Classifications

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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/0005—Vertebrate antigens
  • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/385—Haptens or antigens, bound to carriers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/70539—MHC-molecules, e.g. HLA-molecules
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
  • A61K2039/6031—Proteins
  • A61K2039/605—MHC molecules or ligands thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
  • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/50—Fusion polypeptide containing protease site

Definitions

• the present invention relates to therapeutical uses of non-classical human major histocompatibility complex (MHC) molecules (also named MHC class lb molecules) in combination with peptide antigens for the treatment of multiple sclerosis (MS), MOG antibody disease and MOG antibody positive neuromyelitis optica.
• MHC human major histocompatibility complex
• the invention more specifically relates to recombinant polypeptides comprising peptide antigens and one or more domains of a non-classical MHC class lb molecule.
• the invention also relates to methods of producing such recombinant polypeptides, pharmaceutical compositions comprising the same, as well as their uses for treating multiple sclerosis (MS), MOG antibody disease and MOG antibody positive neuromyelitis optica.
• MS Multiple sclerosis
• MOG encephalomyelitis also known as myelin-oligodendrocyte glycoprotein antibody disease, MOG antibody disease, MOGAD
• MS multiple sclerosis
• MOG encephalomyelitis also known as myelin-oligodendrocyte glycoprotein antibody disease, MOG antibody disease, MOGAD
• T cells of the immune system attack myelin sheaths in the central nervous system, resulting in gradually progressive neurodegeneration.
• the relapsing flare-up of disease activity characteristic of MS can be effectively suppressed by immunomodulatory therapies.
• MOGAD which has long been considered a subtype of MS, there are no approved therapeutics, so therapy initially consists of watching and waiting for the disease to progress. In both diseases, slowly progressive neurodegeneration occurs.
• Some therapeutics effective in MS such as the antibodies natalizumab or ocrelizumab or the orally bioavailable S1P inhibitor fingolimod, even carry the risk that a persistent and normally harmless intracerebral infection with JO virus can no longer be controlled, resulting in an often fatal progressive multifocal leukoencephalopathy.
• the problem of either insufficient immunomodulation or immunomodulation with too severe side effects remains unsolved in both diseases, especially with regard to slow progression.
• Common biologies do not cross the blood-brain barrier and thus cannot have an anti-inflammatory effect in situ.
• regulatory cells have been described to cross it well (Schneider-Hohendorf et al., Eur J Immunol. 2010 Dec; 40(12):3581- 90).
• MS multiple sclerosis
• NMO MOG antibody positive neuromyelitis optica
• MHC class lb molecules such as HLA-G possess the ability to induce antigen-specific tolerance towards presented peptide antigens.
• MHC class lb molecules can advantageously be used according to the invention to suppress immune responses in an antigen-specific manner.
• molecules other than naturally occurring MHC class lb molecules and in particular polypeptides which only comprise at least one domain of an MHC class lb molecule, preferably at least an [alpha]3 domain of an MHC class lb molecule, can be used:
• the [alpha] 1 and [alpha]2 domains of variable class I a molecules can be combined with the [alpha]3 domain of a human MHC class lb molecule in order to suppress immune responses towards peptides presented by these antigens.
• Antigen-loaded HLA-G molecules can be unstable.
• the inventors designed soluble recombinant polypeptides comprising a peptide antigen, an MHC class lb molecule such as HLA-G and p2-microglobulin (b2m), and connected these three components covalently (e.g., via covalent linkers).
• the antigen-binding a1 and a2 domains of an MHC class lb molecule such as HLA-G were exchanged by the respective domains of other MHC molecules to enhance the flexibility and versatility of these recombinant polypeptides (see, for instance, Figure 2).
• These alternative recombinant polypeptides can be designed with antigen-binding domains of other human HLA molecules.
• constructs comprising the a1 and a2 domains of murine H2-K b can present the ovalbumin-derived peptide SIINFEKL to OT-1 T cells.
• OT-1 T cells express a transgenic T cell receptor that specifically recognizes this antigen) (WO 2018/215340).
• the inventors have found that by using the recombinant polypeptides of the invention, immune responses against human myelin-oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1) can be suppressed.
• multiple sclerosis (MS), myelin-oligodendrocyte glycoprotein antibody disease (MOG antibody disease) and MOG antibody positive neuromyelitis optica can be treated by the recombinant polypeptides of the invention.
• the recombinant polypeptides of the invention are expected to be highly advantageous in the immunoterapeutic treatment of multiple sclerosis (MS), MOG antibody disease and MOG antibody positive neuromyelitis optica, because are expected to show an improved safety profile as compared to conventional drugs aganst these diseases, which may cause severe side effects such as progressive multifocal leukoencephalopathy.
• MS multiple sclerosis
• MOG antibody disease MOG antibody positive neuromyelitis optica
• the inventors have surprisingly found that the recombinant polypeptides of the invention do not only modulate T-cell responses but also prevent the formation of MOG-specific autoantibodies in model experiments. It is expected that this finding will translate into a clinical improvement in patients having multiple sclerosis (MS), MOG antibody disease and MOG antibody positive neuromyelitis optica, because MOG- specific autoantibodies are involved in the pathology of these diseases.
