MHC Ib-mediated islet antigen-specific immunosuppression as a novel treatment for type 1 diabetes

Abstract

The present invention relates to the therapeutic use of non-classical human major histocompatibility complex (MHC) molecules (also referred to as MHC class Ib molecules) in combination with peptide antigens for the treatment of type 1 diabetes (T1D). The invention more particularly relates to recombinant polypeptides comprising a peptide antigen and one or more domains of a non-classical MHC class Ib molecule. The invention also relates to methods for producing such recombinant polypeptides, pharmaceutical compositions comprising same, and their use for treating type 1 diabetes (T1D).

Description

[Technical field]

[0001] The present invention relates to the therapeutic use of non-classical human major histocompatibility complex (MHC) molecules (also referred to as MHC class Ib molecules) in combination with peptide antigens for the treatment of type 1 diabetes (T1D). The present invention more specifically relates to recombinant polypeptides comprising a peptide antigen and one or more domains of a non-classical MHC class Ib molecule. The present invention also relates to methods for producing such recombinant polypeptides, pharmaceutical compositions comprising same, and their use for treating type 1 diabetes (T1D). [Background technology]

[0002] In all autoimmune diseases, type 1 diabetes is caused by an excessive immune response against the body's own tissues, which are mistaken for foreign and attacked. T cells play a crucial role, as they are able to recognize individual target structures (antigens) very selectively through their receptors. Currently, the disease is mainly treated with anti-inflammatory drugs or antibodies that gradually suppress the immune response and suppress symptoms or slow the progression of the disease. At the same time, however, functional T cells are necessary for the survival of patients with autoimmune diseases, as they can recognize and fight dangerous viruses, bacteria, parasites, and mutated cells. Therefore, systemic immunosuppression has only a limited scope of use. Therefore, in the case of type 1 diabetes, one attempt is to administer insulin to compensate for the damage that has occurred. However, it is difficult to check blood sugar levels and administer insulin accurately. Therefore, there are many unmet medical needs, and various sequelae reduce the life expectancy of type 1 diabetes patients by about 11-13 years (Livingstone et al,JAMA.2015 Jan 6;313(1):37-44).

[0003] CD8 T cells that attack pancreatic islet cells play an important role in type 1 diabetes (Tsai S, Shameli A, Santamaria P. CD8+ T cells in type 1 diabetes. Adv Immunol. 2008;100:79-124). However, one of the important tools for diagnosing type 1 diabetes is the detection of autoantibodies in serum, such as islet cytoplasmic autoantibodies (ICA), glutamic acid decarboxylase 65 autoantibodies (GADA), pancreatic endocrine tumor-associated protein II autoantibodies (IA-2A), or insulin autoantibodies (IAA). With this method, patients at risk can be identified before significant islet cell destruction occurs. Furthermore, suppression of both CD8 T cell responses and autoantibody formation significantly improves the outcome of the disease.

[0004] WO 2018 / 215340 relates to the combination of MHC class Ib molecules with peptides for targeted therapeutic immune modulation. Overall, there remains a need for improved drugs for the treatment of type 1 diabetes (T1D). Summary of the Invention [Problem to be solved by the invention]

[0005] The inventors have found that human MHC class Ib molecules, such as HLA-G, have the ability to induce antigen-specific tolerance to peptide antigens presented thereto. Thus, MHC class Ib molecules can be advantageously used according to the invention to suppress immune responses in an antigen-specific manner, despite having a similar structure and sequence to classical human MHC class Ia molecules, which induce antigenic peptide-specific immune responses. Furthermore, the inventors have found that for the suppression of immune responses according to the invention, molecules other than naturally occurring MHC class Ib molecules can be used, in particular polypeptides comprising at least one domain of an MHC class Ib molecule, preferably at least the [α]3 domain of an MHC class Ib molecule only: the [α]1 and [α]2 domains of variable class Ia molecules can be combined with the [α]3 domain of human MHC class Ib molecules to suppress immune responses to peptides presented by these antigens. The inventors have further found that antigens housed within the peptide-binding cleft of HLA-G induce selective tolerance to allogeneic T cells. The inventors have observed two mechanisms in particular: induction of apoptosis in highly activated cytotoxic CD8+ T cells, and induction of regulatory T cells in allogeneic naive T cells. Thus, the present invention allows the induction of selective tolerance to unique antigens without compromising protective immune responses against pathogens.

[0006] Antigen-loaded HLA-G molecules 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 MHC class Ib molecules such as HLA-G were exchanged with the respective domains of other MHC molecules to increase the flexibility and versatility of these recombinant polypeptides (see, e.g., FIG. 2). These alternative recombinant polypeptides can be designed taking into account the antigen-binding domains of other human HLA molecules. It has already been found that a construct containing the α1 and α2 domains of mouse H2-Kb can present the ovalbumin-derived peptide SIINFEKL to OT-1 T cells, which express a transgenic T cell receptor that specifically recognizes this antigen (WO 2018 / 215340).

[0007] Surprisingly, the inventors have found that the recombinant polypeptides of the invention can be used to suppress immune responses against human proinsulin / human insulin (INS), human glutamic acid decarboxylase 65 (GAD65), human islet amyloid polypeptide (IAPP), and human zinc transporter 8 (ZNT8). Thus, according to the invention, the recombinant polypeptides of the invention can be used to treat type 1 diabetes (T1D).

