 8 to 10 amino acids.

[0015] 3. The recombinant polypeptide according to item 1 or 2, wherein the peptide antigen described in i) consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 34, 42 to 51, and 82.

[0016] 4. The recombinant polypeptide according to any one of items 1 to 3, wherein the peptide antigen described in i) consists of the amino acid sequence of SEQ ID NO: 34.

[0017] 5. The recombinant polypeptide according to any one of items 1 to 3, wherein the peptide antigen described in i) consists of the amino acid sequence of SEQ ID NO: 48.

[0018] 6. The recombinant polypeptide according to any one of items 1 to 5, wherein the [α]1 domain described in (v) and the [α]2 domain described in (vi) are derived from a human MHC class Ia molecule, preferably a human HLA-A2 molecule, or derived from a human MHC class Ib molecule, preferably a human HLA-G molecule.

[0019] 7. The recombinant polypeptide according to any one of items 1 to 6, wherein the [α]3 domain of the MHC class Ib molecule described in (vii) is the [α]3 domain of human HLA-E, human HLA-F or human HLA-G, preferably human HLA-G.

[0020] 8. A recombinant polypeptide according to any one of items 1 to 7, wherein the [α]3 domain or derivative described in (vii) is identical to the [α]3 domain having the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 21, or SEQ ID NO: 53, or has at least 80% amino acid sequence identity, at least 90% amino acid sequence identity, at least 92% amino acid sequence identity, at least 94% amino acid sequence identity, at least 96% amino acid sequence identity, at least 98% amino acid sequence identity, or at least 99% amino acid sequence identity, or is identical to the [α]3 domain having the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 21, or SEQ ID NO: 53.

[0021] 9. A recombinant polypeptide according to any one of items 1 to 8, wherein the [α]3 domain or derivative described in (vii) is identical to the [α]3 domain having the amino acid sequence of SEQ ID NO: 9.

[0022] 10. A recombinant polypeptide according to any one of items 1 to 8, wherein the [α]3 domain or derivative described in (vii) is identical to the [α]3 domain having the amino acid sequence of SEQ ID NO: 21.

[0023] 11. A recombinant polypeptide according to any one of items 1 to 8, wherein the [α]3 domain or derivative described in (vii) is identical to the [α]3 domain having the amino acid sequence of SEQ ID NO: 53.

[0024] 12. The recombinant polypeptide according to any one of items 1 to 11, wherein the linker sequence described in (ii) and / or the linker sequence described in (iv) comprises an amino acid sequence (GGGGS)n, where n is an integer of 1 or more, and n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, preferably selected from the group consisting of 2, 3, 4, and 5.

[0025] 13. The recombinant polypeptide according to any one of items 1 to 12, wherein the sequence of the human polypeptide domain described in (iii) is at least 95% identical to the amino acid sequence of SEQ ID NO: 5, preferably at least 98% identical to the amino acid sequence of SEQ ID NO: 5, and more preferably identical to the amino acid sequence of SEQ ID NO: 5.

[0026] 14. A recombinant polypeptide according to any one of items 1 to 13, wherein the polypeptide is a dimer or a polymer.

[0027] 15. A recombinant polypeptide as described in any one of items 1 to 14, wherein the polypeptide contains or consists of all of components i) to vii), and the polypeptide does not contain components vii) to x), or the polypeptide contains or consists of all of components i) to x).

[0028] 16. A recombinant polypeptide described in any one of items 1-15, further comprising an N-terminal secretory signal peptide sequence.

[0029] 17. The recombinant polypeptide, in order from the N-terminus to the C-terminus, is as follows ((a) and (b)): (a) A peptide antigen selected from the group consisting of amino acid sequences of SEQ ID NOs. 34, 42-51 and 82, and (b) Amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 55-60 A recombinant polypeptide consisting of an amino acid sequence, as described in any one of items 1 to 15.

[0030] 18. A recombinant polypeptide described in any one of items 1 to 17, wherein the recombinant polypeptide is soluble.

[0031] 19. A nucleic acid encoding one or more polypeptides as described in any one of items 1 to 18, wherein the nucleic acid is preferably a vector.

[0032] 20. A pharmaceutical composition or kit comprising at least one nucleic acid as described in item 19.

[0033] 21. A pharmaceutical composition or kit comprising at least one recombinant polypeptide described in any one of items 1 to 18.

[0034] 22. The pharmaceutical composition or kit according to item 21, wherein the pharmaceutical composition or kit comprises at least two different recombinant polypeptides as described in any one of items 1 to 18, each of which comprises a different peptide antigen as defined in item 3.

[0035] 23. A pharmaceutical composition or kit according to any one of items 20-22, for use in the treatment of neuromyelitis optica in human patients.

[0036] 24. A pharmaceutical composition or kit for use as described in item 23, wherein the treatment is an immunotherapy, and the treatment is preferably by inducing immune tolerance to human aquaporin 4.

[0037] 25. A pharmaceutical composition or kit for use as described in any one of items 23-24, wherein the treatment is for reducing plasma or cerebrospinal fluid levels of autoantibodies against human aquaporin 4, and the human patient is a patient who had plasma or cerebrospinal fluid autoantibodies against human aquaporin 4 prior to the initiation of treatment.

[0038] 26. A pharmaceutical composition or kit for use as described in any one of items 23-25, wherein the treatment is by inducing myelin-specific regulatory T cells.

[0039] 27. Recombinant host cells comprising the nucleic acid or vector described in item 19, and expressing a recombinant polypeptide described in any one of items 1 to 18.

[0040] 28. A method for obtaining a pharmaceutical composition comprising a polypeptide described in any one of items 1 to 18, the method comprising: (a) culturing recombinant host cells described in item 27 under conditions that enable the expression of recombinant polypeptide from nucleic acid molecules; (b) recovering recombinant polypeptide; (c) purifying recombinant polypeptide; and (d) formulating recombinant polypeptide into a pharmaceutical composition. [Brief explanation of the drawing]