• MS multiple sclerosis
• MOG antibody disease MOG antibody positive neuromyelitis optica
• the invention relates to the following preferred embodiments:
• a recombinant polypeptide capable of presenting a peptide antigen comprising, in an N- to C-terminal order,
• a peptide antigen presented by said recombinant polypeptide wherein the peptide antigen is a peptide of human myelin-oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1); ii) optionally a linker sequence; ill) optionally a sequence of a human polypeptide domain comprising a sequence of a human p2 microglobulin, or an amino acid sequence at least 90% identical to the amino acid sequence of human p2 microglobulin represented by SEQ ID NO: 5; iv) optionally a linker sequence; v) optionally an [alpha] 1 domain of an MHC molecule; vi) optionally an [alpha] 2 domain of an MHC molecule; vii) an [alpha] 3 domain of an MHC class lb molecule or a derivative of an [alpha] 3 domain of an MHC class
• the recombinant polypeptide according to item 1 wherein said peptide antigen according to i) is 7 to 11 amino acids in length, preferably 8-10 amino acids in length.
• the recombinant polypeptide according to item 1 or 2 wherein said peptide antigen according to i) consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33.
• MOG myelin-oligodendrocyte glycoprotein
• the recombinant polypeptide according to any one of items 1-2 and 4-5, wherein said peptide antigen consists of the amino acid sequence of SEQ ID NO: 2.
• the recombinant polypeptide according to item 20 wherein the linker sequence according to (ii) comprises the amino acid sequence (GGGGS)n, and wherein n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 and is preferably selected from the group consisting of 2, 3, 4 and 5.
• the recombinant polypeptide according to any one of the preceding items wherein said sequence of a human polypeptide domain according to (ill) 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.
• a peptide antigen selected from the group consisting of the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33; and
• the pharmaceutical composition or kit according to item 34 wherein the pharmaceutical composition or kit comprises at least two different recombinant polypeptides according to any one of items 1-30, and wherein each of the different polypeptides comprises a different peptide antigen as defined in any one of items 3 to 6.
• the pharmaceutical composition or kit for use according to item 36 wherein the treatment is a treatment of multiple sclerosis (MS).
• the pharmaceutical composition or kit for use according to item 36 wherein the treatment is a treatment of MOG antibody disease.
• the pharmaceutical composition or kit for use according to item 36 wherein the treatment is a treatment of MOG antibody positive neuromyelitis optica.
• a recombinant host cell comprising a nucleic acid or a vector according to item 31 or 32 and expressing the recombinant polypeptide according to any one of items 1-30.
• a method for obtaining pharmaceutical composition comprising a polypeptide according to any one of items 1-30, the method comprising the steps of (a) culturing the recombinant host cell of item 45 under conditions allowing expression of the recombinant polypeptide from the nucleic acid molecule, (b) recovering the recombinant polypeptide, (c) purifying the recombinant polypeptide, and (d) formulating the recombinant polypeptide into a pharmaceutical composition.
• Figure 1 Depiction of a peptide-loaded soluble MHO lb molecule suitable to achieve therapeutic antigenspecific immunomodulation.
• An optional linker connecting the antigenic peptide with the beta2microglobulin molecule is displayed in grey stick style, and an optional disulfide trap is depicted in black spheres.
• This figure was generated using Pymol and is adapted from structures published in Clements et al., Proc Natl Acad Sci U S A. 2005 Mar 1 ; 102(9):3360-5 and Hansen et al., Trends Immunol. 2010 Oct;31 (10):363-9.
• Figure 2 Example for a vector-based construct encoding a single chain MHO lb molecule suitable for therapeutic peptide-specific immunomodulation.
• HLA-G1 and HLA-G5 each consist of 3 [alpha] domains (here in black), a non-covalently associated beta 2- microglobulin subunit (here in dark grey) and the antigenic peptide presented on HLA-G (short black arrow).
• HLA-G1 further contains a transmembrane domain and a short intracellular chain (not shown here).
• the [alpha]-3 domain is capable of binding to the receptors ILT2 (see Shiroishi et al., Proc Natl Acad Sci U S A. 2003 July 22; 100(15):8856-8861) and ILT4 (see Shiroishi et al., Proc Natl Acad Sci U S A.
• MHC class 1 a non-covalently linked MHC class 1 complex.
• protein tags such as SpotTag, myc tag and/or His(6x) tag
• they may be introduced in such a way as to enable their later optional removal via cleavage using an optional Factor Xa or Furin cleavage site.
• the antigenic peptide, beta 2-microglobulin and MHC lb [alphajchain can be linked in order to increase the stability.
• the vector map was generated using Snapgene Viewer Software.
• Figure 3 Surrogates of recombinant polypeptides of the invention induce IL10 secreting Treg in mice.
• 10Opig of surrogate molecules consisting of a viral (Gp34) or Ovalbumin (Ova) model peptide antigen, murine H2-K b alphal and 2 domains, and human HLA-G alpha 3 domain and beta-2- microglobulin were injected i.p. into 12 week old C57BL/6 mice.
• mice were sacrificed and splenocytes isolated via Ficoll gradient were rechallenged with 5pig/ml of either Gp34 or Ova peptide in an 48h standard murine IL-10 ELI Spot assay (Mabtech mouse IL-10 HRP ELI Spot Kit) (A).
• FIG. 4 Surrogates of recombinant polypeptides of the invention prevent CD8+ T-cell driven EAE in mice.
• the model antigen ovalbumin (OVA) is expressed in oligodendrocytes under the control of the myelin basic protein (MBP) promoter (ODC-OVA). This leads to 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 recognizes exactly this peptide-MHC combination.