[0008] In addition, the present invention provides that the recombinant polypeptide of the present invention not only modulates T cell responses, but also prevents the formation of autoantibodies against human proinsulin and human insulin (INS), human glutamic acid decarboxylase 65 (GAD65), human islet amyloid polypeptide (IAPP), and human zinc transporter 8 (ZNT8). Since such autoantibodies, in addition to CD8+ T cells, are involved in the pathology of type 1 diabetes (T1D), it is expected that this advantage will contribute to the clinical improvement of human patients with type 1 diabetes (T1D). [Means for solving the problem]

[0009] Thus, the present invention relates to the following preferred embodiments: 1. A recombinant polypeptide capable of presenting a peptide antigen, the recombinant polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen presented by the recombinant polypeptide, the peptide antigen being a peptide of human proinsulin or human insulin, human glutamic acid decarboxylase 65, human islet amyloid polypeptide, or human zinc transporter 8; ii) optionally, a linker sequence; iii) optionally a sequence of a human polypeptide domain that includes a 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 as set forth in SEQ ID NO:5; iv) optionally, a linker sequence; v) optionally, the [α]1 domain of an MHC molecule; vi) optionally, the [α]2 domain of an MHC molecule; vii) an [α]3 domain of an MHC class Ib molecule, or a derivative of an [α]3 domain of an MHC class Ib molecule, which is capable of binding to ILT2 or ILT4; viii) optionally, a protease cleavage site; ix) optionally, a spacer sequence; and x) Possibly, an affinity tag. 2. The recombinant polypeptide according to item 1, wherein the peptide antigen according to i) is 7 to 11 amino acids in length, preferably 8 to 10 amino acids in length. 3. The recombinant polypeptide according to item 1 or 2, wherein the peptide antigen according to i) consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 2, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27. 4. The recombinant polypeptide according to any one of items 1-3, wherein the peptide antigen consists of an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:2, SEQ ID NO:22, and SEQ ID NO:23. 5. The recombinant polypeptide according to any one of items 1-3, wherein the peptide antigen is a peptide antigen of human proinsulin or human insulin, preferably consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 25.

[0010] 6. The recombinant polypeptide according to any one of items 1-3, wherein the peptide antigen is a peptide antigen of human glutamic acid decarboxylase 65, preferably consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:23, SEQ ID NO:26, and SEQ ID NO:27. 7. The recombinant polypeptide according to any one of items 1 to 3, wherein the peptide antigen is a peptide antigen of human glutamic acid decarboxylase 65 and consists of the amino acid sequence of SEQ ID NO: 26. 8. The recombinant polypeptide according to any one of items 1 to 3, wherein the peptide antigen is a peptide antigen of human zinc transporter 8, preferably consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 and 24. 9. The recombinant polypeptide according to any one of items 1-3, wherein the peptide antigen is a peptide antigen of human islet amyloid polypeptide. 10. The recombinant polypeptide according to any one of items 1 to 9, 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 or a human MHC class Ib molecule.

[0011] 11. The recombinant polypeptide according to item 10, 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. 12. The recombinant polypeptide according to item 11, wherein the [α]1 domain described in (v) and the [α]2 domain described in (vi) are derived from a human HLA-A2 molecule. 13. The recombinant polypeptide according to item 10, wherein the [α]1 domain described in (v) and the [α]2 domain described in (vi) are derived from a human MHC class Ib molecule. 14. The recombinant polypeptide according to item 13, wherein the [α]1 domain described in (v) and the [α]2 domain described in (vi) are derived from a human HLA-G molecule. 15. A recombinant polypeptide according to any one of items 1 to 14, wherein the [α]3 domain of the MHC class Ib molecule according to (vii) is the [α]3 domain of human HLA-E, human HLA-F or human HLA-G.

[0012] 16. A recombinant polypeptide according to any one of items 1 to 15, wherein the [α]3 domain of an MHC class Ib molecule according to (vii) is the [α]3 domain of human HLA-G. 17. A recombinant polypeptide according to any one of items 1-16, wherein the [α]3 domain or derivative according to (vii) is identical to or has at least 80% amino acid sequence identity, preferably at least 90% amino acid sequence identity, to an [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 21. 18. The recombinant polypeptide according to item 17, wherein the [α]3 domain or derivative according to (vii) is identical to or has at least 92% amino acid sequence identity with an [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 2. 19. The recombinant polypeptide according to item 17, wherein the [α]3 domain or derivative according to (vii) is identical to or has at least 94% amino acid sequence identity with an [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 21. 20. A recombinant polypeptide according to item 17, wherein the [α]3 domain or derivative according to (vii) is identical to or has at least 96% amino acid sequence identity with an [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 2.

[0013] 21. A recombinant polypeptide according to item 17, wherein the [α]3 domain or derivative according to (vii) is identical to or has at least 98% amino acid sequence identity with an [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 21. 22. A recombinant polypeptide according to item 17, wherein the [α]3 domain or derivative according to (vii) is identical to or has at least 99% amino acid sequence identity with an [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 21. 23. The recombinant polypeptide according to item 17, wherein the [α]3 domain described in (vii) is identical to the [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 21. 24. A recombinant polypeptide according to any one of items 1 to 23, wherein the linker sequence according to (ii) and / or the linker sequence according to (iv) comprises the amino acid sequence (GGGGS)n, where n is an integer equal to or greater than 1. 25. The recombinant polypeptide according to item 24, wherein the linker sequence according to (ii) comprises the amino acid sequence (GGGGS)n, where 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.