[0041] [Figure 1] A depiction of a peptide-loaded soluble MHC Ib molecule suitable for achieving therapeutic antigen-specific immunomodulation. The presented peptide antigen is depicted as a dotted sphere, the HLA-G alpha 1-3 domains are sketched in light gray, and the beta-2 microglobulin domain is shown in dark gray. Any linkers connecting the antigenic peptide to the beta-2 microglobulin molecule are shown in gray stick style, and any disulfide traps are depicted as black spheres. This drawing was generated using Pymol and adapted from structures published in Clements et al., Proc Natl Acad Sci USA. 2005 Mar 1;102(9):3360-5 and Hansen et al., Trends Immunol. 2010 Oct;31(10):363-9. [Figure 2]Examples of constructs based on vectors encoding single-chain MHC Ib molecules suitable for therapeutic peptide-specific immunomodulation. HLA-G1 and HLA-G5 each consist of three [alpha] domains (shown here in black), a non-covalently bound beta-2-microglobulin subunit (shown here in dark gray), and an antigenic peptide presented on HLA-G (short black arrow). HLA-G1 further contains a transmembrane domain and a short intracellular chain (not shown here). As shown here, the [alpha]-3 domain can bind to receptors ILT2 (see Shiroishi et al., Proc Natl Acad Sci USA. 2003 July 22;100(15):8856-8861) and ILT4 (see Shiroishi et al., Proc Natl Acad Sci USA. 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 the complex MHC Ib molecule, one or more protein tags (such as SpotTag, myc tag, and / or His(6x) tag) may be introduced. They can be introduced in a manner that allows for subsequent optional removal by cleavage using an optional factor Xa cleavage site. Furthermore, antigenic peptides, beta-2-microglobulin, and the MHC Ib[alpha] chain can be linked to increase stability. Vector maps were generated using Snapgene Viewer Software. [Figure 3-1]The recombinant polypeptide surrogate of the present invention induces IL-10 secreting Tregs in mice. In this experiment, 100 μg of surrogate molecules consisting of viral (Gp34) or ovalbumin (Ova) model peptide antigen, mouse H2-Kb alpha 1 and 2 domains, and human HLA-G alpha 3 domain and beta-2-microglobulin were injected via ip into 12-week-old C57BL / 6 mice. After 14 days, the mice were sacrificed, and splenocytes were rechallenged with 5 μg / ml of Gp34 or Ova peptide in a 48-hour standard mouse IL-10 ELIspot assay (Mabtech mouse IL-10 HRP ELIspot kit; cells cultured in RPMI 10% FCS 10 ng / ml IL2) (A). A significant increase in regulatory T cells secreting only IL-10 was detectable in response to rechallenge with the peptides to which tolerance had been induced by surrogate molecule injection. (A) Experimental design; (B) Results. [Figure 3-2] This is a continuation of Figure 3-1. [Figure 4-1]The recombinant polypeptide surrogate of the present invention prevents CD8+ T cell-driven EAE in mice. In this EAE mouse model, the model antigen ovalbumin (OVA) is expressed in oligodendrocytes under the control of a myelin basic protein (MBP) promoter (ODC-OVA). This results in the presentation of the OVA257-264 peptide on H-2Kb MHC molecules on oligodendrocytes. OT-I mice express a T cell receptor (OT-I) on their CD8+ T cells, which accurately recognizes this peptide-MHC combination. When CD8+ T cells derived from these mice are introduced into 10-day-old ODC-OVA mice, these mice develop experimental autoimmune encephalomyelitis (EAE), which in many respects resembles the onset and symptoms of MS (Na et al., Brain, Volume 131, Issue 9, September 2008, Pages 2353-2365). In this experiment, 500 μg of surrogate molecules or PBS consisting of viral (Gp34) or ovalbumin (Ova) model peptide antigen, mouse H2-Kb alpha 1 and 2 domains, and human HLA-G alpha 3 domain and beta-2-microglobulin were injected on the same day. EAEs were scored according to Bittner et al., J Vis Exp. 2014 Apr 15;(86):51275. Only the ovalbumin tolerance-inducing surrogate molecule almost completely prevented EAE symptoms. (A) Experimental design; (B) Results. [Figure 4-2] This is a continuation of Figure 4-1. [Figure 5-1]Several surrogates of the recombinant polypeptides of the present invention selectively prevent CD4+ T cell-driven EAE in mice. In this model, administration of MOG 35-55 peptides combined with a complete Freund adjuvant that activates CD4+ Th17 cells and pertussis toxin that enhances blood-brain barrier permeability induces a potent, myelin-specific autoimmune response (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). Furthermore, mice were injected with either a 100 μg / mouse surrogate molecule or PBS containing either the virus (Gp34) or two Mog peptide antigens (Mog37 or Mog44), mouse H2-Db alpha-1 and alpha-2 domains, and human HLA-G alpha-3 domain and beta-2-microglobulin, on the first day. Mog44 peptide containing the surrogate molecule significantly reduced EAE symptoms and weight loss. (A) Experimental design; (B) EAE score; (C) Body weight. [Figure 5-2] This is a continuation of Figure 5-1. [Figure 5-3] This is a continuation of Figure 5-1. [Figure 6-1]The Mog44 surrogate of the recombinant polypeptide of the present invention prevented inflammation and CD8 T cell infiltration in the spinal cord. 10 μm fresh frozen sections were stained with commercially available toluidine 1x staining reagent at room temperature for 1 hour. Strong infiltration of immune cells was detected in EAE but prevented by Mog44_Db_G. 10 μm fresh frozen sections 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, HRP-bound secondary antibody, and DAB solution (detailed method: Karikari et al., Brain Behav Immun. 2022 Jan 12;101:194-210). EAE induced by Mog35-55 resulted in strong infiltration of CD8+ cells into the spinal cord, which was prevented by the MOG44_Db_G surrogate molecule. (A) Toluidine; (B) CD8-DAB. [Figure 6-2] This is a continuation of Figure 6-1. [Figure 7] Detection of anti-MOG35-55 antibodies in Mog-EAE mice treated with the recombinant polypeptide surrogate of the present invention ("AIM Bio"). After sacrificing mice, mouse serum was collected from cardiac puncture. After using 10 μg / ml of Mog35-55 for overnight coating, the wells were blocked with 1% BSA, and anti-Mog35-55 antibodies were detected using the indicated secondary HRP-conjugated antibody. Mog35-55-induced EAE correlated with high levels of Mog35-55-specific IgG autoantibodies, which were undetectable in animals treated with 100 μg of the MOG44_Db_G surrogate molecule. [Figure 8]A list of exemplary human NMO recombinant polypeptide candidates. The following are exemplary peptide antigen sequences of the NMO recombinant polypeptide candidates shown in the figure: Construct Peptide Antigen Sequence ID: hAQP4 42-50_HLAG Myc / His Tag FLAMLIFVL Sequence ID: 31 hAQP4 45-53_HLAG Myc / His Tag MLIFVLLSL Sequence ID: 32 hAQP4 65-72_HLAG Myc / His Tag PLPVDMVL Sequence ID: 33 hAQP4 71-79_HLAG Myc / His Tag VLISLCFGL Sequence ID: 22 hAQP4 126-135_HLAG Myc / His Tag AIIGAGILYL Sequence ID: 34 hAQP4 127-135_HLAG Myc / His Tag IIGAGILYL Sequence ID: 23 hAQP4 45-53_A2G Myc / His Tag MLIFVLLSL Sequence ID: 32 hAQP4 65-72_A2G Myc / His Tag PLPVDMVL SEQ ID NO: 33 hAQP4 71-79_A2G Myc / His Tag VLISLCFGL SEQ ID NO: 22 hAQP4 126-135_A2G Myc / His Tag AIIGAGILYL SEQ ID NO: 34 hAQP4 127-135_A2G Myc / His Tag IIGAGILYL SEQ ID NO: 23 hAQP4 156-164_A2G Myc / His Tag AGHGLLVEL SEQ ID NO: 35 hAQP4 238-247_A2G Myc / His Tag IIGAVLAGGL SEQ ID NO: 24 hAQP4 45-HLAG SPOTtag MLIFVLLSL SEQ ID NO: 32 hAQP4 36-43_HLAG SPOTtag KAVTAEFL SEQ ID NO: 36 hAQP4 71-79_HLAG SPOTtag VLISLCFGL SEQ ID NO: 22 hAQP4 64-72_HLAG SPOTtag KPLPVDMVL SEQ ID NO: 2 hAQP4 71-79_A2G SPOTtag VLISLCFGL SEQ ID NO: 22 hAQP4 127-A2G SPOTtag IIGAGILYL SEQ ID NO: 23 hAQP4 238-A2G SPOTtag IIGAVLAGGL SEQ ID NO: 24 hAQP4 137-145_HLAG SPOTtagFigure 8 further shows which combinations of the AQP4 peptide and antigen-presenting MHC class Iα1 and α2 domains (HLAg=HLA-G, A2G=HLA-A2 presenting domain + HLA-Gα3 domain) yield successful (high), intermediate (average, av), or (low) results in terms of production, quality control, and prioritization in healthy blood donors (shown in Figure 9). [Figure 9-1]Upregulation of CD8 Tregs in healthy blood donors by the recombinant polypeptide of the present invention ("AQP_64") containing the KPLPVDMVL antigen. In vitro Treg induction via a peptide-HLA-G-containing construct (AIM Biologicals) was performed as follows: PBMCs derived from healthy donors were purified by density centrifugation of leukocytes from a leukocyte reduction chamber using Ficoll. The cells were centrifuged at 1200×g for 20 minutes without a brake, the interphase ring was collected, and washed with 1×PBS (5 minutes, 300×g). The PBMCs were frozen until further use. The PBMCs were thawed one day before PBMC pulse treatment (d-1) and left overnight at 37°C in 5 ml of 5% human AB serum-containing X-VIVO 15 medium in a 6-well plate. On the following day (d0), the cells were counted and resuspended in X-VIVO 15 complete medium (5% hAB serum and cytokine cocktail: 20 ng / ml hIL-2, 20 ng / ml hGM-CSF, 10 ng / ml hIL-4, and 10 ng / ml hTGF-b1) at a cell density of 3 × 10⁶ cells / ml. For the experiment, 3 × 10⁶ cells were seeded into each well of a 12-well plate containing a final volume of 1000 μl of X-VIVO complete medium (containing the cytokine cocktail and 5 μg / ml of AIM Bio molecules or each control). On day 3, 1 ml of complete medium (containing cytokines) was added, and on day 6, a second pulse treatment was performed with 5 μg / ml of AIM Bio molecules (after removing the medium). On days 7, 10, and 12, 1 ml of complete medium (containing cytokines) was added. On day 13, the PVDF membrane of the ELISpot plate was activated with 50 μl / well EtOH (35% v / v) for 1 minute, and then washed five times with 200 μl sterile distilled water. The plate was coated overnight at 4°C with 100 μl / well anti-hIL10 (clone 9D-7, 1:500 dilution in PBS, sterile filtration). The following day, unbound coating antibody was removed, and the plate was washed five times with 200 μl PBS, 200 μl blocking buffer (X-VIVO 15 5% hAB serum) was added, and the plate was incubated at room temperature for 30 minutes to 2 hours.On day 14, 200,000 cells per well were seeded for 48 hours in a double-row ELISpot plate, including a negative control (cells + PBS) and a positive control (e.g., LPS). Secondary antibodies: 1 μg / ml aIL-10 biotinylated antibody (1:1000 dilution) and horseradish peroxidase conjugate streptavidin (0.5% BSA / PBS, 1:750 dilution) were prepared in 0.5% BSA / 1×PBS. The tetramethylbenzidine solution was filtered using a 0.45 μm filter and stored at 4°C until use. The cell supernatant was removed and the wells were washed five times with 100 μl PBS. The last excess buffer was removed using a paper towel. 25 μl diluted HRP-streptavidin (1:750) was added per well and incubated at room temperature in the dark for 1 hour, followed by five washes with sterile 1×PBS. 100 μl of filtered TMB substrate was added per well for 15–25 minutes until blue spots appeared. The reaction was stopped by thoroughly washing the wells with water. The plastic underdrain of the plate was removed, and the bottom and sides of the plate were also washed with tap water and dried. AQP4_64_G_Spt induced at least 30% more IL-10 secreting T regs in 65% of all healthy blood donors. [Figure 9-2] Percentage increase in IL10 spots in PBMCs shown for HLA-A2+ and HLA-A2- donors (in response to processing with AQP4_64_G_Spt). [Figure 10] The control experiment for the sample shown in Figure 7 demonstrates that total IgG is not reduced by treatment with the MOG47_Db_G surrogate molecule. Total IgG was quantified using the Easy-Titer® Human IgG (γ-chain) Assay Kit (Thermo Fisher) according to the manufacturer's instructions. This experiment, combined with Figure 7, demonstrates that selective antibody responses can be suppressed using single-chain MHC Ib molecules. [Figure 11-1]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 minutes. For this purpose, A) Coomasiegel staining of a 12% polyacrylamide gel using 2 μg AIM Bio and B) aHLA-G Western blotting using 1 μg protein and 2A12aHLA-G antibody (1:1000) blot were performed under non-reducing conditions. Both monomers and dimers were detectable. [Figure 11-2] This is a continuation of Figure 11-1. [Figure 12] Single-stranded MHC Ib molecules are thermally stable. For the Thermal Shift Assay (TSA), 3 μg of each single-stranded MHC Ib molecule, or motabizumab as a control molecule, was diluted to 25 μl in PBS and 5× SYPRO Orange dye (stock 5000×, final concentration: 5×). A melting curve program was set up in StepOnePlus Instrument using StepOnePlus Software 2.3. The starting temperature was 25°C for 1 minute, followed by a 1°C temperature increase per minute until the final temperature reached 95°C over 2 minutes, during which autofluorescence was measured as an arbitrary unit. The data was exported and graphed using Prism V7.04. The Boltzmann sigmoid function was used to determine the melting temperature (Tm). [Figure 13]Single-stranded MHC Ib molecules induce Tregs in a dose-dependent manner. OT-I mice were intraperitoneally injected with the indicated amounts of single-stranded H2_Kbα1+2 and HLA-Gα3 domain constructs (containing human β-2-microglobulin and the indicated peptide) or a carrier (PBS). Ova is a homologous peptide of the OT-I TCR in these mice, and Gp34 is an unrelated virus-derived control peptide. After 14 days, the mice were sacrificed, and splenocytes were examined for IL-10 secreting cells using the Recall Mouse IL-10 ELISpot kit (200,000 cells per well, MabTech Mouse IL-10 ELISpot kit, 5 μg / ml of the indicated peptide or PBS only, 48 hours). Clear induction of IL-10 secreting cells in response to the Ova peptide was observed when injected with 50 μg and 500 μg of mouse-compatible Ova_KbG. [Figure 14] Single-stranded MHC Ib molecules inhibit T cell lysis in a dose-dependent manner. Splenocytes were harvested from sacrificed OT1 / BL6 mice and washed once in RPMI 5% FCS. Erythrocytes were removed for 3 minutes using 2 ml of 1× sterile erythrocyte lysis buffer. Cells were cultured at high density (10⁷ cells / ml) for 72 hours in RPMI 10% FCS medium containing GMCSF 20 ng / mL, IL-2 20 ng / mL, and IL-4 10 ng / mL, with increasing doses of Ova_KbG. Cells were then scraped from plates, and CD8+ cells were purified using magnetic beads. Sterile 96-well white plates were used. Luciferase-expressing Panc02 target cells were loaded with 20 μg / mL of Ova peptide (SIINFEKL) at 37°C for 60 minutes with shaking at 500 rpm. CD8+ effector T cells were added in a 50:1 ratio, as well as luciferin. Luminescence was measured after 24 hours and 48 hours. [Figure 15]Serum cytokines from EAE-ODC Ova mice. Serum cytokines from EAE-ODC Ova mice were measured using the Th1 / Th2 10plex Flowcytomix kit (eBioscience) according to the manufacturer's instructions. This kit was used for the simultaneous detection of 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. This array kit provides a mixture of eight different capture beads with varying fluorescence intensities, each coated with a capture antibody specific to that cytokine. A mixture of beads coated with 10 different specific capture antibodies was used. Subsequently, 25 μL of the mixed capture beads, 25 μL of an unknown serum sample or standard diluent, and 25 μL of phycoerythrin (PE) detection reagent were successively added to each well of a 96-V bottom-well plate and incubated in the dark at room temperature for 2 hours. The samples were washed with 1 mL of washing buffer for 5 minutes and centrifuged. After discarding the supernatant, the bead pellet was resuspended in 200 μL of buffer. The samples were measured using an Attune® NxT flow cytometer and analyzed with Attune Cytometric Software (Thermo Fisher Scientific). [Figure 16]An exemplary reference mouse-fitted AIM Bio molecular structure map. The corresponding exemplary amino acid sequence is as follows: MSRSVALAVLALLSLSGLEAVTTVHGNLGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSF RVDLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC (Sequence ID 38) [Figure 17] Kooma Siegel of purified and stability-tested single-stranded MHC Ib molecules of NMO surrogates. After purification, the stability of the single-stranded 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 minutes. See Figure 11 for the methods. [Figure 18] Western blot of purified and stability-tested NMO surrogate single-strand MHC Ib molecules (2A12 HLA-G5 antibody 1:5000). All experiments showed that these molecules are very stable, but AQP203_H2KbG showed a stronger tendency to polymerize. [Figure 19] Thermal shift assay (TSA). The results obtained in the previous figure were also confirmed by TSA, where the polymer exhibited high autofluorescence at low temperatures. [Figure 20]EAE score. AQP147_KbG provided complete protection from EAE in treated 2D2 mice. Mice were injected with either the treatment or the control AIM Bio on D0, D15, and D30. Mice were evaluated daily based on the 0-10 EAE scoring system described in Table 1. Untreated mice developed severe EAE symptoms throughout the experiment. One untreated mouse reached a score of 6 on D14 and was euthanized. Its score was estimated to be 6 for the remaining days of the evaluation period. The control treatment with gp34_KbG did not provide the desired protection. (*p<0.05, **p<0.01, ***p<0.001) [Figure 21-1] Immune cell infiltration in the optic nerve of wild-type and 2D2 mice. AQP147_KbG treatment inhibited immune cell infiltration in the optic nerve. CD3+ and CD8+ fluorescence signals obtained from each treatment group are shown in panels A (optic nerve) and B (spinal cord). DAPI images show all nuclei present in each section. The number of CD3+ and CD8+ T cells was significantly higher in the optic nerve (panel C) and spinal cord (panel D) of control and untreated mice. AQP203_KbG inhibited immune infiltration better than the control and untreated groups. Data are expressed as mean + / - SEM. Scale bar 25 μm. One-sided t-test. [Figure 21-2] This is a continuation of Figure 21-1. [Figure 21-3] This is a continuation of Figure 21-2. [Figure 21-4] This is a continuation of Figure 21-3. [Figure 22-1] Quantification of truncated caspase-3 in the optic nerve, spinal cord, and retina of wild-type and 2D2 mice. AQP147_KbG completely inhibits apoptosis in the optic nerve, spinal cord, and retina. Fluorescence signals obtained from truncated caspase-3 (apoptosis marker) staining in the optic nerve (panels A, B), spinal cord (panels C, D), and retina (panels E, F) are shown. Untreated and control-treated mice had the highest levels of truncated caspase-3 and therefore the highest levels of apoptosis. Data are represented as mean + / - SEM. Scale bar 25 μm. One-sided t-test. [Figure 22-2]This is a continuation of Figure 22-1. [Figure 22-3] This is a continuation of Figure 22-2. [Figure 22-4] This is a continuation of Figure 22-3. [Figure 22-5] This is a continuation of Figure 22-4. [Figure 22-6] This is a continuation of Figure 22-5. [Figure 23-1] CD8+CD122+ and CD4+FoxP3+ regulatory T cells in the optic nerve. This figure shows that while AIM-treated mice have fewer CD8 T cells, they clearly have more regulatory T cells in the optic nerve, albeit fewer (using a one-sided t-test). [Figure 23-2] This is a continuation of Figure 23-1. [Figure 24-1] Quantification of retinal inner layer thickness in wild-type and 2D2 mice. AQP147_KbG completely rescues IRL cells from degradation. Hematoxylin-eosin staining results show that retinal inner layer thickness was preserved in AQP147_KbG mice, while untreated and control-treated mice had thinner IRLs. Data are presented as mean + / - SEM. Scale bar 25 μm. Two-sided t-test. [Figure 24-2] This is a continuation of Figure 24-1. [Figure 25-1] A, B, D: Simple Western results; C, E: Supernatant concentrations measured by ELISA. The constructs used in this figure are as specified in the "Sequence" section below and in Example 8 below. Simple Western was performed as described in Example 8; ELISA was performed as a sandwich ELISA as described in Example 8. [Figure 25-2] This is a continuation of Figure 25-1. [Figure 25-3] This is a continuation of Figure 25-2. [Figure 25-4] This is a continuation of Figure 25-3. [Figure 25-5] This is a continuation of Figure 25-4. [Figure 26-1]ELISpot using patient PBMCs. A: Photograph of the wells in the assay. The number of counted spots is shown in the figure. B: Quantification. ELISpot was performed as described in Example 8. In 4 out of 5 NMO patient PBMCs treated with AIM Bio AQP65P65K_G, the number of IL-10 spots increased compared to untreated PBMCs. [Figure 26-2] This is a continuation of Figure 26-1. [Figure 27] Optimization of anchor residues in hAQPA_HLA-G AIM Bio. A: HLA-G peptide binding motif. B: The HLA-G binding peptide motif was described by Di Marco et al. (Moreno Di Marco, Heiko Schuster, Linus Backert, Michael Ghosh, Hans-Georg Rammensee, Stefan Stevanovic; Unveiling the Peptide Motifs of HLA-C and HLA-G from Naturally Presented Peptides and Generation of Binding Prediction Matrices. J Immunol 15 October 2017; 199 (8): 2639-2651. https: / / doi.org / 10.4049 / jimmunol.1700938). Their analysis suggests preferred amino acids at specific positions for tethering the peptide to the groove formed by the a1 and a2 domains of naturally occurring HLA-G. The inventors believed that these findings could provide a basis for optimizing the recombinant polypeptide of the present invention. They found that optimization of anchor residues could be advantageously carried out for the polypeptide of the present invention (also referred to herein as "AIM Bio"), resulting in a more stable molecule with improved production levels. [Figure 28] Size exclusion chromatography analysis of purified AQP4126-135_A2G [Figure 29]Thermal shift assay: Melting curve analysis of AIM Bio AQP126-135_A2G. [Figure 30] Jess Simple Western run of AIM Bio optimized for AQP65-72_P65K_G and AQP65-72_P65R_G anchor residues. [Figure 31] ILT4-Fc binding ELISA [Modes for carrying out the invention]