• EAE autoimmune encephalomyelitis
• Figure 5 Surrogates of recombinant polypeptides of the invention can result in effective bystander immunosuppression.
• Figure 6 Some surrogates of recombinant polypeptides of the invention selectively prevent CD4 + T cell driven EAE in mice.
• 100pig/mouse of surrogate molecules consisting of a viral (Gp34) or two Mog peptide antigens (Mog37 or Mog44), murine H2-D b alphal and 2 domains, and human HLA-G alpha 3 domain and beta-2-microglobulin or just PBS were injected the first day.
• Gp34 a viral
• Mog37 or Mog44 two Mog peptide antigens
• murine H2-D b alphal and 2 domains murine H2-D b alphal and 2 domains
• human HLA-G alpha 3 domain and beta-2-microglobulin or just PBS were injected the first day.
• the Mog44 peptide containing surrogate molecule significantly reduced EAE symptoms (B) and weight loss (C).
• FIG. 7 Mog44 surrogates of recombinant polypeptides of the invention prevented inflammation and CD8 T cell infiltration in the spinal cord.
• A Toluidine
• B CD8-DAB
• Murine serum was collected from heart puncture after mice were sacrificed and diluted 1 :50 in PBS. 10pg/ml Mog35-55 in PBS were used for coating over night, wells were then blocked using 1%BSA for 2h, before diluted sera were added for 1 h. Anti-Mog35-55 antibodies were detected using the indicated secondary HRP coupled antibodies (dilution 1:5000 in PBS). Mog35-55 induced EAE correlated with high levels of Mog35-55 specific IgG autoantibodies, which were not detectable in animals treated with 10Opg MOG44_Db_G surrogate molecule.
• Figure 9 List of the human MS & MOGAD recombinant polypeptide candidates.
• the myelin (MAG, MBP, MOG, PLP) peptide and MHC class I presenting molecules are as follows:
• Figure 10 Upregulation of CD8 Treg in healthy blood donors by a recombinant polypeotide of the invention containing the VLLAVLPVL antigen ("Mog157_A2G”)
• PBMCs from healthy donors were purified via density centrifugation was performed on white blood cells from a leukocyte reduction chamber using Ficoll. Cells were centrifuged for 20 min at 1200 x g without brake followed by collection of the interphase ring that was washed with 1x PBS (5 min, 300 x g). PBMC were frozen till further use.
• PBMCs were thawed 1 day prior to PBMC pulsing (d-1) and kept over night in 5 ml X-VIVO 15 medium containing 5% human AB serum in a well of a 6 well plate at 37°C.
• X-VIVO 15 complete medium 5% hAB serum & cytokine cocktail: 20 ng/ml hlL-2, 20 ng/ml hGM-CSF, 10 ng/ml hlL-4 & 10 ng/ml hTGF-b1
• 3x10 6 cells were seeded in the respective wells of a 12-well plate with a final volume of 1000 pl X-VIVO complete medium with cytokine cocktail and 5 pg/ml of an AIM Bio molecule or the respective controls.
• ELISPOT plate PVDF membrane was activated with 50 pl/well EtOH (35% v/v) for 1 min followed by 5x washing with 200 pl distilled sterile water. Plate was coated with 100 pl/well anti-hlL10 (clone 9D-7, 1 :500 dilution in PBS, sterile filtered) at 4°C over night. On the next day, unbound coating antibody was removed, 5 washing steps were performed with 200 pl PBS and 200 pl blocking buffer (X-VIVO 155% hAB serum) was added and the plate incubated for 30 min - 2 h at room temperature.
• 200,000 cells were seeded per well on the ELISPOT plates in duplicates, including negative controls (cells plus PBS) and a positive control (e.g. LPS).
• negative controls cells plus PBS
• a positive control e.g. LPS
• Secondary antibody was prepared: 1 pg/ml al L-10-biotinylated antibody in 0.5% BSA/1x PBS (1 :1000 dilution) and horseradish peroxidase-conjugated streptavidin (1 :750 in 0.5% BSA/PBS), tetramethylbenzidine solution was filtered using a 0.45 pm filter and stored at 4°C till use.
• MOG157_A2G_Spt induced at least 30% more IL-10 secreting T reg in 75% of of all healthy blood donors.
• Figure 11 Control experiment for Figure 8 and showing that total IgG is not reduced by MOG47_Db_G surrogate molecule treatment.
• Easy-TiterTM Human IgG (gamma chain) Assay Kit (Thermo Fisher) was used to quantify total IgG according to the manjufacturers instructions. This experiments in conjunction with Figure 8 indicates that selective antibody responses can be suppressed using single-chain MHC lb molecules.
• Figure 12 Stability of purified single-chain MHC lb molecules. After purification of the single chain MHC lb molecules, their stability was analysed after 1 and 3 freeze-thawing cycles, storage for 5 days at room temperature and heating up to a temperature of 50°C for 30 min. For this, A) a Coomassie gel staining of a 12% polyacrylamide gel using 2 pig AIM Bio and B) an aHLA-G Western blot using the 2A12aHLA-G antibody (1 :1000) blot using 1 pig protein was performed under non-reducing conditions. Both monomers and dimers are detectable.
• FIG. 13 Single-chain MHC lb molecules are thermally stable.
• TSA Thermal Shift Assay
• 3 pig of the respective single chain MHC lb molecule or Motavizumab as control molecule were diluted with PBS and 5x SYPRO Orange dye (stock 5000x, final concentration: 5x) to a volume of 25 pil.