[0014] 26. A recombinant polypeptide according to item 24 or 25, wherein the linker sequence according to (iv) comprises the amino acid sequence (GGGGS)n, where 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. 27. The recombinant polypeptide according to any one of items 1 to 26, wherein the sequence of the human polypeptide domain according to (iii) is at least 95% identical to the amino acid sequence of SEQ ID NO: 5, preferably at least 98% identical to the amino acid sequence of SEQ ID NO: 5, more preferably identical to the amino acid sequence of SEQ ID NO: 5. 28. The recombinant polypeptide according to any one of items 1 to 27, wherein the polypeptide is a dimer or multimer. 29. A recombinant polypeptide according to any one of items 1 to 28, wherein the polypeptide comprises or consists of all of components i) to vii). 30. The recombinant polypeptide according to any one of items 1 to 29, wherein the polypeptide does not contain components viii) to x).

[0015] 31. A recombinant polypeptide according to any one of items 1 to 29, wherein the polypeptide comprises or consists of all of components i) to x). 32. The recombinant polypeptide according to any one of items 1 to 31, further comprising an N-terminal secretory signal peptide sequence. 33. The recombinant polypeptide according to any one of items 1 to 31, wherein the recombinant polypeptide consists of an amino acid sequence consisting of, in order from N-terminus to C-terminus, the following ((a) and (b)): (a) a peptide antigen selected from the group consisting of the amino acid sequences of SEQ ID NO:2, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:2; and (b) The amino acid sequence of SEQ ID NO:16. 34. The recombinant polypeptide according to any one of items 1-33, wherein the recombinant polypeptide is soluble. 35. A nucleic acid encoding one or more polypeptides according to any one of items 1-34.

[0016] 36. The nucleic acid according to item 35, wherein the nucleic acid is a vector. 37. A pharmaceutical composition comprising at least one nucleic acid according to item 35 or 36. 38. A pharmaceutical composition comprising at least one recombinant polypeptide according to any one of items 1 to 34. 39. The pharmaceutical composition or kit according to item 38, wherein the pharmaceutical composition or kit comprises at least two different recombinant polypeptides according to any one of items 1 to 34, each of the different recombinant polypeptides comprising a different peptide antigen as defined in any one of items 3 to 9. 40. At least (A) to (C) below: (A) a recombinant polypeptide, wherein the peptide antigen is a peptide antigen of human proinsulin or human insulin; (B) a recombinant polypeptide, wherein the peptide antigen is a peptide antigen of human glutamic acid decarboxylase 65; (C) Recombinant polypeptide in which the peptide antigen is a peptide antigen of human zinc transporter 8. and optionally (D) A recombinant polypeptide, wherein the peptide antigen is a peptide antigen of human islet amyloid polypeptide. 40. The pharmaceutical composition or kit according to item 38 or 39, comprising:

[0017] 41. The pharmaceutical composition or kit according to any one of items 38-40, comprising at least three different recombinant polypeptides according to any one of items 1-34, wherein the peptide antigen of a first recombinant polypeptide among the at least three different recombinant polypeptides consists of the amino acid sequence of SEQ ID NO: 2, the peptide antigen of a second recombinant polypeptide among the at least three different recombinant polypeptides consists of the amino acid sequence of SEQ ID NO: 22, and the peptide antigen of a third recombinant polypeptide among the at least three different recombinant polypeptides consists of the amino acid sequence of SEQ ID NO: 23. 42. A pharmaceutical composition or kit according to any one of items 37-41 for use in the treatment of type 1 diabetes in a human patient. 43. The pharmaceutical composition or kit according to item 42, wherein the treatment is immunotherapy. 44. The pharmaceutical composition or kit according to any one of items 42-43, wherein the treatment is by inducing immune tolerance to human proinsulin and / or human insulin, human glutamic acid decarboxylase 65, human islet amyloid polypeptide, and / or human zinc transporter 8. 45. The pharmaceutical composition or kit according to any one of items 42-44, wherein the treatment is for reducing the amount in plasma of autoantibodies against insulin (insulin autoantibody IAA), glutamic acid decarboxylase 65 (GAD-65), islet antigen-2A (IA-2A), or zinc transporter ZnT8, as assessed by a radioactive binding assay or a non-radioactive electrochemiluminescence-based antigen binding assay.

[0018] 46. ​​The pharmaceutical composition or kit according to any one of items 42-45, wherein the human patient is a patient who has plasma autoantibodies against insulin (insulin autoantibody IAA), glutamic acid decarboxylase (GAD-65), islet antigen-2A (IA-2A), or zinc transporter ZnT8 before starting treatment. 47. A recombinant host cell comprising a nucleic acid or vector according to item 35 or 36 and expressing a recombinant polypeptide according to any one of items 1 to 34. 48. A method for obtaining a pharmaceutical composition comprising a polypeptide according to any one of items 1 to 34, comprising the steps of: (a) culturing a recombinant host cell according to item 47 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.

[0019] The pharmaceutical composition or kit used in the present invention can also be used to treat type 1 diabetes in human patients in conjunction with stem cell therapy for regeneration of pancreatic tissue. Such co-treatment is beneficial because the recombinant polypeptides of the present invention promote the regeneration of pancreatic tissue by stem cell therapy through their specific immunosuppressive effect.