[0042] Definitions and General Techniques Unless otherwise defined below, terms used in this invention should be understood in accordance with their general meanings known to those skilled in the art. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. Publications referred herein may be cited by specifying the complete references within the text.

[0043] All proteins according to the present invention, including the recombinant polypeptide of the present invention, can be obtained by methods known in the art. Such methods include methods for the production of recombinant polypeptides. The recombinant polypeptide of the present invention can be expressed in recombinant host cells according to the present invention. Recombinant host cells of the present invention are mammalian cells such as CHO and HEK cells.

[0044] It will be understood that the recombinant polypeptide of the present invention may optionally include a secretion signal peptide sequence. Similarly, it will be understood that the recombinant polypeptide of the present invention may include, for example, an affinity tag to facilitate purification, and an optional protease cleavage site between the tag and the polypeptide to facilitate removal of the tag by, for example, protease cleavage.

[0045] Furthermore, it should be understood that references to amino acid sequences referred to herein include not only unmodified amino acid sequences but also typical post-translational modifications of these amino acid sequences that occur in cell expression systems known in the art, including mammalian cells such as CHO and HEK cells (e.g., glycosylation or deamidation of amino acids, clipping of specific amino acids, or other post-translational modifications).

[0046] Similarly, it will be understood that the recombinant polypeptides of the present invention may contain their respective propeptides.

[0047] It will also be understood that the recombinant polypeptides of the present invention may be in a soluble form or a membrane-bound form. Whether a recombinant polypeptide is "soluble" under these conditions can be determined by methods known in the art, for example, by measuring the turbidity of the recombinant polypeptide under the reference conditions shown above. As used herein, solubility means that at least 95% of the recombinant polypeptide is determined to be soluble under these reference conditions.

[0048] Single-chain MHC molecules can be stored, for example, in PBS at -80°C (with or without 0.1% human albumin as a carrier, depending on the protein concentration) or in 50% glycerol at 20°C.

[0049] According to the present invention, the MHC molecule is preferably a human MHC molecule.

[0050] The recombinant polypeptide of the present invention is preferably an isolated recombinant polypeptide.

[0051] A method for preparing recombinant polypeptides capable of binding to and presenting peptide antigens according to the present invention will be understood. For example, peptide antigen-binding domains such as the [alpha]1 and [alpha]2 domains are well known, and these domains can be modified. The ability of the polypeptides and peptide antigens according to the present invention to bind to 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 in silico bioinformatics prediction, as well as confirmatory methods such as MHC peptide polymerization and stimulation assays.

[0052] According to the present invention, the recombinant polypeptide, pharmaceutical composition, and kit of the present invention are preferably suitable for use in human patients.

[0053] According to the present invention, the recombinant polypeptide, pharmaceutical composition, and kit of the present invention are preferably suitable for use in the treatment of neuromyelitis optica in human patients.

[0054] According to the present invention, the recombinant polypeptide, pharmaceutical composition, and kit are preferably suitable for inducing immune tolerance to human aquaporin 4 in, for example, human patients.

[0055] According to the present invention, these recombinant polypeptides, pharmaceutical compositions, and kits of the present invention, wherein the [alpha]1 domain in (v) and the [alpha]2 domain in (vi) are derived from a human HLA-A2 molecule, are preferably for use in the treatment of neuromyelitis optica (NMO) in HLA-A2 positive human patients. This use is expected to be associated with a more favorable (lower immunogenicity) immunogenicity profile than in HLA-A2 negative patients.

[0056] In contrast, HLA-G does not exhibit the high levels of polymorphism observed with respect to classical HLA alleles. The HLA-G-based polypeptides of the present invention are expected to have a favorable immunogenicity profile and half-life across the human population.

[0057] According to the present invention, it is understood that the recombinant polypeptide, pharmaceutical composition, and kit of the present invention are stable.

[0058] In relation to peptide antigens used in accordance with the present invention, it will be understood that any length of these peptide antigens referred to herein (e.g., "7-11 amino acid length") refers to the length of the peptide antigen itself. Therefore, the length of the peptide antigen referred to herein does not include the length conferred by additional amino acids that are not part of the peptide antigen, such as additional amino acids derived from possible linker sequences, etc.

[0059] According to the present invention, each occurrence of the term "contains" may be replaced with the term "consisting of".

[0060] Methods and Techniques Generally, unless otherwise defined herein, the methods used in the present invention (e.g., cloning methods or methods related to antibodies) are carried out in accordance with procedures known in the art, such as those described in Sambrook et al. ("Molecular Cloning: A Laboratory Manual," 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1989), Ausubel et al. ("Current Protocols in Molecular Biology," Greene Publishing Associates and Wiley Interscience; New York 1992), and Harlow and Lane ("Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1988) (all incorporated herein by reference).

[0061] Protein-protein binding, such as antibody binding to each of these target proteins, can be evaluated by methods known in the industry. Protein-protein binding is preferably evaluated by surface plasmon resonance spectroscopy.

[0062] For example, the binding of MHC class Ib molecules or recombinant polypeptides according to the present invention to their receptors (e.g., ILT2 and ILT4) is preferably evaluated by surface plasmon resonance spectroscopy. More preferably, the binding of MHC class Ib molecules or recombinant polypeptides according to the present invention to their receptors is evaluated by surface plasmon resonance spectroscopy at 25°C. Suitable conditions for such surface plasmon resonance spectroscopy are described by Shiroishi et al., Proc Natl Acad Sci USA. 2003 July 22;100(15):8856-8861.

[0063] Sequence alignment of sequences 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. p. 403-410; Altschul et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402). Appropriate parameters for sequence alignment of short peptides using the BLAST algorithm, suitable for peptide antigens according to the present invention, are known in the art. Many software tools using the BLAST algorithm automatically adjust the parameters for sequence alignment for short input sequences. In one embodiment, the following parameters are used: maximum target sequence 10; word size 3; BLOSUM62 matrix; gap cost: present 11, extended 1; conditional composition score matrix adjustment. Therefore, when used in relation to sequences, terms such as "identity" or "identical" preferably refer to the identity value obtained by using the BLAST algorithm.

[0064] Preparation of the pharmaceutical composition of the present invention The pharmaceutical composition of the present invention is prepared according to known standards for the preparation of pharmaceutical compositions.

[0065] For example, pharmaceutical compositions are prepared in a manner that allows them to be stored and administered appropriately. The pharmaceutical compositions of the present invention may therefore contain pharmaceutically acceptable components such as carriers, excipients and / or stabilizers.

[0066] Such pharmaceutically acceptable components are not toxic in the amounts used when the pharmaceutical composition is administered to human patients. The pharmaceutically acceptable components added to a pharmaceutical composition depend on the chemical properties of the active ingredients present in the composition, the specific intended use of the pharmaceutical composition, and the route of administration.

[0067] Generally, pharmaceutically acceptable components used in connection with the present invention are used in accordance with the knowledge available in the industry, for example, from Remington's Pharmaceutical Sciences, Ed. AR Gennaro, 20th edition, 2000, Williams & Wilkins, PA, USA. Pharmaceutical compositions comprising nucleic acids (e.g., RNA) of the present invention can also be formulated in accordance with the knowledge available in the industry, for example, using liposomal formulations targeting dendritic cells.

[0068] The peptide antigen according to the present invention Peptide antigens that can be used in accordance with the present invention, including the peptide antigens defined above, are not particularly limited other than their ability to be presented on MHC molecules. The “peptide antigen presented by the recombinant polypeptide” as referred to in connection with the present invention is understood to be the peptide antigen presented to human T cells by the recombinant polypeptide, provided that such T cells are present such that the peptide antigen binds to T cell receptors on human T cells.

[0069] Peptides that can be presented on MHC molecules can be generated as is well known in this industry (see, for example, Rammensee, Bachmann, Emmerich, Bachor, Stevanovic. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics. 1999 Nov;50(3-4):213-9; Pearson et al., MHC class I-associated peptides derive from selective regions of the human genome. J Clin Invest. 2016 Dec 1;126(12):4690-4701; and Rock, Reits, Neefjes. Present Yourself! By MHC Class I and MHC Class II Molecules. Trends Immunol. 2016 Nov;37(11):724-737).

[0070] Peptide antigens are generally known in the art. Generally, the peptide antigens according to the present invention can bind to MHC class I proteins. Those skilled in the art will understand that, for each MHC class Ib molecule or polypeptide capable of presenting the peptide according to the present invention, a peptide antigen capable of binding to the said MHC class Ib molecule or recombinant polypeptide is preferably used. These peptide antigens can be selected based on methods known in the art.

[0071] The attachment of a peptide antigen to an MHC class Ib molecule, or to a polypeptide capable of peptide antigen binding according to the present invention, can be done by methods known in the art, for example, Rammensee, Bachmann, Emmerich, Bachor, Stevanovic. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics. 1999 Nov;50(3-4):213-9; Pearson et al., MHC class I-associated peptides derive from selective regions of the human genome. J Clin Invest. 2016 Dec 1;126(12):4690-4701; and Rock, Reits, Neefjes. Present Yourself! By MHC Class I and MHC Class II Molecules. Trends Immunol. 2016 Nov;37(11):724-737 It can be evaluated by the following method.

[0072] Such methods include experimental methods and methods for predicting peptide antigen binding.

[0073] Anchor residues that are useful for immobilizing peptide antigens on MHC class I molecules and for ensuring the binding of peptide antigens to MHC class I molecules are well known in the art.

[0074] In preferred embodiments according to all embodiments of the present invention, the peptide antigen used in accordance with the present invention contains either an anchor or a preferred amino acid residue at a predicted position for an MHC class I molecule.