• a melting curve program was set up on a StepOnePlus Instrument using the StepOnePlus Software 2.3. The start temperature was 25°C for one minute followed by a temperature increase of 1°C per minute to a final temperature of 95°C for
• FIG. 14 Single-chain MHC lb molecules induce Treg in a dose-dependent manner.
• OT-I mice were injected i.p. with indicated amounts of single-chain H2_K b alphal +2 and HLA-G alpha3 domain constructs with human beta-2-microglobulin and the indicated peptide or carrier (PBS).
• Ova is the cognate peptide for the OT-I TOR in these mice, Gp34 is an irrelevant, virus derived control peptide.
• mice were sacrificed and splenocytes tested for IL10 secreting cells in a recall mouse IL-10 ELISpot (200,000 cells per well, MabTech mouse IL-10 ELISpot kit, 5 pig/ml of the indicated peptide or only PBS were added, 48h).
• a recall mouse IL-10 ELISpot 200,000 cells per well, MabTech mouse IL-10 ELISpot kit, 5 pig/ml of the indicated peptide or only PBS were added, 48h.
• a clear induction of IL-10 secreting cells reactive to Ova peptide was observed when 50 and 500 pig mouse adapted Ova_KbG were injected.
• Figure 15 Single-chain MHC lb molecules inhibit T cell lysis in a dose-dependent manner. 10 mio 0T-1/ml for
• Splenocyte isolation and treatment 0T1/BL6 Mice were sacrificed and splenocytes were collected and washed once in RPMI 5% FCS. Red blood cells were removed with 2ml 1x sterile RBC lysis buffer for 3 min. Cells were cultured in high density culture (10mio cells/ml) for 72h in RPM1 10% FCS medium with GMCSF 20 ng/mL, IL-2 20ng/ml and IL-4 10 ng/ml and increasing doses of Ova_KbG. Cells are then scraped from the plates, CD8+ cells are then purified via magnetic beads.
• FIG. 16 Serum cytokines from EAE-ODC Ova mice. Serum cytokines from EAE-ODC Ova mice were measured with Th 1/Th2 10plex Flowcytomix Kit (eBioscience) according to the manufacturer's instruction. The kit was used for the simultaneous detection of mouse granulocyte-macrophage colony-stimulating factor (GMCSF), interleukin 1 alpha (IL-1 a), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin- 10 (IL-10), t interleukin-17 (IL-17), and tumor necrosis factor (TNF) in a single sample.
• GMCSF mouse granulocyte-macrophage colony-stimulating factor
• IL-1 a interleukin-1 alpha
• IL-2 interleukin-2
• IL-4 interleukin-4
• IL-6 interleukin-6
• IL-10 interleukin- 10
• TNF tumor
• This array kit provides a mixture of ten capture beads with distinct fluorescent intensities that have been coated with capture antibodies specific for each cytokine. Beads coated with ten specific capture antibodies were mixed. Subsequently, 25 pL of the mixed captured beads, 25 pL of the unknown serum sample or standard dilutions, and 25 pL of phycoerythrin (PE) detection reagent were added consecutively to each well in 96-V bottom well plates and incubated for 2 h at room temperature in the dark. The samples were washed with 1 mL of wash buffer for 5 min and centrifuged. The bead pellet was resuspended in 200 pL buffer after discarding the supernatant. Samples were measured on the AttuneTM NxT Flow Cytometer and analyzed Attune Cytometric Software (Thermo Fisher Scientific).
• Figure 17 Immunofluorescence analyses of a spinal cord of a MOG-induced EAE model
• A, B Caspase 3 infiltration in MOG-induced EAE spinal cord.
• C Images; D: Quantitation E, F : CD3 infiltration in MOG-induced EAE spinal cord.
• Figure 18 Increase in IL10 spots after treatment with MOG157_A2G
• the recombinant polypeptides of the invention are meant to optionally include a secretion signal peptide sequence.
• the recombinant polypeptides of the invention are meant to also optionally include affinity tags, e.g. in order to facilitate purification, and optional protease cleavage sites between the tag and the polypeptide, e.g. in order to facilitate removal of the tags by protease cleavage.
• any reference to amino acid sequences referred to herein is meant to encompass not only the unmodified amino acid sequence but also typical posttranslational modifications of these amino acid sequences (e.g., glycosylation or deamidation of amino acids, the clipping of particular amino acids or other posttranslational modifications) occurring in cellular expression systems known in the art, including mammalian cells such as CHO and HEK cells.
• polypeptides of the invention are meant to optionally include the respective pro-peptides.
• the recombinant polypeptides of the invention can be in form of their soluble or their membrane-bound form. Whether a recombinant polypeptide is "soluble” under these conditions can be determined by methods known in the art, e.g., by measuring the turbidity of the recombinant polypeptide under the above-indicated reference conditions. As used herein, soluble means that at least 95% of the recombinant polypeptide is determined to be soluble under these reference conditions.
• Single chain MHC molecules can be stored, for instance, in PBS at -80°C (with or without 0.1% human albumin as carrier, depending on the protein concentration) or in 50% glycerol at -20°C.
• MHC molecules are preferably human MHC molecules.
• the recombinant polypeptides of the invention are preferably isolated recombinant polypeptides.
• a recombinant polypeptide capable of binding and presenting an peptide antigen according to the invention can be prepared.