[0020] The pharmaceutical composition or kit used in the present invention may also be used to treat type 1 diabetes in human patients in conjunction with stem cell therapy for regeneration of pancreatic tissue or human beta cell regenerative drug therapy. Human beta cell regenerative drug therapy for diabetes is reviewed in P Wang, E Karakose, L Choleva, K Kumar, RJ DeVita., A Garcia-Ocana, AF Stewart Andrew. Human Beta Cell Regenerative Drug Therapy for Diabetes: Past Achievements and Future Challenges. Frontiers in Endocrinology 12, 2021. DOI=10.3389 / fendo.2021.671946. [Brief description of the drawings]

[0021] [Figure 1] Schematic diagram of peptide-loaded soluble MHC Ib molecules suitable for achieving therapeutically effective antigen-specific immune modulation. The presented peptide antigens are represented as dotted spheres, the HLA-Gα1-3 domains are depicted in light grey and the β2 microglobulin domains are shown in dark grey. The optional linkers connecting the antigenic peptides to the β2 microglobulin molecules are shown in grey stick form and the optional disulfide traps are depicted as black spheres. The diagram was created using Pymol and is based on structures published in Clements et al.,Proc Natl Acad Sci US A. 2005 Mar 1;102(9):3360-5 and Hansen et al.,Trends Immunol.2010 Oct;31(10):363-9. [Diagram 2]Examples of vector constructs encoding single-chain MHC Ib molecules suitable for therapeutic peptide-specific immunomodulation. HLA-G1 and HLA-G5 each consist of three [α] domains (shown in black in the figure), a non-covalently bound β2 microglobulin subunit (shown in dark grey in the figure), and an antigenic peptide (short black arrow) presented on HLA-G. HLA-G1 further contains a transmembrane domain and a short intracellular chain (not shown in the figure). As shown in the figure, the [α]3 domain can bind to the receptors ILT2 (see Shiroishi et al., Proc Natl Acad Sci US A. 2003 July 22; 100(15): 8856-8861) and ILT4 (see Shiroishi et al., Proc Natl Acad Sci US A. 2006 October 31; 103(44): 16412-7) on immune cells. Physiologically, these sequences form the MHC class 1 complex by non-covalent binding. To facilitate purification of the complex MHC Ib molecule, one or more protein tags (such as Spot tag, myc tag and / or His(6x) tag) may be introduced. These can be introduced for subsequent removal, if desired, by cleavage using an optional factor Xa cleavage site. Furthermore, the antigen peptide, β2 microglobulin and MHC Ib[α] chain can be linked to increase stability. Vector maps are generated using Snapgene Viewer Software. [Figure 3A]Surrogates of the invention induce IL10-secreting Tregs in mice. In this experiment, 100 μg of surrogates containing virus-derived (Gp34) or ovalbumin (Ova) model peptide antigens, mouse-derived H2-Kb-[α]1 and 2 domains, and human HLA-G[α]3 domain and β2 microglobulin were administered intraperitoneally to 12-week-old C57BL / 6 mice. 14 days later, mice were sacrificed and isolated splenocytes were rechallenged with 5 μg / ml of Gp34 or Ova peptides in a 48-h mouse IL10 ELIspot assay. A significant increase in IL-10-secreting regulatory T cells was detectable only in response to peptide rechallenge when tolerance was induced by injection of the surrogate molecules. (A) Experimental design, (B) Results. [Figure 3B] Continued from Figure 3A. [Figure 4A]The recombinant polypeptide surrogate of the present invention prevents EAE caused by CD8+ T cells in mice. In this MS mouse model, the model antigen ovalbumin (Ova) was expressed in oligodendrocytes under the control of the myelin basic protein (MBP) promoter (ODC-OVA). This results in the presentation of the OVA257-264 peptide on H-2Kb-MHC molecules in oligodendrocytes. OT-I mice express a T cell receptor (OT-I) that precisely recognizes this peptide-MHC combination in CD8+ T cells. When CD8+ T cells from these mice were transferred to 10-day-old ODC-OVA mice, experimental autoimmune encephalomyelitis (Na et al., Brain, Volume 131, Issue 9, September 2008, Pages 2353-2365) developed that mimicked MS in many ways in pathology and symptomology. In this experiment, 500 μg of virus-derived (Gp34) or ovalbumin (Ova) model peptide antigens, mouse-derived H2-Kb-[α]1 and 2 domains, and surrogates containing human HLA-G[α]3 domain and β2 microglobulin, or PBS alone were injected on the same day. EAE was scored according to Bittner et al.,J Vis Exp.2014 Apr 15;(86):51275. Only the surrogates that induced ovalbumin tolerance were able to almost completely prevent EAE symptoms. (A) Experimental design, (B) Results. [Figure 4B] Continued from Figure 4A. [Figure 5A]Some of the surrogates of the recombinant polypeptides of the present invention selectively prevent CD4+ T cell-induced EAE in mice. In this model, administration of MOG 35-55 peptide in combination with complete Freund's adjuvant, which activates CD4+ Th17 cells, and pertussis toxin, which increases the permeability of the blood-brain barrier, induces a strong myelin-specific autoimmune response (Protocol: Bittner et al., J Vis Exp. 2014 Apr 15; (86): 51275). Not only antibodies but also CD4+ cells play an important role in the development of EAE (Tigno-Aranjuez et al., J Immunol November 1, 2009, 183 (9) 5654-5661). In addition, 100 μg / mouse of surrogate molecules consisting of viral antigen (Gp34) or two Mog peptide antigens (Mog37 or Mog44), mouse H2-Dbα1 and 2 domains, and human HLA-Gα3 domain and β2 microglobulin, or PBS alone were injected on the first day. Mog44 peptide-containing surrogate molecules significantly reduced EAE symptoms and weight loss. (A) Experimental design; (B) EAE score; (C) Body weight. [Figure 5B] Continued from Figure 5A. [Figure 5C] Continued from Figure 5B. [Figure 6A] Mog44 surrogate of the recombinant polypeptide of the present invention prevented inflammation and CD8 T cell infiltration in the spinal cord. Fresh frozen sections of 10 μm were stained with a commercially available toluidine 1x staining reagent at room temperature for 1 hour. Strong infiltration of immune cells was detected in EAE, but was prevented by Mog44_Db_G. Fresh frozen sections of 10 μm were dried briefly at room temperature, fixed with acetone, blocked with 5% BSA 10% normal goat serum in PBS, and stained with 1:100 anti-CD8 antibody, HRP-conjugated secondary antibody, and DAB solution (detailed method: Karikari et al., Brain Behav Immun. 2022 Jan 12; 101: 194-210). [Figure 6B] Continued from Figure 6A. [Figure 7A]Detection of anti-MOG35-55 antibodies in Mog-EAE mice administered recombinant polypeptide surrogates of the present invention ("AIM Bio"). Briefly, mice were sacrificed and mouse serum was collected by cardiac puncture. After coating with 10 μg / ml Mog35-55 overnight, wells were blocked with 1% BSA, and anti-Mog35-55 antibodies were detected with the indicated HRP-conjugated secondary antibodies. Figure 7B shows that the total amount of IgG is not reduced by the MOG47DbG surrogate molecule in these samples. Total IgG was quantified using the Easy-Titer™ Human IgG (Gamma Chain) Assay Kit (Thermo Fisher). [Figure 7B] Continued from Figure 7A. [Figure 8] List of human T1D recombinant polypeptide candidates. Correct protein folding correlates with good, or at least acceptable, expression. Demonstrated induction of Tregs in at least 30% of PBMCs as detected by ELIspot and predicted folding by AlphaFold2. Recombinant polypeptides shown in the figures are:

[0022] TIFF2025510149000001.tif204139 [Figure 9-1] Upregulation of CD8 Tregs in healthy blood donors by recombinant polypeptides of the invention. The figure shows upregulation of CD8+ T cells by recombinant polypeptides containing the zinc transporter 8 peptide antigen ILKDFSILL (A), the insulin peptide antigen ALWGPDPAAA (B), and the glutamic acid decarboxylase 65 peptide antigen EWESNGQPE (C), respectively. [Figure 9-2] Continued from Figure 9-1. [Figure 10A]The purified single-chain MHC Ib molecules are stable monomers or dimers. After purification of the single-chain MHC Ib molecules for Figures 3 and 4, their stability was analyzed after one and three freeze-thaw cycles, after storage at room temperature for 5 days, and after heating at 50°C for 30 min. For this, A) Coomassie gel staining of a 12% polyacrylamide gel with 2 μg AIM Bio and B) aHLA-G Western blot using 2A12aHLA-G antibody (1:1000) blot with 1 μg protein was performed under non-reducing conditions. Both monomers and dimers are detectable. [Figure 10B] Continued from Figure 10A. [Figure 11] Single-chain MHC Ib molecules are heat stable. For the Thermal Shift Assay (TSA), 3 μg of each single-chain MHC Ib molecule or Motavizumab as a control molecule was diluted in PBS and 5x SYPRO Orange dye (5000x stock, 5x final concentration) to a volume of 25 μl. A melting curve program was set up in the StepOnePlus Instrument using StepOnePlus Software 2.3. The starting temperature was 25°C for 1 min, then increased by 1°C per min, to a final temperature of 95°C for 2 min. Autofluorescence was measured in arbitrary units. Data was exported and graphed in Prism V7.04. A Boltzmann sigmoid function was used to determine the melting temperature (Tm). [Figure 12]Single chain MHC Ib molecules induce Tregs in a dose-dependent manner. OT-I mice were injected intraperitoneally with the indicated amounts of single chain H2_Kbα1+2 and HLA-Gα3 domain constructs (with human β2 microglobulin and the indicated peptides) or vehicle (PBS). Ova is the cognate peptide of the OT-I TCR in these mice, and Gp34 is a control peptide derived from an irrelevant virus. After 14 days, mice were sacrificed and splenocytes were tested for IL-10 secreting cells in a recall mouse IL-10 ELISpot (200,000 cells per well, MabTech mouse IL-10 ELISpot kit, 5 μg / ml of the indicated peptide or PBS alone was added, 48 hours). A clear induction of IL-10 secreting cells in response to Ova peptide was observed when 50 μg and 500 μg of mouse-matched Ova_KbG were injected. [Figure 13] Single-chain MHC Ib molecules inhibit T cell lysis in a dose-dependent manner. OT1 / BL6 mice were sacrificed, splenocytes were harvested, and washed once in RPMI 5% FCS. Red blood cells were removed with 2 ml of 1x sterile red blood cell lysis buffer for 3 min. Cells were cultured at high density (107 cells / ml) for 72 h in RPMI 10% FCS medium containing 20 ng / ml GMCSF, 20 ng / ml IL-2, and 10 ng / ml IL-4, and increasing doses of Ova_KbG. Cells were then scraped off the plate and CD8+ cells were purified with magnetic beads. Sterile 96-well white plates were used. Luciferase-expressing Panc02 target cells were loaded with 20 μg / ml Ova peptide (SIINFEKL) and shaken at 500 rpm for 60 min at 37°C. CD8+ effector T cells were added with luciferin at a ratio of 50:1. Luminescence was measured after 0, 24 and 48 hours. [Figure 14]Single-chain MHCIb molecules induce IL-10 expression in EAE-ODC Ova mice. Serum cytokines in EAE-ODC Ova mice were measured using a Th1 / Th2 10plex Flowcytomix Kit (eBioscience) according to the manufacturer's instructions. This kit was used to simultaneously detect mouse granulocyte-macrophage colony-stimulating factor (GMCSF), interleukin 1α (IL-1a), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 17 (IL-17), and tumor necrosis factor α (TNFα) in one sample. Eight specific capture antibody-coated beads were mixed. Then, 25 μL of mixed capture beads, 25 μL of unknown serum sample or standard dilution, and 25 μL of phycoerythrin (PE) detection reagent were added consecutively to each well of a 96-V bottom well plate and incubated for 2 hours at room temperature in the dark. Samples were washed with 1 mL of wash buffer for 5 minutes and centrifuged. After removing the supernatant, the bead pellet was resuspended in 200 μL buffer. Samples were run on an Attune™ NxT flow cytometer and analyzed with Attune Cytometric Software (Thermo Fisher Scientific). [Figure 15] Increase in IL-10 secreting T cells in response to treatment with single chain MHCIb molecules (recombinant polypeptide of the invention). The % increase in IL-10 secreting T cells in response to treatment with single chain MHCIb molecules is shown. Black line indicates HLA-A2 positive, grey negative donors. Significantly increased Treg responses were observed in both HLA-A2 positive and negative donors (response rates are shown in legend). [Figure 16]Thermal shift assay. In the thermal shift assay (TSA), 3 μg of each single-chain MHC Ib molecule was diluted to 25 μl with PBS and SPYRO Orange dye (5000x stock, final concentration: 5x). The melting curve program was set up in the StepOnePlus Instrument using StepOnePlus Software 2.3. The starting temperature was 25°C for 1 min, then increased by 1°C per min, and finally to 95°C for 2 min, whereby the autofluorescence was measured using arbitrary units. The data was exported and graphed using Prism V7.04. The Boltzmann sigmoid function was used to determine the melting temperature (Tm). A higher melting temperature indicates better protein stability in therapeutic applications. [Figure 17A] Thermostability assay. A,C: Western blots showing the recombinant polypeptides. B,D: Coomassie gels of the indicated recombinant polypeptides (by the same technique). The data show that the T1D single chain MHC Ib molecule (recombinant polypeptide of the invention) can be purified, stored, and can withstand freeze-thaw cycles. [Figure 17B] Continued from Figure 17A. [Figure 17C] Continued from Figure 17B. [Figure 17D] Continued from Figure 17C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Definitions and General Techniques Unless otherwise defined below, the terms used in the present invention shall be understood according to the general meaning known to those skilled in the art. All publications, patents and patent applications cited herein are incorporated by reference in their entirety for all purposes. Publications mentioned herein may be cited by specifying the complete literature reference in the text.