[0075] Such predictions can preferably be made as described in one of the following publications: - Rammensee et al., SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics (1999) 50: 213-219 - Nielsen et al., Protein Sci (2003) 12:1007-1017 - Neefjes et al., Nat Rev Immunol. 2011 Nov 11;11(12):823-36 - Diehl et al., Curr Biol. 1996 Mar 1;6(3):305-14 - Lee et al., Immunity. 1995 Nov;3(5):591-600 - Desai & Kulkarni-Kale, T-cell epitope prediction methods: an overview. Methods Mol Biol. 2014;1184:333-64 - Jumper et al., Highly accurate protein structure prediction with AlphaFold. Nature 2021;596:583-589.

[0076] In this invention, the peptide antigen is derived from human aquaporin 4.

[0077] It is understood that the non-anchored amino acid residues of the peptide antigen of the present invention may or may not contain conservative substitutions, preferably two or fewer, and more preferably one, with respect to the corresponding amino acid sequence of the peptide antigen derived from human aquaporin 4.

[0078] The peptide antigen of the present invention preferably consists of naturally occurring amino acids. However, 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 model of the shown peptide antigen amino acid sequence of human aquaporin 4.

[0079] Methods for synthesizing peptide antigens, including the peptide antigen according to the present invention, are well known in the art.

[0080] Therapeutic application of the present invention The recombinant polypeptide of the present invention can be used for the treatment of neuromyelitis optica.

[0081] The treatment may involve inducing myelin-specific regulatory T cells. Since such regulatory T cells (e.g., CD8-positive regulatory T cells) are activated in myelinated structures, they provide target cell protection against cytotoxic T cells that recognize the same or a different myelin antigen. Regulatory T cells (e.g., CD8-positive regulatory T cells) are well known in the art and can be detected, for example, by their secretion of IL-10.

[0082] CD8-positive regulatory T cells are not as well-known as CD4-CD25 regulatory T cells, but they have even been described as being more potent. For example, Junfeng Liu , Dacan Chen, Golay D. Nie and Zhenhua Dai CD8+CD122+ T-Cells: A Newly Emerging Regulator with Central Memory Cell Phenotypes. Front. Immunol. doi: 10.3389 / fimmu.2015.00494; and Niederlova, V., Tsyklauri, O., Chadimova, T. and Stepanek, O. (2021), CD8 + Tregs revisited: A heterogeneous population with different phenotypes and properties. Eur. J. Immunol., 51: 512-530. https: / / doi.org / 10.1002 / eji.202048614 Please refer to the following.

[0083] These are characterized by the expression of CD122 and CD8 in mice, but their human counterparts are described as being positive for CD8 and CXCR3. For example, 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) Please refer to the following.

[0084] The treatment according to the present invention may be a treatment to reduce the plasma level or cerebrospinal fluid (CSF) level of autoantibodies against human aquaporin 4. The human patient may be a patient who had plasma autoantibodies or cerebrospinal fluid (CSF) autoantibodies against human aquaporin 4 prior to the initiation of treatment.

[0085] According to the present invention, autoantibodies can be detected by various methods known in the art. A preferred approach is a cell-based assay (CBA) in which an antigen suspected to be the target of an autoantibody (e.g., aquaporin 4) 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. Mock-transfected sister cells serve as a control. Autoantibodies that bind to cells are detected with different fluorescently labeled anti-human specific secondary antibodies that recognize whole human IgG (heavy and light chains), IgG-Fc (constant chain), or IgG1. Binding is quantified by flow cytometry (CBA-FACS) or by visual scoring by microscopic evaluation of immunofluorescence (CBA-IF), and titers are often determined. Other approaches such as enzyme-linked immunosorbent assay (ELISA) or Western blotting are also possible, but are often less sensitive because conformation-affected antibodies may not be detected by these methods. A preferred approach is: Waters, P., Pettingill, P. & Lang, B. Detection methods for neural autoantibodies. Handb. Clin. Neurol.133, 147-163 (2016) It is described there.

[0086] The method for detecting Aqp4-specific autoantibodies is as follows: 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) It will be listed.

[0087] array The preferred amino acid sequences referred to in this application can be independently selected from the following sequences. The sequences are represented in N-terminus to C-terminus order and are represented by a single-letter amino acid code.

[0088] An example sequence, which is part of the recombinant polypeptide of the present invention: Optionally selected leader peptides (not present in recombinant polypeptides due to processing during cell expression): For example, DNA sequence: MSRSVALAVLALLSLSGLEA (SEQ ID NO: 1) encoded by ATGAGCAGATCTGTGGCCCTGGCTGTTCTGGCTCTGCTGTCTCTGTCTGGCCTGGAAGCC (SEQ ID NO: 41)

[0089] Alternative optional leader peptides: Example of mouse Ig heavy chain peptide: MGWSCIILFLVATATGVHS (SEQ ID NO: 88) Example of human OSM peptide: MGVLLTQRTLLSLVLALLFPSMASM (SEQ ID NO: 89) Example of VSV-G peptide: MKCLLYLAFLFIGVNC (SEQ ID NO: 90) Example of mouse Ig kappa peptide: METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 91) Example of BM40 peptide: MRAWIFFLLCLAGRALA (SEQ ID NO: 92) An example of a secreton peptide: MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO: 93) Example of human IgKVIII peptide: MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 94) Example of a CD33 peptide: MPLLLLLPLLWAGALA (SEQ ID NO: 95) Example of a tPA peptide: MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 96) Example of human chymotrypsinogen peptide: MAFLWLLSCWALLGTTFG (SEQ ID NO: 97) Example of human trypsinogen-2 peptide: MNLLLILTFVAAAVA (SEQ ID NO: 98) Example of human IL-2 peptide: MYRMQLLSCIALSLALVTNS (SEQ ID NO: 99) Example of Gaussia luc peptide: MGVKVLFALICIAVAEA (SEQ ID NO: 100) Example of albumin (HSA) peptide: MKWVTFISLLFSSAYS (SEQ ID NO: 101) An example of influenza hemagglutinin peptide: MKTIIALSYIFCLVLG (SEQ ID NO: 102) Example of human insulin peptide: MALWMRLLPLLALLALWGPDPAAA (SEQ ID NO: 103) Example of silkworm fibroin LC peptide: MKPIFLVLLVVTSAYA (SEQ ID NO: 104)

[0090] Peptide antigen: Any MHC class I peptide corresponding to the MHC class I[α]1 and 2 domains, e.g., KPLPVDMVL (SEQ ID NO: 2), or any peptide antigen according to the present invention (all derived from human AQP4): hAQP4 126-135_A2G Myc / His Tag AIIGAGILYL Sequence ID 34 AQP4_111-119_A2G_fs-_Spt SIAKSVFYI Sequence ID 42 AQP4_242-251_A2G_fs-_Spt VLAGGLYEYV Sequence ID 43 AQP4_292-301_A2G_fs-_Spt LILKPGVVHV Sequence ID 44 AQP4_293-301_A2G_fs-_Spt ILKPGVVHV Sequence ID 45 AQP4_210-218_A2G_fs-_Spt ASMNPARSF Sequence ID 46 AQP134-142_A2G_fs-_Spt YLVTPPSVV Sequence ID 82 AQP4_293-301_G_fs-_Spt ILKPGVVHV Sequence ID 47 AQP4_65-72_P65K_G_fs-_Spt KLPVDMVL Sequence ID 48 AQP4_65-72_P65R_G_fs-_Spt RLPVDMVL Sequence ID 49 AQP4_126-134_A126R_G_fs-_Spt RIIGAGILY Sequence ID 50 AQP4_164-172_L164R_F172L_G_fs-_Spt RIITFQLVL Sequence ID 51

[0091] The first linker: for example, GGGGSGGGGSGGGGS (sequence number 3), GCGASGGGGSGGGGS (sequence number 4), or GCGASGGGGSGGGGS (sequence number 52).

[0092] Beta-2 microglobulin, for example: IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM (Sequence ID 5, Human Beta-2 Microglobulin).

[0093] A second linker, for example: GGGGSGGGGSGGGGSGGGGS(SEQ6).

[0094] The [alpha]1&2 domain, Y84, derived from human HLA-G or any other MHC class I [alpha]1&2 domain suitable for presenting a selected antigen peptide, may be C or A in the DT variant.

[0095] For example, the [alpha]1 and [alpha]2 domains derived from human HLA-G: for example GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRA (Sequence ID 7) Or: Human HLA-A2 [alpha]1 & 2 domains: For example, GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRT(Sequence ID 8)

[0096] Human HLA-G[alpha]3 domain (or any MHC Ib[alpha]3 domain, such as HLA-F that also interacts with ILT2 and ILT4 receptors), e.g.:DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDL (SEQ ID NO: 9; sequence of HLA-G[alpha]3 having a furin cleavage site in the C-terminal amino acid sequence derived from intron 4 of human HLA-G), or DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESGSLSEDL (Sequence code 53; sequence of HLA-G[alpha]3 with the furin cleavage site removed from the C-terminal amino acid sequence derived from intron 4 of human HLA-G).

[0097] Note that the following underlined amino acids in this sequence are suitable for ILT2 or ILT4 receptor interaction: [ka]

[0098] Alternatively, a shorter human HLA-G[alpha]3 domain lacking an optional C-terminal amino acid sequence derived from intron 4 can be used (SKEGDGGIMSVRESRSLSEDL; SEQ ID NO: 20, or SKEGDGGIMSVRESGSLSEDL; SEQ ID NO: 54), i.e.: DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (Sequence ID 21).

[0099] The inventors have found that recombinant polypeptides can be produced using any one of the above sequences of HLA-G[alpha]3. The presence of the sequence of SEQ ID NO: 53 in the recombinant polypeptide, i.e., the presence of the HLA-G[alpha]3 sequence in which the fulin cleavage site in the C-terminal amino acid sequence derived from intron 4 of human HLA-G has been removed, was found to be advantageous for improving the purification of recombinant polypeptides, for example, for the purification of recombinant polypeptides containing a C-terminal tag.

[0100] Factor Xa restriction site: IEGRTGTKLGP (SEQ ID NO: 10) SpotTag:PDRVRAVSHWSSC (Sequence ID 11) Myc tag:EQKLISEEDL(sequence number 12) His tag: HHHHHH * (Sequence ID 13) Spacer array: For example, NSAVD (sequence number 14) or GS.

[0101] Exemplary reference peptide antigens that may be part of a recombinant polypeptide are as follows: Preferably, VLISLCFGL (SEQ ID NO: 22) in a recombinant polypeptide containing HLA-Gα1 and 2 domains Preferably, IIGAGILYL (SEQ ID NO: 23) in a recombinant polypeptide containing HLA-A2α1 and 2 domains Preferably, a recombinant polypeptide containing HLA-A2α1 and 2 domains, IIGAVLAGGL (SEQ ID NO: 24)

[0102] The following are exemplary peptide antigens according to the present invention that may be part of the recombinant polypeptide of the present invention: Preferably, a recombinant polypeptide containing HLA-A2α1 and α2 domains and HLA-G[α]3 domain, and AIIGAGILYL (SEQ ID NO: 34) Preferably, a recombinant polypeptide containing HLA-A2α1 and α2 domains and HLA-G[α]3 domain, containing SIAKSVFYI (SEQ ID NO: 42) Preferably, a recombinant polypeptide containing HLA-A2α1 and α2 domains and HLA-G[α]3 domain, VLAGGLYEYV (SEQ ID NO: 43) Preferably, LILKPGVVHV (SEQ ID NO: 44) in a recombinant polypeptide containing HLA-A2α1 and α2 domains and HLA-G[α]3 domain Preferably, a recombinant polypeptide containing HLA-A2α1 and α2 domains and HLA-G[α]3 domain, containing ILKPGVVHV (SEQ ID NO: 45) Preferably, ASMNPARSF (SEQ ID NO: 46) is present in a recombinant polypeptide containing HLA-A2α1 and α2 domains and HLA-G[α]3 domain. Preferably, a recombinant polypeptide containing HLA-A2α1 and α2 domains and HLA-G[α]3 domain, YLVTPPSVV (SEQ ID NO: 82) Preferably, a recombinant polypeptide containing human HLA-G-derived α1 and α2 domains and an HLA-G[α]3 domain, containing ILKPGVVHV (SEQ ID NO: 47) Preferably, a recombinant polypeptide containing human HLA-G-derived α1 and α2 domains and HLA-G[α]3 domain, containing KLPVDMVL (SEQ ID NO: 48) Preferably, a recombinant polypeptide containing human HLA-G-derived α1 and α2 domains and HLA-G[α]3 domain, RLPVDMVL (SEQ ID NO: 49) Preferably, a recombinant polypeptide containing human HLA-G-derived α1 and α2 domains and an HLA-G[α]3 domain, RIIGAGILY (SEQ ID NO: 50) Preferably, a recombinant polypeptide containing human HLA-G-derived α1 and α2 domains and an HLA-G[α]3 domain, RIITFQLVL (SEQ ID NO: 51)

[0103] Examples of reference recombinant polypeptides (including optional leader peptides): MSRSVALAVLALLSLSGLEAKPLPVDMVLGCGASGGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTE KDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTD RMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC* (Sequence ID 15; Note that the asterisk represents a stop codon)

[0104] The peptide antigen sequence of the full-length recombinant polypeptide described above (here, KPLPVDMVL) can be replaced with any peptide antigen sequence according to the present invention, i.e., any peptide antigen presented by the recombinant polypeptide, and it should be noted that this peptide antigen is a peptide of human aquaporin 4. That is, the recombinant polypeptide of the present invention consists of a sequence of peptide antigens that are peptides of human aquaporin 4 (for example, any one of the peptide antigens of SEQ ID NOs. 2, 22, 23, and 24 according to the present invention, or preferably any one of the peptide antigens of SEQ ID NOs. 34, 42-51, and 82), followed thereafter GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWD RDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGCYNQS The sequence EASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC* (Sequence ID 16; note that the asterisk represents a stop codon), or the sequence following it. CGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDR DMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSE The sequence AGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESGSLSEDLGSPDRVRAVSHWSSC* (Sequence ID 55; note that the asterisk represents a stop codon), or the sequence following it. GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWD RDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQS The sequence EAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESGSLSEDLGSPDRVRAVSHWSSC* (Sequence ID 56; note that the asterisk represents a stop codon), or the sequence following it. GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKI VKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTL RGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHV THHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGGSPDRVRAVSHWSSC* The sequence (sequence number 57; note that the asterisk represents a stop codon), or following CGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAH SQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW The sequence of the HLA-G[α]3 domain in (SEQ ID NO: 58), the C-terminal amino acid sequence derived from intron 4, the variable cleavage site, and the sequence without an optional tag, or the sequence following it. The sequence GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (Sequence ID 59), or the sequence following it. It can consist of the sequence GCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (sequence number 60).