• peptide antigen-binding domains such as [alpha]1 and [alpha]2 domains are well-known, and modifications of these domains can be made.
• the capability of a peptide antigen to bind to the polypeptides and MHC molecules according to the invention can be determined by techniques known in the art, including but not limited to explorative methods such as MHC peptide elution followed by Mass spectrometry and bio-informatic prediction in silico, and confirmative methods such as MHC peptide multimere binding methods and stimulation assays.
• the recombinant polypeptides, pharmaceutical compositions and kits of the invention are preferably suitable for use in a human patient.
• the recombinant polypeptides, pharmaceutical compositions and kits of the invention are preferably suitable for use in the treatment of multiple sclerosis (MS), MOG antibody disease or MOG antibody positive neuromyelitis optica in a human patient.
• MS multiple sclerosis
• MOG antibody disease MOG antibody positive neuromyelitis optica
• the recombinant polypeptides, pharmaceutical compositions and kits of the invention are preferably suitable for inducing immunological tolerance against human myelin-oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1), e.g., in a human patient.
• MOG human myelin-oligodendrocyte glycoprotein
• MBP human myelin basic protein
• MAG human myelin-associated glycoprotein
• PGP1 human myelin proteolipid protein
• any lenghts of these peptide antigens referred to herein are meant to refer to the length of the peptide antigens themselves.
• the lenghts of peptide antigens referred to herein do not include the length conferred by additional amino acids which are not part of the peptide antigens such as additional amino acids from possible linker sequences etc.
• each occurrence of the term “comprising” may optionally be substituted with the term “consisting of'.
• the methods used in the present invention are performed in accordance with procedures known in the art, e.g. the procedures described in Sambrook et al. ("Molecular Cloning: A Laboratory Manual.”, 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1989), Ausubel et al. ("Current Protocols in Molecular Biology.” Greene Publishing Associates and Wiley Interscience; New York 1992), and Harlow and Lane (“Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1988), all of which are incorporated herein by reference.
• Protein-protein binding such as binding of antibodies to their respective target proteins, can be assessed by methods known in the art. Protein-protein binding is preferably assessed by surface plasmon resonance spectroscopy measurements.
• binding of MHC class lb molecules or recombinant polypeptides according to the invention to their receptors, including ILT2 and ILT4, is preferably assessed by surface plasmon resonance spectroscopy measurements. More preferably, binding of MHC class lb molecules or recombinant polypeptides according to the invention to their receptors is assessed by surface plasmon resonance measurements at 25°C.
• Sequence Alignments of sequences according to the invention are performed by 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 alignments of short peptides by the BLAST algorithm which are suitable for peptide antigens in accordance with the invention, are known in the art. Most software tools using the BLAST algorithm automatically adjust the parameters for sequence alignments for a short input sequence.
• the following parameters are used: Max target sequences 10; Word size 3; BLOSUM 62 matrix; gap costs: existence 11, extension 1; conditional compositional score matrix adjustment.
• identity or “identical” preferably refer to the identity value obtained by using the BLAST algorithm.
• compositions of the present invention are prepared in accordance with known standards for the preparation of pharmaceutical compositions.
• compositions are prepared in a way that they can be stored and administered appropriately.
• the pharmaceutical compositions of the invention may therefore comprise pharmaceutically acceptable components such as carriers, excipients and/or stabilizers.
• Such pharmaceutically acceptable components are not toxic in the amounts used when administering the pharmaceutical composition to a human patient.
• the pharmaceutical acceptable components added to the pharmaceutical compositions may depend on the chemical nature of the active ingredients present in the composition, the particular intended use of the pharmaceutical compositions and the route of administration.
• the pharmaceutically acceptable components used in connection with the present invention are used in accordance with knowledge available in the art, e.g. from Remington's Pharmaceutical Sciences, Ed. AR Gennaro, 20th edition, 2000, Williams & Wilkins, PA, USA.
• Pharmaceutical compositions comprising the nucleic acids of the invention e.g., RNAs
• peptide antigens which can be used in accordance with the invention are not particularly limited other than by their ability to be presented on MHC molecules. It is understood that a "peptide antigen presented by said recombinant polypeptide” as referred to in relation to the invention is a peptide antigen that is presented by said recombinant polypeptide to human T cells, if such T cells are present, in a way that it binds to a T cell receptor on the human T-cells.
• MHC molecules which are able to be presented on MHC molecules can be generated as known in the art (see, for instance, 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 l-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).
• Peptide antigens are generally known in the art.
• the peptide antigens in accordance with the invention are capable of binding to MHC class I proteins. It will be understood by a person skilled in the art that for each MHC class lb molecule or polypeptide capable of presenting peptides in accordance with the invention, peptide antigens which are capable of binding to said MHC class lb molecule or recombinant polypeptide will preferably be used. These peptide antigens can be selected based on methods known in the art.
• Binding of peptide antigens to MHC class lb molecules or to polypeptides capable of peptide antigen binding in accordance with the invention can be assessed by methods known in the art, e.g. the methods of:
• Such methods include experimental methods and methods for the prediction of peptide antigen binding.
• Anchor residues which serve to anchor the peptide antigen on the MHC class I molecule and to ensure binding of the peptide antigen to the MHC class I molecule are known in the art.
• the peptide antigen used in accordance with the invention contain any of the anchor or preferred amino acid residues in the positions as predicted for MHC class I molecules.
• the peptide antigen is from human myelin-oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1).