[0024] All proteins of the present invention, including recombinant polypeptides of the present invention, can be obtained by methods known in the art. Such methods include methods for producing recombinant polypeptides. The recombinant polypeptides of the present invention can be expressed in recombinant host cells according to the present invention. The recombinant host cells of the present invention are preferably mammalian cells, such as CHO cells and HEK cells.

[0025] It will be understood that the recombinant polypeptides of the present invention are intended to optionally include a secretory signal peptide sequence. Similarly, the recombinant polypeptides of the present invention are intended to optionally include an affinity tag, e.g., to facilitate purification, and also to optionally include a protease cleavage site between the tag and the polypeptide, e.g., to facilitate removal of the tag by protease cleavage.

[0026] It is also understood that any reference to amino acid sequences referred to herein is intended to encompass not only the unmodified amino acid sequences but also typical post-translational modifications of these amino acid sequences (e.g., glycosylation or deamidation of amino acids, clipping of specific amino acids, or other post-translational modifications) that occur in cellular expression systems known in the art, including mammalian cells such as CHO cells and HEK cells.

[0027] Likewise, it will be understood that the recombinant polypeptides of the present invention are intended to optionally include the respective propeptides.

[0028] It will also be understood that the recombinant polypeptides of the invention may be in soluble or membrane-bound form. The term "soluble" as used herein means that the recombinant polypeptide is soluble at 5 μg / ml to 5 mg / ml in the following reference conditions: PBS, optionally with 0.1% human serum or 50% glycerol. 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 standard conditions indicated above. As used herein, soluble means that at least 95% of the recombinant polypeptide is determined to be soluble under these standard conditions. 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.

[0029] According to the present invention, the MHC molecule is preferably a human MHC molecule.

[0030] The recombinant polypeptide of the present invention is preferably an isolated recombinant polypeptide.

[0031] It will be understood how recombinant polypeptides capable of binding and presenting peptide antigens of the invention may be prepared. For example, peptide antigen binding domains, such as [α]1 and [α]2 domains, are well known and modifications of these domains can be made. The ability of the polypeptides of the invention and peptide antigens to bind to MHC molecules can be determined by techniques known in the art, including but not limited to exploratory methods such as mass spectrometry after MHC peptide elution and in silico bioinformatics prediction, and confirmatory methods such as MHC peptide multimer binding and stimulation assays.