[0105] Examples of preferred recombinant polypeptides of the present invention (shown without an optional leader peptide, without an optional C-terminal amino acid sequence derived from intron 4, and without an optional tag): HLA-A2G structure: AQP 126-135 _A2G; Most preferred Protein sequence: [ka] DNA sequences (Note that the following DNA sequences do not represent the DNA sequences encoding the N-terminal leader peptide): [ka] Coded by

[0106] AQP4_111-119_A2G_fs-_Spt Protein sequence: [ka] DNA sequences (Note that the following DNA sequences do not represent the DNA sequences encoding the N-terminal leader peptide): [ka] Coded by

[0107] AQP4_242-251_A2G_fs-_Spt Protein sequence: [ka] [ka] DNA sequences (Note that the following DNA sequences do not represent the DNA sequences encoding the N-terminal leader peptide): [ka] Coded by

[0108] AQP4_292-301_A2G_fs-_Spt Protein sequence: [ka] [ka] DNA sequences (Note that the following DNA sequences do not represent the DNA sequences encoding the N-terminal leader peptide): [ka] Coded by

[0109] AQP4_293-301_A2G_fs-_Spt Protein sequence: [ka] DNA sequences (Note that the following DNA sequences do not represent the DNA sequences encoding the N-terminal leader peptide): [ka] Coded by

[0110] AQP4_210-218_A2G_fs-_Spt - Monomer Protein sequence: ASMNPARSFGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (Sequence ID 69) DNA sequences (Note that the following DNA sequences do not represent the DNA sequences encoding the N-terminal leader peptide): [ka] Coded by

[0111] AQP134-142_A2G_fs-_Spt Protein sequence YLVTPPSVVGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRW (Sequence ID 83) DNA sequence

[0112] HLA-G structure: AQP4_293-301_G_fs-_Spt Protein sequence: [ka]

[0113] DNA sequences (Note that the following DNA sequences do not represent the DNA sequences encoding the N-terminal leader peptide): [ka] [ka] Coded by

[0114] Optimization of anchor residues AQP4_65-72_P65K_G_fs-_Spt; Most preferred Protein sequence:

[0115] [ka] DNA sequences (Note that the following DNA sequences do not represent the DNA sequences encoding the N-terminal leader peptide): [ka] [ka] Coded by

[0116] AQP4_65-72_P65R_G_fs-_Spt Protein sequence: [ka]

[0117] DNA sequences (Note that the following DNA sequences do not represent the DNA sequences encoding the N-terminal leader peptide): [ka] [ka] Coded by

[0118] AQP4_126-134_A126R_G_fs-_Spt Protein sequence:

[0119] [ka] DNA sequences (Note that the following DNA sequences do not represent the DNA sequences encoding the N-terminal leader peptide): [ka] [ka] Coded by

[0120] AQP4_164-172_L164R_F172L_G_fs-_Spt Protein sequence:

[0121] [ka] DNA sequences (Note that the following DNA sequences do not represent the DNA sequences encoding the N-terminal leader peptide): [ka] [ka] Coded by

[0122] The recombinant polypeptides of the present invention shown above may also contain any one of the following: an optional leader peptide, a C-terminal amino acid sequence derived from intron 4 of the HLA-G[α]3 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.

[0123] The receptors ILT2 (also known as LILRB1) and ILT4 (also known as LILRB2) are well known in the art. Preferred sequences of these receptors according to the present invention are as follows:

[0124] ILT2: MTPILTVLICLGLSLGPRTHVQAGHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRL YREKKTALWITRIPQELVKKGQFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTG AYIKPTLSAQPSPVVNSGGNVILQCDSQVAFDGFSLKEGEDEHPQCLNSQPHARGSSRA IFSVGPVSPSRRWWYRCYAYDSNSPYEWSLPSDLLELLVLGVSKKPSLSVQPGPIVAPEE TLTLQCGSDAGYNRFVLYKDGERDFLQLAGAQPQAGLSQANFTLGPVSRSYGGQYRCYGA HNLSSEWSAPSDPLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKE GAADDPWRLRSTYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLTHPSDPLELVVS GPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLLLLF LILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQ PEDGVEMDTRSPHDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMD TEAAASEAPQDVTYAQLHSLTLRREATEPPPSQEGPSPAVPSIYATLAIH (Sequence ID 17).

[0125] ILT4: MTPIVTVLICLGLSLGPRTHVQTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRL YREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGA YPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEEEHPQCLNSQPHARGSSRAI FSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPGVSKKPSLSVQPGPVVAPGES LTLQCVSDVGYDRFVLYKEGERDLRQLPGRQPQAGLSQANFTLGPVSRSYGGQYRCYGAH NLSSECSAPSDPLDILITGQIRGTPFISVQPGPTVASGENVTLLCQSWRQFHTFLLTKAG AADAPLRLRSIHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSDPYLLSHPSEPLELVVSG PSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVVLLLLLLLLLF LILRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKDTQ PEDGVEMDTRAAASEAPQDVTYAQLHSLTLRRKATEPPPSQEREPPAEPSIYATLAIH(Sequence ID 18).

[0126] The sequence of human aquaporin 4 is known in the art. The preferred amino acid sequence of human aquaporin 4 is as follows: >sp|P55087-2|AQP4_HUMAN Isoform 1 of Aquaporin-4 OS=Homo sapiens OX=9606 GN=AQP4 MVAFKGVWTQAFWKAVTAEFLAMLIFVLLSLGSTINWGGTEKPLPVDMVLISLCFGLSIA TMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPSVV GGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDSKRTDVTGSIALAIGFSVAIGHL FAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRF KEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSS V (SEQ ID NO: 19)

[0127] >sp|P55087|AQP4_HUMAN Aquaporin-4 Isoform 2 OS=Homo sapiens OX=9606 GN=AQP4 PE=1 SV=2 MSDRPTARRWGKCGPLCTRENIMVAFKGVWTQAFWKAVTAEFLAMLIFVLLSLGSTINWG GTEKPLPVDMVLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIA AQCLGAIIGAGILYLVTPPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDS KRTDVTGSIALAIGFSVAIGHLFAINYTGASMNPARSFGPAVIMGNWENHWIYWVGPIIG AVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEVEDNRSQVETDDLILKPGVVH VIDVDRGEEKKGKDQSGEVLSSV (SEQ ID NO: 29)

[0128] >NP_001304313.1 aquaporin-4 isoform M1x [Homo sapiens] MSDRPTARRWGKCGPLCTRENIMVAFKGVWTQAFWKAVTAEFLAMLIFVLLSLGSTINWGGTEKPLPVDM VLISLCFGLSIATMVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVTPPS VVGGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDSKRTDVTGSIALAIGFSVAIGHLFAINYTGA SMNPARSFGPAVIMGNWENHWIYWVGPIIGAVLAGGLYEYVFCPDVEFKRRFKEAFSKAAQQTKGSYMEV EDNRSQVETDDLILKPGVVHVIDVDRGEEKKGKDQSGEVLSSVXLEDRTESRQDSLELSSDFLPPIKETD LL (Sequence ID 30; X may be tryptophan, cysteine, arginine, serine, or a stop codon)

[0129] The present invention is further illustrated by the following non-limiting embodiments: [Examples]

[0130] Example 1 Method for producing recombinant polypeptides according to the present invention - Expi-293F cells (Thermo Fisher) grown in Expi-293® expression medium (Thermo Fisher): 2.5 x 10⁻¹⁶ cells using the Expifectamine® 293 T transfection kit (Thermo Fisher) with Opti-MEM (Thermo Fisher) for complexation of DNA with Expifectamine. 6 Transfection of 1 μg of DNA into cells / ml, addition of enhancer according to protocol after 18-20 hours, 19 mm 2 The supernatant was collected after 4-6 days on an orbital shaker at 125 rpm (37°C, 8% CO2, humidified incubator). - Spot-tagged protein purification: Equilibration of Spot-Cap resin: Transfer the desired amount of slurry into a suitable tube, settle the beads by centrifugation (4°C, 4 min, 2500 g), remove and discard the supernatant, add 10 beds of PBS (cold) to the beads, mix and invert, settle the beads by centrifugation (4°C, 4 min, 2500 g), remove and discard the supernatant, repeat twice. - Add the required volume of beads to the supernatant, incubate on a rotator at 4°C, and wash the beads by repeating centrifugation (4°C, 4 min, 2500 g), then remove the supernatant. - Prepare a 500 μM Spot-peptide solution in PBS, remove the supernatant, and incubate with 1 / 3 of the Spot-peptide solution for 5-10 minutes. The beads are settled by centrifugation. For protein concentration, an Amicon Ultra-4 centrifugation filter (15kDa cutoff) is used, and spot peptides are removed using a 15kDa Amicon cutoff column. After rinsing the Amicon Ultra-4 centrifugation filter (15kDa cutoff) with PBS, wash it with 0.1N NaOH (4000g, centrifugation at 4°C) to remove trace amounts of glycerin.

[0131] ELISPOT: 1)Cell culture A) Isolation of PBMCs (under laminar flow hood) To isolate peripheral blood mononuclear cells (PBMCs), density centrifugation was performed using leukocytes from a leukocyte removal chamber and density gradient media (e.g., Ficoll or ROTI Sep 1077). After centrifugation at 1200xg for 20 min without using a brake, the interphase ring collection was washed with 1xPBS (5 min, 300xg). The PBMCs were frozen until further use.

[0132] B) Pulsing of PBMC (under laminar flow hood) The PBMCs were thawed one day before PBMC pulsing (d-1) and kept overnight in 5 ml of X-VIVO 15 medium containing 5% human AB serum in each well of a 6-well plate at 37°C.

[0133] The next day (d0), count the cells, 3 x 10 6 The cells were resuspended in X-VIVO 15 complete medium (5% hAB serum & cytokine cocktail: 20 ng / ml hIL-2, 20 ng / ml hGM-CSF, 10 ng / ml hIL-4 & 10 ng / ml hTGF-b1) at a cell density of cells / ml.

[0134] For the experiment, 3x10 6 Each cell was seeded in a cytokine cocktail and 5 μg / ml of AIM Bio molecule or their respective controls into each well of a 12-well plate containing 1000 μl of X-VIVO complete medium.

[0135] On day 3, 1 ml of complete medium (containing cytokines) was added, and on day 6, a second pulse was performed using 5 μg / ml of the recombinant polypeptide of the present invention or its surrogate (collectively referred to as the "AIM Bio" molecule) (after removing the medium). On days 7, 10, and 12, 1 ml of complete medium (containing cytokines) was added.

[0136] What is needed: X-VIVO 15 medium + 5% human AB serum X-VIVO 15 complete medium: X-VIVO 15 medium + cytokine cocktail: 5% human AB serum supplemented with 10 ng / ml TGF-b1, 10 ng / ml IL-4, 20 ng / ml IL-2, and 20 ng / ml GM-CSF.

[0137] 2) ELISPOT Laminar flow hood On day 13, ELISPOT plates were coated with anti-hIL10 (clone 9D-7, 1:500 dilution in sterile filtered PBS) and aIL10 (10G8-biotin). On day 14, 200,000 cells per well, including negative control (cells + PBS) and positive control (e.g., LPS), were seeded onto ELISPOT plates in two replicates.