• MOG human myelin-oligodendrocyte glycoprotein
• MBP human myelin basic protein
• MAG human myelin-associated glycoprotein
• PBP1 human myelin proteolipid protein
• non-anchor amino acid residues of the peptide antigen of the invention may or may not contain conservative substitutions, preferably not more than two conservative substitutions, more preferably one conservative subsitution with respect to the corresponding amino acid sequence of a peptide antigen from human myelin-oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG) or human myelin proteolipid protein (PLP1).
• MAG myelin-oligodendrocyte glycoprotein
• MBP human myelin basic protein
• MAG human myelin-associated glycoprotein
• PGP1 human myelin proteolipid protein
• Peptide antigens of the invention preferably consist of naturally occurring amino acids. However, non-naturally occurring amino acids such as modified amino acids can also be used.
• a peptide antigen of the invention encompasses the peptidomimetic of the indicated peptide antigen amino acid sequence of human myelin-oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin-associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1).
• the recombinant polypeptides of the invention can be used for the treatment of multiple sclerosis, MOG antibody disease and MOG antibody positive neuromyelitis optica.
• the treatment can be a treatment by inducing myelin-specific regulatory T cells.
• regulatory T cells e.g., CD8-positive regulatory T cells
• cytotoxic T cells recognizing the same or another myelin antigen.
• Regulatory T-cells e.g., CD8- positive regulatory T cells are known in the art and can be detected, for instance, by their secretion of IL-10.
• CD8-positive regulatory T cells are not as well known as CD4CD25 regulatory T cells, they have even been described to be more potent. See, for instance:
• CD122 and CD8 While these are characterized by expression of CD122 and CD8 in mice, their human counterparts have been described to be CD8 and CXCR3 positive. See, for instance:
• the treatment can be a treatment for reducing plasma or cerebrospinal fluid levels of autoantibodies against human myelin-oligodendrocyte glycoprotein.
• the human patient can be a patient who had plasma or cerebrospinal fluid autoantibodies against myelin-oligodendrocyte glycoprotein prior to the start of the treatment.
• the autoantibodies can be detected by various methods known in the art.
• a preferred approach are cell-based assays (CBAs) where the suspected target antigen of the autoantibodies (e.g., myelin-oligodendrocyte glycoprotein) is overexpressed in HEK293 or CHO cells which are then incubated with serum or cerebrospinal fluid, typically for 1 h at room temperature. Mock-transfected sister cells serve as controls.
• CBAs cell-based assays
• the suspected target antigen of the autoantibodies e.g., myelin-oligodendrocyte glycoprotein
• HEK293 or CHO cells which are then incubated with serum or cerebrospinal fluid, typically for 1 h at room temperature. Mock-transfected sister cells serve as controls.
• Autoantibodies that bind to the cells are detected with different fluorescently labeled anti-human specific secondary antibodies that recognize total human IgG (heavy and light chain), IgG-Fc
• Binding is quantified by either flow cytometry (CBA-FACS) or visual scoring by microscopic evaluation of the immunofluorescence (CBA-IF), which is often titrated.
• CBA-FACS flow cytometry
• CBA-IF immunofluorescence
• ELISAs enzyme-linked immunosorbent assays
• Western Blots are also possible, but often less sensitive, as conformationsensitive antibodies may not be detected by these methods. Suitable approaches have been described in
• Myelin oligodendrocyte glycoprotein (MOG) antibodies can be found in Reindl M, Waters P. Myelin oligodendrocyte glycoprotein antibodies in neurological disease. Nat Rev Neurol. 2019 Feb;15(2):89-102. doi: 10. 1038/s41582-018-0112-x. PMID: 30559466. Sequences
• Preferred amino acid sequences referred to in the present application can be independently selected from the following sequences.
• the sequences are represented in an N-terminal to C-terminal order; and they are represented in the one-letter amino acid code.
• Optional leader Peptide (absent from the recombinant polypeptide due to processing during cellular expression): e.g. MSRSVALAVLALLSLSGLEA (SEQ ID NO: 1)
• Peptide antigen any MHO class I peptide corresponding to MHO class I [alpha] 1&2 domains, e.g.
• VLLAVLPVL (SEQ ID NO: 2) (most preferred)
• First linker For instance GGGGSGGGGSGGGGS (SEQ ID NO: 3) or GCGASGGGGSGGGGS (SEQ ID NO: 4) beta 2 Microglobulin, for instance:
• Second Linker for instance:
• [Alpha] 1 & 2 domain derived either from human HLA-G or from any other MHO class I [alpha]1&2 domain suitable to present the selected antigenic peptide, Y84 may be C in DT variant e.g. [Alpha] 1 & 2 domain derived from human HLA-G: E.g., GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAH AQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAA QISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRA (SEQ ID NO: 7)
• Human HLA-A2 [alpha] 1 & 2 domain E.g.,
• Human HLA-G [alpha]3 domain (or any MHO lb [alpha]3 domain, such as HLA-F, which also interacts with ILT2 and ILT4 receptors), for instance:
• EQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDL a shorter form of a human HLA-G [alpha]3 domain which lacks the optional C- terminal amino acid sequence from intron 4 (SKEGDGGIMSVRESRSLSEDL; SEQ ID NO: 20), i.e.: DPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVWPSGE EQRYTCHVQHEGLPEPLMLRW (SEQ ID NO: 21), Factor Xa restriction site: IEGRTGTKLGP (SEQ ID NO: 10) SpotTag: PDRVRAVSHWSSC (SEQ ID NO: 11) Myc tag: EQKLISEEDL (SEQ ID NO: 12) His tag: HHHHHH* (SEQ ID NO: 13)
• Spacer sequence e.g. NSAVD (SEQ ID NO: 14) or GS
• exemplary peptide antigens which can be part of the recombinant polypeptides of the invention are as follows:
• Example for a recombinant polypeptide of the invention (with the optional leader peptide): MSRSVALAVLALLSLSGLEAVLLAVLPVLGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVS GFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGG GGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAP WVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGCYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYD GKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVT HHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTF
• sequence of the peptide antigen (here: VLLAVLPVL) of the above full length recombinant polypeptide can be substituted by any peptide antigen sequence in accordance with the invention, i.e. by any peptide antigen presented by said recombinant polypeptide, wherein the peptide antigen is a peptide of human myelin-oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin- associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1).