[0032] According to the present invention, the recombinant polypeptides, pharmaceutical compositions and kits of the invention are preferably suitable for use in human patients.

[0033] According to the present invention, the recombinant polypeptides, pharmaceutical compositions and kits of the present invention are preferably suitable for use in the treatment of type 1 diabetes in human patients.

[0034] According to the invention, the recombinant polypeptides, pharmaceutical compositions and kits of the invention are preferably suitable for inducing immune tolerance to human proinsulin or human insulin, human glutamic acid decarboxylase 65, human islet amyloid polypeptide or human zinc transporter 8, for example in a human patient.

[0035] According to the present invention, it is understood that the recombinant polypeptides, pharmaceutical compositions and kits of the present invention are stable.

[0036] It will be understood that any length of these peptide antigens referred to herein (e.g., "7-11 amino acids long") in relation to the peptide antigens used in accordance with the present invention is intended to refer to the length of the peptide antigen itself. Thus, the length of the peptide antigen referred to herein does not include length contributed by additional amino acids that are not part of the peptide antigen, such as additional amino acids from potential linker sequences, etc.

[0037] In accordance with the present invention, each occurrence of the term "comprising" may optionally be replaced with the term "consisting of."

[0038] Methods and Techniques Generally, unless otherwise defined herein, the methods used in the present invention (e.g., cloning methods or antibody-related methods) are carried out according to procedures known in the art, e.g., as described in Sambrook et al. ("Molecular Cloning: A Laboratory Manual.", 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1989), Ausubel et al. ("Current Protocols in Molecular Biology." Greene Publishing Associates and Wiley Interscience; New York 1992), and Harlow and Lane ("Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 1988), all of which are incorporated herein by reference.

[0039] Protein-protein binding, such as the 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.

[0040] For example, the binding of MHC class Ib molecules or recombinant polypeptides of the invention to their receptors, such as ILT2 and ILT4, is preferably assessed by surface plasmon resonance spectroscopy. More preferably, the binding of MHC class Ib molecules or recombinant polypeptides of the invention to their receptors is assessed by surface plasmon resonance at 25° C. Suitable conditions for such surface plasmon resonance measurements are described in Shiroishi et al., Proc Natl Acad Sci US A. 2003 July 22; 100(15): 8856-8861.

[0041] Sequence alignment of the sequences of the present invention is performed using the BLAST algorithm (see Altschul et al. (1990) "Basic local alignment search tool." Journal of Molecular Biology 215. p. 403-410.: Altschul et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402). Suitable parameters for sequence alignment of short peptides by the BLAST algorithm, suitable for the peptide antigens of the present invention, are known in the art. Most software tools using the BLAST algorithm automatically adjust the parameters for sequence alignment of short input sequences. In one embodiment, the following parameters are used: Max target sequences 10; Word size 3; BLOSUM 62 matrix; gap costs; existence 11, extension 1; conditional compositional score matrix adjustment. Thus, when used in the context of sequences, terms such as "identity" or "identical" preferably refer to the identity value obtained using the BLAST algorithm.

[0042] Preparation of the Pharmaceutical Composition of the Invention The pharmaceutical compositions of the present invention are prepared in accordance with known standards for the preparation of pharmaceutical compositions.

[0043] For example, pharmaceutical compositions are prepared so that they can be appropriately stored and administered. Thus, the pharmaceutical compositions of the present invention may contain pharma- ceutical acceptable components, such as carriers, excipients, and / or stabilizers.

[0044] Such pharma- ceutically acceptable ingredients are non-toxic in the amounts used when the pharmaceutical composition is administered to a human patient. The pharma- ceutically acceptable ingredients added to a pharmaceutical composition may depend on the chemical nature of the active ingredients present in the composition, the particular intended use of the pharmaceutical composition, and the route of administration.

[0045] Generally, pharma- ceutically acceptable ingredients used in connection with the present invention are used according to the knowledge available in the art, e.g., information from Remington's Pharmaceutical Sciences, Ed. AR Gennaro, 20th edition, 2000, Williams & Wilkins, PA, USA. Pharmaceutical compositions containing the nucleic acids (e.g., RNA) of the present invention can also be formulated according to the knowledge available in the art, e.g., using liposomal formulations that target dendritic cells.

[0046] Peptide antigens of the present invention Peptide antigens that can be used according to the present invention, including those defined above, are not particularly limited except by their ability to be presented on MHC molecules. The "peptide antigen presented by said recombinant polypeptide" referred to in the context of the present invention is understood to be a peptide antigen that is presented by said recombinant polypeptide to human T cells, if human T cells are present.

[0047] Peptides that can be presented on MHC molecules can be generated as known in the art (see, e.g., 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).

[0048] Peptide antigens are widely known in the art. Generally, the peptide antigens of 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 that can present the peptide of the present invention, preferably, peptide antigens that can bind to said MHC class Ib molecule or recombinant polypeptide are used. These peptide antigens can be selected based on methods known in the art.

[0049] Binding of a peptide antigen to an MHC class Ib molecule, or to a polypeptide capable of binding a peptide antigen according to the invention, can be assessed by methods known in the art, for example by the following method: - 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; - Rock,Reits,Neefjes.Present Yourself! By MHC Class I and MHC Class II Molecules.Trends Immunol.2016 Nov;37(11):724-737.