[0138] The PFDF membrane was activated with 50 μl / well EtOH (35% v / v) for 1 min, and then washed five times with 200 μl of distilled sterile water. The plate was coated with 100 μl / well antibody solution overnight at 4°C. The following day, unbound coating antibody was removed, and the plate was washed five times with 200 μl of PBS, 200 μl of blocking buffer (X-VIVO 15 5% hAb serum) was added, and the plate was incubated at room temperature for 30 min to 2 hours.

[0139] Each antigenic peptide or control DMSO was prepared in DMSO, and a final volume of 5 μg peptide / ml was added to the final volume of 100 μl / well. 150,000 cells per well were seeded in X-VIVO 15 medium containing 5% human AB serum. The blocking buffer (X-VIVO 15 medium + 5% hAB serum) was carefully removed, and medium containing PBS as a negative control and a stimulant (total volume of 5 μg / ml in each well) was added to the other wells, and incubated overnight at 37°C.

[0140] Outside of the laminar flow hood Secondary antibodies were prepared: a solution of 1 μg / ml aIL-10 biotinylated antibody in 0.5% BSA / 1x PBS (1:1000 dilution), horseradish peroxidase-conjugated streptavidin (1:750 in 0.5% BSA / PBS), and tetramethylbenzidine was filtered using a 0.45 μm filter and stored at 4°C until use.

[0141] The cell supernatant was removed and washed five times with 100 μl of PBS. Finally, excess buffer was removed using a paper towel.

[0142] 25 μl of diluted HRP-streptavidin (1:750) was added per well, incubated at room temperature in the dark for 1 h, and then 5 washing steps were performed using sterile 1x PBS.

[0143] 100 μl of filtered TMB substrate was added per well for 15~25 min until blue spots developed. The reaction was stopped by thoroughly washing the wells with tap water.

[0144] The plastic underdrain of the plate was removed, the bottom and sides of the plate were washed with tap water and dried.

[0145] Plates were read using an ImmunoSpot S6 Ultra-V Analyzer (Cellular Technology Limited), analyzed in Excel, and graphing / statistics were performed in GraphPad Prism.

[0146] Requirements: Capture antibodies: anti-hIL10 (Clone: 9D-7, Mabtech #3430-3-250; 1:500 dilution), anti-hIL10-biotinylated (Mabtech, #3430-6-250) 1x PBS (sterile) 35% EtOH (v / v) Blocking buffer: X-vivo 5% hAB serum (sterile) [Blocking is performed in the same medium as cell culture] Dilution buffer: 0.5% BSA in PBS Washing buffer: 1x PBS Medium: For T cells, X-VIVO 15 Medium (Lonza) Filter syringe: Millex GV ELISPOT PVDF Plate (#MSIP4510, Millipore) TMB substrate.

[0147] Example 2 (Reference): Surrogates of the recombinant polypeptides of the present invention induce IL10-secreting Tregs in mice To induce tolerance to the OVA peptide or the viral Gp34 peptide respectively, wild-type C57BL / 6 mice were injected with 100 μg of a recombinant polypeptide (also referred to as "AIM Bio") having the following sequences: Ova_KbG SIINFEKLGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC (SEQ ID NO: 25), and Gp34_KbG AVYNFATMGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSLRYFVTAVSRPGLGEPRYMEVGYVDDTEFVRFDSDAENPRYEPRARWMEQEGPEYWERETQKAKGNEQSFRVDLRTLLGCYNQSKGGSHTIQVISGCEVGSDGRLLRGYQQYAYDGCDYIALNEDLKTWTAADMAALITKHKWEQAGEAERLRAYLEGTCVEWLRRYLKNGNATLLRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDL GSPDRVRAVSHWSSC (SEQ ID NO: 26). The Gp34 peptide is a well-characterized T cell epitope derived from the lymphocytic choriomeningitis virus (LCMV) glycoprotein. This antigen was traditionally designated Gp33, but it was later found that the epitope presented on H2-K b contains only amino acids 34 to 41 (the epitope starting at amino acid 33, in contrast, is presented on H2-K d ). Therefore, the present inventors refer to the H2-K b epitope as Gp34 in accordance with the most recent recommendations. However, there is still ambiguous use of the Gp33 and Gp34 nomenclature in the literature. The first 8 amino acids of SEQ ID NO: 26 indicate the correct sequence. Two weeks later, the mice were sacrificed, and splenocytes were re-challenged with matching or mismatching peptides. IL-10 secreting cells were quantified by ELIspot. The results are shown in Figure 3.

[0148] Example 3 (Reference): Surrogates of the recombinant polypeptides of the present invention selectively prevent CD8+ T cell-driven EAE in mice As described in (Na et al., Brain. 2008 Sep;131(Pt 9):2353-65), H2-K was introduced into mice expressing ovalbumin in oligodendrocytes. b CD8 recognizes ovalbumin epitopes under these conditions. + Adoptive transfer of OT-I T cells results in experimental autoimmune encephalomyelitis that replicates many MS and late-stage NMO symptoms. In this animal model, a single injection of 500 μg of recombinant polypeptide surrogate molecule (also known as "AIM Bio") that induces tolerance to a targeted ovalbumin epitope nearly completely prevented EAE symptoms, while surrogate molecules presenting a control peptide did not have a significant protective effect (Figure 4). The sequences of the recombinant polypeptide surrogate molecules are listed in Example 2.

[0149] Example 4 (Reference): Some surrogates of the recombinant polypeptides of the present invention are CD4 in mice. + Selectively prevents EAEs driven by T cells. On the day of ip injection of 33 μg or 100 μg of the recombinant polypeptide surrogate molecule of the present invention ("AIM Bio"), 100 μl of MOG35-55 peptide / CFA (complete Freund adjuvant; final concentrations of Mycobacterium Tuberculosis H37RA and peptide, 1 mg / ml each) emulsion was sc-injected into the left and right flanks, respectively, and 250 ng of pertussis toxin (in 200 μl of PBS) was administered intraperitoneally. A second injection of pertussis toxin was given 3 days later. In this animal model, the single-injection AIM Bio surrogate molecule that induces 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 have a significant protective effect (Figure 5). In this model, Mog44 AIM Bio also prevented inflammation and CD8 T cell infiltration in the spinal cord (Figure 6). The sequence of the recombinant polypeptide surrogate molecule was either those listed in Example 2 or the following:

[0150] Mog44_DbG FSRVVHLYRNGGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSMRYFETAVSRPGLEEPRYISVGYVDNKEFVRFDSDAENPRYEPRAPWMEQEGPEYWERETQKAKGQEQWFRVSLRNLLGCYNQSAGGSHTLQQMSGCDLGSDWRLLRGYLQFAYEGRDYIALNEDLKTWTAADMAAQITRRKWEQSGAAEHYKAYLEGECVEWLHRYLKNGNATLLRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC (SEQ ID NO: 27)

[0151] Mog37_DbG VGWYRSPFSRGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLS QPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGPHSMRYFETAVSRPGLEEPRYISVGYVDNKEFVRFDSDAENPRYEPRAPWMEQEGPEYWERETQKAKGQEQWFRVSLRNLLG CYNQSAGGSHTLQQMSGCDLGSDWRLLRGYLQFAYEGRDYIALNEDLKTWTAADMAAQITRRKWEQSGAAEHYKAYLEGECVEWLHRYLKNGNATLLRTDPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDLGSPDRVRAVSHWSSC (Sequence ID 28)

[0152] In this model, Mog44 AIM Bio also completely prevented the formation of MOG-specific autoantibodies in serum tested by ELISA (see also confirmation in Figure 7; Figure 10). This strongly suggests that the recombinant polypeptide of the present invention is an effective therapeutic agent in NMO, which is often characterized by an antibody response to human aquaporin 4. Thus, the patient population is defined by common autoimmune-associated antigens. Certain MHC molecules are also associated with NMO.

[0153] Mog-reactive antibodies in the serum of AIM Bio (33 or 100 μg) treated mice were detected by a standard ELISA protocol, with three washes between each step. Briefly, ELISA plates were coated with 10 μg / ml Mog35-55 peptide, blocked with PBS 1% BSA, and then mouse serum diluted 1:25 in PBS 1% BSA was added over 1 hour. Anti-mouse IgG-HRP or anti-mouse heavy and light chain-HRP antibodies diluted 1:5000 were used for detection.

[0154] Example 5 Human Recombinant Polypeptide Candidates of the Invention for NMOs The recombinant polypeptide of the present invention is a newly developed protein complex derived from the pregnancy-associated immunosuppressive MHC molecule HLA-G. HLA-G is thought to enable the embryo to influence the maternal immune system so that it becomes tolerant of embryonic antigens but further antagonizes antigens derived from pathogens. The recombinant polypeptide of the present invention, containing variable peptides, was able to selectively eliminate peptide-specific cytotoxic effector T cells and induce peptide-specific regulatory T cells in vitro.

[0155] Figure 8 shows a list of human MS&MOGAD recombinant polypeptide candidates.

[0156] Our findings demonstrate that a single-chain protein containing the AQP4 peptide antigen and the HLA-G alpha-3 domain can induce tolerance-genic T cells in healthy donors. Consequently, CD8 Treg cells were upregulated by at least 30% in 65% of all healthy blood donors (Figure 9).

[0157] Example 6 (Reference): Further proof of the stability and effectiveness of the recombinant polypeptide of the present invention Furthermore, in order to obtain further proof of the stability and efficacy of the recombinant polypeptides and their surrogates of the present invention, the inventors undertook to obtain and test recombinant polypeptides having the general structure of the recombinant polypeptides of the present invention but containing various different peptide antigens. As shown in Figures 11 and 12, respectively, the tested recombinant polypeptides were stable during lyophilization and storage and were heat-stable. Furthermore, they induced Tregs in a dose-dependent manner (Figure 13) and inhibited T cell lysis in a dose-dependent manner (Figure 14). The effect of recombinant polypeptides on serum cytokine profiles in EAE-ODC Ova mice is shown in Figure 15. There is induction of IL-10 and possibly IL-4, both of which are known to be immunosuppressive cytokines that downmodulate the immune response in an inflammatory setting. This requires the HLA-G alpha-3 domain + allopeptide. IL-2 is thought to be induced in response to the presentation of an allopeptide unrelated to the alpha-3 domain to cells. IL-2 is required for T cell activation and survival.

[0158] Example 7 (Reference): Therapeutic effects of recombinant peptides containing mouse aquaporin antigen peptides in 2D2 TCR transgenic mice

[0159] Materials and methods Experimental model 2D2 TCR transgenic mice were divided into four cohorts. Each cohort consisted of at least five animals. Lactation-matched WT (C57BL / 6) mice were designated as the fifth cohort. The experimental period began when 2D2 mice spontaneously developed optic neuritis. Disease onset was determined by ocular abnormalities or elevated serum anti-MOG IgG levels. The day after disease onset, mice were intravenously injected with either the single-stranded MHC Ib molecule AQP147_KbG (antigen peptide: VTTVHGNL; SEQ ID NO: 39) / AQP203_KbG (antigen peptide: FAINYTGASM; SEQ ID NO: 40), which was adapted for treatment mice, or the control AIM Bio Gp34_KbG. Control mice received PBS only. The dose was 5 mg / kg body weight in 250 μl. Either the control injection or the treatment injection was administered intravenously (D0). Untreated mice received PBS. Injections were repeated on days 15 and 30. Mice were weighed daily and monitored for EAE symptoms (Table 1). Mice that reached an EAE score of 6 were sacrificed before the end of the study period. All animals were sacrificed with CO2 on day 42. Optic nerves, eyeballs, spinal cord, brain, serum, and lymph nodes were collected and frozen at -20°C. Cardiac blood was collected and processed for serum analysis.

[0160] EAE was scored according to a 10-point scoring system (Bittner et al., Journal of visualized experiments: JoVE, (86), 51275).

[0161] immunohistochemistry Serial sections of the eyeball, 20 μm thick, were excised and stained with hematoxylin and eosin. Briefly, the frozen sections were dried at room temperature for 10 minutes and stained in hematoxylin solution for 10 minutes. The tissue sections were washed with running water for 10 minutes and stained in eosin solution for 30 seconds. Subsequently, the sections were sequentially dehydrated in 70%, 96%, and 100% ethanol for 30 seconds each. After this, the sections were incubated with xylol for 10 minutes and mounted in a vitro cloud.