• MOG human myelin-oligodendrocyte glycoprotein
• MBP human myelin basic protein
• MAG human myelin-associated glycoprotein
• PGP1 human myelin proteolipid protein
• recombinant polypeptides of the invention may consist of a sequence consisting of a peptide antigen which is a peptide of human myelin-oligodendrocyte glycoprotein (MOG), human myelin basic protein (MBP), human myelin- associated glycoprotein (MAG), or human myelin proteolipid protein (PLP1)(e.g., any one of the peptide antigens of SEQ ID NO: 2, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33), followed by the sequence of
• polypeptides of the invention may also contain the optional leader peptide as exemplified above.
• the receptors ILT2 also known as LILRB1
• ILT4 also known as LILRB2
• Preferred sequences of these receptors in accordance with the invention are as follows:
• PEDGVEMDTRAAASEAPQDVTYAQLHSLTLRRKATEPPPSQEREPPAEPSIYATLAIH SEQ ID NO: 18
• human myelin-oligodendrocyte glycoprotein MOG
• human myelin basic protein MBP
• human myelin-associated glycoprotein MAG
• MBP human myelin basic protein
• MAG human myelin-associated glycoprotein
• PBP1 human myelin proteolipid protein
• Expi-293F cells (Thermo Fisher), grown in Expi-293TM expression medium (Thermo Fisher): transfection of 1 pig DNA into 2.5x10 6 cells/ml using the ExpifectamineTM 293 Transfection kit (Thermo Fisher) using Opti-MEM (Thermo Fisher) for complexation of DNA with Expifectamine, after 18-20 h, addition of enhancer according to the protocol, harvesting of the supernatant after 4-6 days (37°C, 8% CO2, humidified incubator), 19 mm 2 orbital shaker 125 rpm
• Spot-tag protein purification equilibration of Spot-Cap resin: transfer of desired slurry amount into an appropriate tube, sediment beads by centrifugation (4°C, 4 min, 2500 g), remove & discard supernatant, add 10 bed volumes PBS (cold) to beads, invert to mix, sediment beads by centrifugation (4°C, 4 min, 2500 g), remove & discard supernatant, repeat 2 times
• PBMC peripheral blood mononuclear cells
• PBMC pulsing under a laminar flow hood
• PBMCs were thawed 1 day prior to PBMC pulsing (d-1) and kept over night in 5 ml X-VIVO 15 medium containing 5% human AB serum in a well of a 6 well plate at 37°C.
• X-VIVO 15 complete medium X-VIVO 15 medium +2% human AB serum supplemented with cytokine cocktail: 10 ng/ml TGF-b1, 10 ng/ml IL-4, 20 ng/ml IL-2, 20 ng/ml GM-CSF
• ELISPOT plates were coated using anti-hlLW (clone 9D-7, 1 :500 dilution in PBS, sterile filtered) and alL10 (10G8-biotin) and on day 14, 200,000 cells were seeded per well on the ELISPOT plates in duplicates, including negative controls (cells plus PBS) and a positive control (e.g. LPS).
• anti-hlLW clone 9D-7, 1 :500 dilution in PBS, sterile filtered
• alL10 10G8-biotin
• the PFDF membrane was activated with 50 pl/well EtOH (35% v/v) for 1 min followed by 5x washing with 200 pl distilled sterile water. Plate was coated with 100 pl/well antibody solution at 4°C over night. On the next day, unbound coating antibody was removed, 5 washing steps were performed with 200 pl PBS and 200 pl blocking buffer (X-VIVO 15 5% hAB serum) was added and the plate incubated for 30 min - 2 h at room temperature.
• the respective antigenic peptide (e.g.MOG157) in DMSO or DMSO as a control were prepared, and a final amount of 5 pg peptide/ml was added to the final volume of 100 pl/well.
• 150,000 cells were seeded per well in X-VIVO 15 medium with 5% human AB serum.
• Blocking buffer (X VIVO 15 medum +5% hAB serum) was carefully removed, and medium with PBS as negative control and stimulants (5 pg/ml total volume in each well) were added to the other wells and incubated at 37°C over night.
• Secondary antibody was prepared: 1 pg/ml al L-10-biotinylated antibody in 0.5% BSA/1x PBS (1 :1000 dilution) and horseradish peroxidase-conjugated streptavidin (1 :750 in 0.5% BSA/PBS), tetramethylbenzidine solution was filtered using a 0.45 pm filter and stored at 4°C till use.