[0162] Immunofluorescence Longitudinal serial sections with a thickness of 10 μm of the optic nerve were cut out for immunohistochemistry. The tissue sections were fixed with 4% PFA in PBS for 10 minutes; subsequently, blocking was performed for 2 hours at room temperature using a buffer containing 5% BSA, 0.2% Triton-X100, and 5% NGS in 1×PBS. The tissues were stained overnight at 4°C with a single primary antibody or a suitable combination of primary antibodies in a staining buffer containing 1% BSA, 1% NGS, and 0.2% Triton-X100. The primary antibodies were as follows: i) Rat anti-mouse CD3 (1:200, Invitrogen); ii) Rat anti-mouse CD8 (1:200, Biorad); iii) Rabbit anti-cleaved caspase-3 (1:400, Cell Signaling). The sections were washed 3 times with PBS, and stained with the corresponding fluorescently labeled secondary antibodies: i) Anti-rabbit-Cy3 (1:300, Dianova); ii) Anti-rat-AF488 (1:300, Invitrogen) for 1 hour at room temperature in the dark. The sections were washed with PBS, and DNA was stained with DAPI (1:500, Sigma-Aldrich) for 10 minutes at room temperature in the dark. Finally, the sections were washed and mounted with aquapolymount (Polysciences). Optical sections were obtained at 20× or 40× magnification using a Zeiss Axiocam.

[0163] Quantification of Immunostaining Immunofluorescence images were quantified using ImageJ-Fiji Version 1.53t. Three to four sections were analyzed for each animal. The images were split into individual channels and converted to 8-bit images. The threshold of the 8-bit image for cleaved caspase-3 staining was set to 5 MFI, and that for DAPI staining was set to 100. The area covered by cleaved caspase-3 / the area covered by DAPI indicates the proportion of cleaved caspase-3 in the section.

[0164] Quality Control and Stability After purifying single-chain MHC Ib molecules, their stability was analyzed after one and three freeze-thaw cycles, storage at room temperature for 5 days, and heating to 50°C for 30 minutes. For this purpose, A) Coomasiegel staining of a 12% polyacrylamide gel using 2 μg of single-chain MHC-Ib molecules, and B) HLA-G Western blotting using 1 μg of protein with 2A12a HLA-G antibody (1:1000) were performed under non-reducing conditions. Both monomers and dimers were detectable.

[0165] result Quality control and stability Quality control and stability testing demonstrated that all NMO surrogate single-stranded MHC Ib molecules were highly stable (Figures 17-19). However, AQP203_H2KbG_spt appeared to be more quantifiable, which may explain the lower efficacy in some assays.

[0166] Therapeutic effect Notably, treatment with AQP147_KbG provided complete protection from EAEs in treated mice (Figure 20), inhibited immune cell infiltration in the optic nerve (Figure 21), and completely inhibited apoptosis in the optic nerve, spinal cord, and retina (Figure 22). Furthermore, while AIM-treated mice had fewer CD8 T cells, they clearly had more regulatory T cells in the optic nerve, albeit fewer (Figure 23). In addition, AQP147_KbG completely rescued IRL (inner retinal lamina) cells from degradation (Figure 24).

[0167] These data confirm that the recombinant polypeptide and its surrogate of the present invention have therapeutic effects. Therefore, according to the present invention, the recombinant polypeptide of the present invention can be used to treat neuromyelitis optica in human patients.

[0168] Example 8: Expression and testing of a polypeptide ("AIM Bio") having the polypeptide structure defined in claim 1 and containing the peptide antigen according to the present invention. Sandwich ELISA protocol 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), blocked at RT for 2 hours, blocked for 1 hour with 150 μl of 1% BSA (blocking buffer) in PBS, and then washed three times with 200 μl of PBS / T. Expi293F expression cultures were diluted with blocking buffer (1:10) and tested in two replicates. Biotinylated anti-β2 microglobulin antibody (MA1-19506, Thermo Fisher) was diluted in blocking buffer (1:1000) and 100 μl was added to each well over 1 hour. After three washes with 200 μl of PBS / T, 100 μl of horseradish peroxidase (HRP) (405210, Biolegend; diluted 1:100 in blocking buffer) conjugated with streptavidin was added for 20 minutes. The binding of the biotinylated anti-β2 microglobulin detection antibody was then visualized by washing three times for 10–15 minutes with 200 μl of PBS / T, followed by 100 μl of TMB substrate solution (BioLegend). The reaction was stopped with 100 μl of 1N H2SO4. Absorbance was measured at 450 nm using a Tecan Sunrise® ELISA plate reader, corrected at 620 nm, and the results were evaluated using Magellan® software.

[0169] Transfection protocol Expi-293F cells (Thermo Fisher) were grown 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, and the number was 4.5-5.7 x 10⁶. 6 Transfected at a density of individual live cells / ml (over 99% viability).

[0170] For a final culture volume of approximately 12 ml, 3 x 10 7 1000 viable cells were diluted in 10 ml of Expi293® expression medium containing GlutaMAX (Gibco, A14351-01). 10 μg of DNA per 1 ml of total transfection volume 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 complexing buffer (Thermo Fisher). After 3–5 min, the DNA was added to the Expifectamine-293® / Opti-MEM® transfection mix and incubated at RT for 15–20 mins for the formation of the DNA-Expifectamine-293F® reagent complex. Next, 1200 μl of DNA-Expifectamine-293® reagent complex was added dropwise to 50 ml bioreactors containing 30 million cells in 10 ml of culture medium. The cells were then shaken using an orbital shaker, 19 mm 2 The samples were incubated at a diameter of 225 rpm in a humidified incubator at 37°C and 8% CO2.

[0171] After incubation for 18-20 hours, cells were enhanced by adding 60 μl of Enhancer I and 600 μl of Enhancer II (ExpiFectamine® 293 Transfection Enhancer 1+2, Catalog A14524, part of the ExpiFectamine® 293 Transfection Kit) while shaking the bioreactor. After spinning down the cells at 300 g for 5 minutes at room temperature, the supernatant was harvested on day 5 by centrifugation at 4000 g for 20 minutes at 4°C.

[0172] Simple Western Protocol Simple Western electrophoresis of Jess cells was performed under non-reducing conditions using EZ Standard Pack 1 (bio-techne, Ref PS-ST01E7) reagents, employing a 12-230 kDa cartridge (bio-techne, Ref SM W004-1), 2A12αHLA-G antibody as the primary antibody (Thermo Fisher, MAI-10358, 1:50 in bio-techne antibody dilution 2, part of the anti-mouse detection kit), and the anti-mouse detection kit (bio-techne Ref DM-002). The supernatant from transfected Expi293F cells was pre-diluted 1:10 with 1x PBS, then separately diluted 1:10 with 0.1x sample buffer. After further dilution 1:5 with 5x fluorescence master mix, 3 μl was applied to a single cartridge well along with a molecular weight marker. 5 μl of protein ladder (EZ Standard Pack 1) was used for molecular weight determination in one well of the first row. In the next row, 10 μl of antibody dilution 2 (bio-techne, part of the anti-mouse detection kit) was loaded per well as a blocking reagent. In the third row, 10 μl of 1:50 diluted primary antibody 2A12αHLA-G was loaded. In the fourth row, 10 μl of mouse secondary antibody from the anti-mouse detection kit was loaded. In the fifth row, 15 μl of luminol-peroxide mix was loaded. In the last three rows, 500 μl of washing buffer was loaded per compartment. The plate was centrifuged at 1000 g for 5 mins in RT to remove air bubbles, and the remaining cover foil was removed. The cartridge and plate were then loaded into the Jess system (bio-techne) using Compass 6.2.0 software, and the instrument was started.

[0173] The separation time was 25 minutes at 375V, followed by a 5-minute blocking time with antibody diluent (bio-techne), 30 minutes of primary antibody incubation, 30 minutes of secondary antibody incubation, and then detection.

[0174] ELISpot Protocol ELISpot analysis was performed according to the following protocol:

[0175] Phase 1: PBMC: AIM Bio Culture reagent Human PBMCs derived from patients with NMOSD (Neuromyelitis Optica Spectrum Disorder) • AIM Bio: AQP64_G, AQP65 P65K _G • Complete culture medium: 5% human AB serum and human cytokines -rhIL-2 (20ng / ml); Immunotools, 11340027 -rhGM-CSF (20ng / ml); Immunotools, 11343128 -rhIL-4 (10ng / ml); Immunotools, 11340047 -rhTGFβ (10 ng / ml); X-vivo 15 (Lonza, Biozym 881026) supplemented with Immunotools, 11343160 • 24-well culture plate ·PBS (sterile)

[0176] PBMC thawing (-1 day) The frozen PBMC is thawed by rotating it in a water bath for approximately 20-30 seconds. Immediately transfer the cells to a 15 ml Falcon tube filled with PBS and centrifuge at 300 g for 5 minutes at RT. Wash the cells two more times with PBS. Resuspend the cells in 5 ml of X-vivo 15 medium supplemented with 5% hAB serum, and allow them to rest overnight in a 6-well culture plate at 37°C.

[0177] Treg induction: Culture PBMCs with AIM Bio for 14 days (sterile conditions). • Test conditions: ○ Negative control (AIM Bio treatment untreated / untreated) [ka] [ka] • Number of PBMCs: 1.2 x 10 in 400 μl of complete culture medium 6 pieces. • Count the cells that have been allowed to rest overnight and calculate the number of cells required for the assay. Wash the cells and place them in 3x10 6 Resuspend in complete culture medium at the final concentration of cells / ml. Add 400 μl (1.2 x 10⁶ cells) of cell suspension to each well in a 24-well plate. • 5 μg / ml AIM Bio AQP64_G and AQP65 P65K Pulsing cells with _G (Day 0). • Supply the cells with 400 μl of complete culture medium every two days. • On day 6, 5 μg / ml of AIM Bio AQP64_G and AQP65 P65K Pulse with _G. • On day 14, seed the cells onto the ELISpot plate.

[0178] Phase 2: ELISpot assay (Perform the EliSpot assay according to the guidelines provided in the Human IL-10 ELISpot (Basic) Kit (Mabtech; 3430-2H)) reagent • ELISPOT PVDF plate (Millipore) • Human IL-10 EliSpot basic kit (Product code: 3430-2H) 35% ethanol • Blocking medium: X-vivo 5% hAB serum • Dilution buffer: 0.5% BSA in PBS or 0.5% FCS in PBS • EliSpot medium: X-VIVO 15 (Lonza, Biozym 881026) supplemented with 5% human AB serum and hIL-2 (20 ng / ml) • Streptavidin-HRP for detection (Biolegend #405210; 1:750 dilution) • ELISpot substrate: TMB for HRP (Mabtech Code: 3651-10) ·PBS (sterile)

[0179] Cell coating (sterilization conditions) • Activate the PVDF wells with 25 μl of ethanol (35% v / v) for a maximum of 1 minute. Wash the plate five times with sterile water (200 μg / well). Dilute the coated antibody anti-hIL-10 (9D7) in sterile PBS to a final concentration of 2 μg / ml in sterile PBS. Add 50 μl of the diluted antibody per well (guideline: 15 μg / ml; 100 μl / well). Incubate overnight at 4-8°C.

[0180] Cell incubation in a plate (under sterile conditions) • Prepare the plate layout. The sample was tested twice in a repeating manner. ·sample: ○ Negative control: Untreated cells (no stimulation) ○Positive control: Untreated cells + 1 μg / ml α-CD3 / CD28 stimulating cocktail ○Test samples: AQP64_G treated cells and AQP65 P65K _G treated cells • Count PBMCs from 14D cultures. Calculate the required number of cells based on all conditions. • The minimum cell viability is greater than 50%. Otherwise, discard the sample. Remove any excess coated antibody under a hood and wash the plate five times with 200 μl of sterile PBS. Add 50 μl of blocking medium per well. Incubate at room temperature for 2 hours. ·1.5x10 in Elispot medium 6 Resuspend the PBMCs derived from all test conditions at the final concentration of / ml. • Resuspend the positive control cells at a higher concentration and supplement the volume with a stimulation cocktail. • Carefully remove excess blocking buffer under the hood. • Place 100 μl of PBMC (1.5 x 10) derived from each condition into each well. 5 Add the suspension of individual cells. Incubate at 37°C for 12-48 hours.

[0181] Spot detection Dilute the biotinylation detection antibody (12G8-biotin) to a concentration of 1 μg / ml (1:1000) in dilution buffer. Remove the cell supernatant and wash the plate five times in 200 μl of PBS. Remove the final excess buffer by pressing it onto a paper towel. Add 50 μl of detection antibody to each well. Incubate overnight at 4°C. Wash five times with 200 μl of PBS. Remove excess liquid by pressing onto a paper towel. Prepare HRP-conjugated streptavidin (1:750 in dilution buffer). Add 50 μl of streptavidin-HRP conjugate to each well. Incubate at room temperature for 1 hour. Wash five times with 200 μl of PBS. Remove excess liquid by pressing onto a paper towel. Add 50 μl of filtered TMB substrate to each well and incubate in a dark room for 10-20 minutes until blue spots appear. • Stop the reaction by rinsing the well with water (running tap water can be used). Remove the plastic underdrain from the plate and thoroughly rinse the bottom and sides of the plate with tap water.

[0182] The analysis was performed using S6 Universal M2 readers (Immunospot, Cleaveland, Ohio).

[0183] result: Example 9: Figure 25 shows that the AIM Bio of the present invention is expressed in Hek293T cells, and monomers (approximately 50 kDa) are detectable for all compounds; expression is further confirmed and quantified in the supernatant by ELISA. This suggests that these molecules can be successfully produced. It also shows that anchor residue-optimized sequences, such as AQP65-72_6(P65K), may have higher production yields.

[0184] Figure 26: ELISpot using patient PBMCs. A: Photograph of the wells in the assay, with duplicates always arranged in a single row. The number of counted spots is shown in the figure. B: Quantification.

[0185] ELISpot was carried out as described in Example 9, but differed in that only 1.2 million PBMCs were used, the cells were seeded in a 24-well plate, and all culture medium volumes were reduced accordingly.

[0186] AIM Bio AQP65 P65K In 4 out of 5 NMO patient PBMCs treated with _G, IL-10 spots increased compared to untreated PBMCs.

[0187] Furthermore, by using a surrogate construct that conforms to the claims except for containing a surrogate peptide, the inventors have found that constructs that can be expressed at favorable expression levels are functional, i.e., capable of inducing antigen-specific immune tolerance. Therefore, given the favorable expression levels of the constructs of the present invention, it is expected that the constructs of the present invention are functional and usable to treat the medical indications defined by the claims.

[0188] Example 10: Characterization of the human recombinant polypeptide ("AIM Bio") of the present invention Size exclusion chromatography To characterize the purified protein, size exclusion chromatography runs were performed at room temperature using a Superdex 200 increase 10 / 300 column on an Akta FPLC (Amersham Biosciences) and Unicorn 5.31 software (GE Healthcare). The buffer was changed from 20% EtOH (storage solution) to 2CV H2O before the run. For analysis, the column was rinsed with 2CV PBS (Sigma, D8537-500ml) at a flow rate of 0.5 ml / min. Subsequently, 10-20 μg of AIM Bio (volume 500 μl) was passed through a Superdex 200 increase 10 / 300 column (Cytiva), and the column was run at a flow rate of 0.5 ml / min per CV to collect the protein fraction.

[0189] Figure 28 shows the purified AQP4. 126-135 The size exclusion chromatography analysis of A2G is shown. Conveniently, this protein was found to be a monomeric protein free of impurities. Notably, this A2G construct was expressed at higher levels than the corresponding purely HLA-G based polypeptide.

[0190] The "AQP4" used in this experimental example 126-135 The _A2G construct contained the amino acid sequence of SEQ ID NO: 87, followed by an optional C-terminal amino acid sequence derived from intron 4 with the furin cleavage site removed (SKEGDGGIMSVRESGSLSEDL; SEQ ID NO: 54), and an optional tag (as specified below).

[0191] Thermal shift assay A thermal shift assay (TSA) was performed to analyze the protein stability of purified AIM Bio. For the TSA, 3 μg of AIM Bio was dissolved in 25 μl of PBS (Sigma, D8537-500 ml) containing SYPRO Orange dye (Merck, S5592, final dilution in PBS 1:2000). The melting curve was recorded using a StepOnePlus Instrument (ThermoFisher) by starting at 25°C and increasing the temperature by 1°C every minute until the final temperature reached 95°C in 2 minutes. The melting curve was analyzed using StepOnePlus 2.3 software. Boltzmann sigmoid curve fitting was performed to determine Tm, and Tm was calculated accordingly.

[0192] Figure 29 shows the thermal shift assay: AIM Bio AQP 126-135 The melting curve analysis of _A2G is shown.

[0193] Jess's Modern Western Run Jess simple Western runs were performed under non-reducing conditions using reagents from EZ Standard Pack 1 (bio-techne, Ref PS-ST01E7), a 12-230 kDa cartridge (bio-techne, Ref SM W004-1), 2A12α HLA-G antibody as the primary antibody (Thermo Fisher, MAI-10358, 1:50 in bio-techne antibody diluent 2, part of the anti-mouse detection kit), and the anti-mouse detection kit (bio-techne Ref DM-002). The supernatant was pre-diluted 1:10, the purified protein was pre-diluted 1:100 with 1× PBS, and then further diluted 1:10 with 0.1× sample buffer. After further dilution 1:5 with 5× fluorescent master mix, 3 μl was added to a single cartridge well with a molecular weight marker. In one well of the first row, 5 μl of protein ladder (EZ Standard Pack 1) was used for molecular weight determination. The next row was loaded with 10 μl of antibody dilution 2 (bio-techne, part of the anti-mouse detection kit) per well as a blocking reagent, the third row with 10 μl of 1:50 diluted primary antibody 2A12αHLA-G, the fourth row with 10 μl of mouse secondary antibody from the anti-mouse detection kit, the fifth row with 15 μl of luminol-hydrogen peroxide mixture, and the last three rows with 500 μl of washing buffer per compartment. To remove air bubbles, the plate was centrifuged at 1000 g for 5 minutes at room temperature, and any remaining cover foil was removed. The cartridge and plate were then loaded into the Jess system (bio-techne) using Compass 6.2.0 software, and the instrument was started.

[0194] The separation time was 25 minutes at 375V, followed by a 5-minute blocking period using antibody diluent (bio-techne), a 30-minute primary antibody incubation, and a 30-minute secondary antibody incubation, after which detection was performed.

[0195] Figure 30 shows AQP 65-72_P65K _G and AQP 65-72_P65RThis shows the Jess run of AIM Bio optimized for G-anchor residues.

[0196] As shown in the figure, optimizing the anchor residue reduced the tendency to form multimers.

[0197] ILT4-Fc binding ELISA For the ILT4 binding assay, 125 ng of AIM Bio (25 μl of 5 μg / ml AIM Bio solution) was coated overnight at 4°C onto a 96-well half-area microplate (Greiner, 675061) covered with sealing foil, followed by three washing steps with 120 μl of PBS / 0.05% Tween 20 (PBST). PBS without AIM Bio was used as a control (AIM Bio was buffered in PBS). Blocking was performed for 1.5 hours with 100 μl of 1% BSA (Albumin Fraction V, Roth, #0163.2) in PBS, followed by three washings with 120 μl of PBS / T on a shaking table (the same procedure was followed for further blocking steps). Human ILT4-Fc (stock concentration 250 μg / ml in PBS) (Sino Biological, #14132-H02H) was diluted with PBS / T and titrated at room temperature for 2 hours using 25 μl of the solution (100 μg / ml, 20 μg / ml, 4 μg / ml, 0.8 μg / ml, 0.16 μg / ml). PBS / T without ILT4-Fc was used as a control. The plates were washed three times with 120 μl of PBS / T. For detection, 25 μl of α-hIgGγ antibody (Thermo, #628420) per well was diluted 1:2000 in PBS / T, 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 for 3 minutes until a color change to blue occurred. The reaction was stopped using 25 μl of 1N H2SO4. The plate was then analyzed at 450 nm using a Sunrise plate reader (Tecan) and Magellan 7.5 software (Tecan), with a reference wavelength of 620 nm. Corrections were made for the control well. The data were calculated using Excel 365 (Microsoft), and the graphs were plotted using Prism 10.2 (GraphPad). The results are shown in Figure 31.

[0198] As shown in the figure, the use of His-tagged or untagged variants of the protein is equivalent in IC 50This results in a concentration, which indicates that the (optional) tag is not related to the binding properties of AIM Bio. [Industrial applicability]

[0199] The pharmaceutical compositions, polypeptides, nucleic acids, cells, and products used in this invention are industrially applicable. For example, they can be used in the manufacture of pharmaceuticals or as pharmaceuticals themselves.

Claims

1. A recombinant polypeptide capable of presenting a peptide antigen, wherein the recombinant polypeptide is arranged in order from the N-terminus to the C-terminus. i) A peptide antigen presented by the recombinant polypeptide, wherein the peptide antigen is a peptide of human aquaporin 4, ii) Depending on the case, the linker sequence, iii) A sequence of a human polypeptide domain containing the sequence of human β2 microglobulin, or an amino acid sequence that is at least 90% identical to the amino acid sequence of human β2 microglobulin represented by Sequence ID No.

5. iv) Depending on the case, the linker array, v) The [α]1 domain of the MHC molecule, vi) [α]2 domain of MHC molecule, vii) The [α]3 domain of an MHC class Ib molecule, or a derivative of the [α]3 domain of an MHC class Ib molecule, wherein the derivative is capable of binding to ILT2 or ILT4, viii) Depending on the case, the protease cleavage site, ix) Depending on the case, the spacer arrangement, and x) Depending on the case, affinity tags The recombinant polypeptide comprising the above.

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

3. The recombinant polypeptide according to claim 1 or 2, wherein the peptide antigen described in i) comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NOs. 34, 42-51, and 82.

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

34.

5. i) The recombinant polypeptide according to any one of claims 1 to 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 claims 1 to 5, wherein the [α]1 domain described in (v) and the [α]2 domain described in (vi) are derived from a human MHC class Ia molecule, preferably a human HLA-A2 molecule, or a human MHC class Ib molecule, preferably a human HLA-G molecule.

7. The recombinant polypeptide according to any one of claims 1 to 6, wherein the [α]3 domain of the MHC class Ib molecule described in (vii) is the [α]3 domain of human HLA-E, human HLA-F, or human HLA-G, preferably human HLA-G.

8. The recombinant polypeptide according to any one of claims 1 to 7, wherein the [α]3 domain or derivative described in (vii) is identical to the [α]3 domain having the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 21, or SEQ ID NO: 53, or has at least 80% amino acid sequence identity, at least 90% amino acid sequence identity, at least 92% amino acid sequence identity, at least 94% amino acid sequence identity, at least 96% amino acid sequence identity, at least 98% amino acid sequence identity, or at least 99% amino acid sequence identity, or is identical to the [α]3 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 to 8, wherein the [α]3 domain or derivative described in (vii) is identical to the [α]3 domain having the amino acid sequence of SEQ ID NO:

9.

10. The recombinant polypeptide according to any one of claims 1 to 8, wherein the [α]3 domain or derivative described in (vii) is identical to the [α]3 domain having the amino acid sequence of SEQ ID NO:

21.

11. The recombinant polypeptide according to any one of claims 1 to 8, wherein the [α]3 domain or derivative described in (vii) is identical to the [α]3 domain having the amino acid sequence of SEQ ID NO:

53.

12. The recombinant polypeptide according to any one of claims 1 to 11, wherein the linker sequence described in (ii) and / or the linker sequence described in (iv) comprises an amino acid sequence (GGGGS)n, where n is an integer of 1 or more, and n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, preferably selected from the group consisting of 2, 3, 4, and 5.

13. The recombinant polypeptide according to any one of claims 1 to 12, wherein the sequence of the human polypeptide domain described in (iii) is at least 95% identical to the amino acid sequence of SEQ ID NO: 5, preferably at least 98% identical to the amino acid sequence of SEQ ID NO: 5, and more preferably identical to the amino acid sequence of SEQ ID NO:

5.

14. The recombinant polypeptide according to any one of claims 1 to 13, wherein the polypeptide is a dimer or a polymer.

15. A recombinant polypeptide according to any one of claims 1 to 14, wherein the polypeptide contains or consists of all of components i) to vii), the polypeptide does not contain components viii) to x), or the polypeptide contains or consists of all of components i) to x).

16. The recombinant polypeptide according to any one of claims 1 to 15, further comprising an N-terminal secretory signal peptide sequence.

17. The recombinant polypeptide is arranged in the following order from the N-terminus to the C-terminus: (a) and (b) (a) A peptide antigen selected from the group consisting of amino acid sequences of SEQ ID NOs. 34, 42-51 and 82, and (b) Amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 55-60 A recombinant polypeptide according to any one of claims 1 to 15, comprising an amino acid sequence consisting of the following.

18. The recombinant polypeptide according to any one of claims 1 to 17, wherein the recombinant polypeptide is soluble.

19. A nucleic acid encoding one or more polypeptides according to any one of claims 1 to 18, wherein the nucleic acid is preferably a vector.

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

21. A pharmaceutical composition or kit comprising at least one recombinant polypeptide as described in any one of claims 1 to 18.

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

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

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

25. A pharmaceutical composition or kit for use according to any one of claims 23 to 24, wherein the treatment is for reducing plasma or cerebrospinal fluid levels of autoantibodies against human aquaporin 4, and the human patient is a patient who had plasma or cerebrospinal fluid autoantibodies against human aquaporin 4 prior to the initiation of treatment.

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

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

28. A method for obtaining a pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 18, the method comprising: (a) culturing recombinant host cells according to claim 27 under conditions that enable the expression of recombinant polypeptide from nucleic acid molecules; (b) recovering recombinant polypeptide; (c) purifying recombinant polypeptide; and (d) formulating recombinant polypeptide into a pharmaceutical composition.

Images