• Capture antibodies anti-hlLW (Clone: 9D-7, Mabtech #3430-3-250; 1 :500 dilution), anti-hlLW- biotinylated (Mabtech, #3430-6-250), 1x PBS (sterile), 35% EtOH (v/v), Blocking buffer: X-vivo 5% hAB serum (sterile) [blocking is done in the same medium as cell culture], Dilution buffer: 0.5% BAS 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
• Example 2 Surrogates of recombinant polypeptides of the invention induce IL10 secreting Treg in mice Wild type black 6 mice were injected with 10Opig recombinant polypeptides (also referred to as "AIMBio”) having the following sequences,
• Gp34 is a well- characterized T cell epitope derived from Lymphocytic Choriomeningitis virus (LCMV) Glycoprotein. While this epitope was traditionally named Gp33, the epitope presented on H2-K b was later found to comprise just amino acids 34-41. (An epitope beginning at amino acid 33 is, in contrast, presented on H2-K d .) Therefore, we call the H2-K b epitope Gp34, which is in line with the most recent recommendations. Still, there is an ambiguous use of the Gp33 and Gp34 nomenclature in the literature. The first eight amino acids of SEQ ID NO: 47 show the respective peptide sequence. After 2 weeks, mice were sacrificed, and splenocytes re-challenged either with the matching or a mismatching peptide. IL-10 secreting cells were quantified by ELIspot. The results are shown in Figure 3.
• Example 3 Surrogates of recombinant polypeptides of the invention prevent CD8+ T-cell driven EAE in mice As described in (Na et al, Brain. 2008 Sep;131 (Pt 9):2353-65.), the adoptive transfer of CD8 + OT-I T cells that recognize an ovalbumin epitope in the context of H2-K b into mice which express ovalbumin in oligodendrocytes leads to experimental autoimmune encephalomyelitis which recapitulates many MS and MOGAD symptoms.
• sequences of the recombinant polypeptide surrogate molecules were as follows: Mog44_DbG FSRWHLYRNGGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERI EKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGG GGSGPHSMRYFETAVSRPGLEEPRYISVGYVDNKEFVRFDSDAENPRYEPRAPWMEQEGPEYWERETQKA KGQEQWFRVSLRNLLGCYNQSAGGSHTLQQMSGCDLGSDWRLLRGYLQFAYEGRDYIALNEDLKTWTAAD MAAQITRRKWEQSGAAEHYKAYLEGECVEWLHRYLKNGNATLLRTDPPKTHVTHHPVFDYEATLRCWALGF YPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAWVPSGEEQ
• AIM Bio 33 g or 10O g recombinant polypeptide of the invention surrogate molecule
• Mog-reactive antibodies in sera of AIM Bio (33 or 10O g) treated mice were detected via standard ELISA protocol, with 3 washes in between each step. Briefly, ELISA plates were coated with 10 g/ml Mog35-55 peptide, blocked with PBS 1% BSA, before mouse sera diluted 1 :25 in PBS 1% BSA were added for 1 h. Antimouse IgG-HRP or anti-mouse heavy and light chain - HRP antibodies diluted 1:5000 were used for detection.
• Example 5 Human recombinant polypeptide candidates of the invention for MS and MOGAD
• the recombinant polypeptides of the invention are newly developed protein complexes derived from the pregnancy-associated immunosuppressive MHC molecule HLA-G. It is likely that HLA-G enables an embryo to influence the maternal immune system to tolerate embryonic antigens but further antagonize antigens from pathogens.
• the recombinant polypeptides of the invention containing variable peptides were able to selectively eliminate peptide-specific cytotoxic effector T cells as well as induce peptide-specific regulatory T cells in the test tube.
• Figure 9 shows a list of the human MS & MOGAD recombinant polypeptide candidates.
• CD8 Treg were upregulated by at least 30% in 75% of all healthy blood donors by a recombinant polypeotide of the invention containing the VLLAVLPVL antigen (also referred to as "Mog157_A2G”; see Figure 10).
• Example 6 Further proof-of-principle of stability and effects of the recombinant polypeptides of the invention. Additionally, the inventors set out to obtain and test recombinant polypeptidies having the general structure of the recombinant polypeptides of the invention but containing various different peptide antigens, in order to obtain further proof-of-principle that recombinant polypeptidies of the invention and surrogates thereof are stable and efficacious. As shown in Figures 12 and 13, respectively, the tested recombinant polypeptidies are stable during freeze-thawing and storage and are thermally stable. Further, they induce Treg in a dosedependent manner (Figure 14) and inhibit T cell lysis in a dose-dependent manner ( Figure 15).
• Example 7 MOG-tolerance inducing recombinant polypeptides of the invention prevent apoptosis (cleaved caspase 3), CD3 + immune cell infiltration and myelin lesions in the spimal cord.
• the mouse model is described in Figure 6.
• Block 1h 5% BSA+0.2% Triton-X100+5% NGS in 1X PBS
• FIG. 17 A, B Caspase 3 activation (apoptosis) can be prevented in MOG-induced EAE spinal cord.
• Example 8 Increase in IL10 spots after treatment with MOG157_A2G
• Figure 18 shows an increase in IL10 spots after treatment with MOG157_A2G, which indicates that tolerogenic Treg have been induced. This is an alternative depiction of Figure 10.
• compositions, polypeptides, nucleic acids, cells, and products for use in the invention are industrially applicable. For example, they can be used in the manufacture of, or as, pharmaceutical products.

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