[0050] Such methods include experimental methods and methods that predict peptide-antigen binding.

[0051] Anchor residues that serve to anchor peptide antigens onto MHC class I molecules and ensure binding of peptide antigens to MHC class I molecules are known in the art.

[0052] In a preferred embodiment consistent with all embodiments of the invention, the peptide antigens used in accordance with the invention contain either anchor residues or preferred amino acid residues at positions predicted for MHC class I molecules.

[0053] Such prediction can preferably be performed as described in any 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 596,583-589(2021).

[0054] In the present invention, the peptide antigen is derived from human proinsulin or human insulin, human glutamic acid decarboxylase 65, human islet amyloid polypeptide, or human zinc transporter 8.

[0055] It is understood that the non-anchor amino acid residues of the peptide antigens of the present invention may or may not contain conservative substitutions, preferably no more than two conservative substitutions, more preferably one conservative substitution, with respect to the corresponding amino acid sequence of a peptide antigen derived from human proinsulin or human insulin, human glutamic acid decarboxylase 65, human islet amyloid polypeptide, or human zinc transporter 8.

[0056] The peptide antigens of the present invention are preferably composed of naturally occurring amino acids. However, non-naturally occurring amino acids can also be used, such as modified amino acids. For example, in one embodiment, the peptide antigens of the present invention include peptidomimetics of the indicated peptide antigen amino acid sequences of human proinsulin or human insulin, human glutamic acid decarboxylase 65, human islet amyloid polypeptide, or human zinc transporter 8.

[0057] Methods for the synthesis of peptidic antigens, including those of the present invention, are well known in the art.

[0058] array Preferred amino acid sequences referred to in this application can be independently selected from the following sequences: The sequences are represented in N-terminal to C-terminal order; in addition, the sequences are represented in the one-letter amino acid code.

[0059] Examples of sequences which are part of the recombinant polypeptides of the invention: Optional leader peptide (which is deleted from the recombinant polypeptide due to processing during cellular expression): e.g., MSRSVALAVLALLSLSGLEA (SEQ ID NO: 1) Peptide antigen: any MHC class I peptide corresponding to the MHC class I [α] 1 and 2 domains, e.g., ALWGPDPAAA (SEQ ID NO: 2) First linker: for example, GGGGSGGGSGGGGS (SEQ ID NO: 3) or GCGASGGGGSGGGGS (SEQ ID NO: 4)

[0060] β2 microglobulin, for example: TIFF2025510149000002.tif12156 (SEQ ID NO:5, human β2 microglobulin) A second linker, for example: GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 6)

[0061] [α]1 and 2 domains derived from either human HLA-G or any other MHC class I [α]1 and 2 domains suitable for presenting a selected antigenic peptide, Y84 may be C in the DT variant For example, the [α]1 and 2 domains from human HLA-G: TIFF2025510149000003.tif18156 (SEQ ID NO: 7) Or: human HLA-A2[α] 1 and 2 domains: e.g. TIFF2025510149000004.tif18156 (SEQ ID NO:8)

[0062] Human HLA-G[α]3 domain (or any MHC Ib[α]3 domain, such as HLA-F, which also interacts with the ILT2 and ILT4 receptors), for example: TIFF2025510149000005.tif13156 (sequence number 9; sequence of HLA-G[α]3).

[0063] Of note, the following underlined amino acids in this sequence are relevant for ILT2 or ILT4 receptor interaction: TIFF2025510149000006.tif11157

[0064] Alternatively, a truncated human HLA-G[α]3 domain may be used, lacking the optional C-terminal amino acid sequence from intron 4 (SKEGDGGIMSVRESRSLSEDL; SEQ ID NO: 20), i.e.: TIFF2025510149000007.tif11156 (SEQ ID NO: 21),

[0065] 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: for example NSAVD (SEQ ID NO: 14) or GS.

[0066] Examples of the most preferred peptide antigens which may be part of the recombinant polypeptide of the present invention are:

[0067] [Table 1]

[0068] Further preferred examples of peptide antigens which may be part of the recombinant polypeptide of the invention are:

[0069] [Table 2] TIFF2025510149000010.tif87161

[0070] Examples of recombinant polypeptides of the present invention (including optional leader peptides): TIFF2025510149000011.tif44156 (SEQ ID NO: 15; note that the asterisk represents the position of the stop codon).

[0071] It should be noted that the sequence of the peptide antigen of the full-length recombinant polypeptide described above can be replaced with any peptide antigen sequence of the present invention, i.e., any peptide antigen presented by the recombinant polypeptide, which is a peptide of human proinsulin or human insulin, human glutamic acid decarboxylase 65, human islet amyloid polypeptide, or human zinc transporter 8. That is, the recombinant polypeptide of the present invention can be composed of a sequence consisting of a peptide antigen that is a peptide of human proinsulin or human insulin, human glutamic acid decarboxylase 65, human islet amyloid polypeptide, or human zinc transporter 8 (e.g., any one of the peptide antigens of SEQ ID NO:2, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27), followed by the following sequence: TIFF2025510149000012.tif19157TIFF2025510149000013.tif25157 (SEQ ID NO: 16; note that the asterisk represents the position of the stop codon). These recombinant polypeptides of the invention may also optionally include a leader peptide, as exemplified above.

[0072] The receptors ILT2 (also known as LILRB1) and ILT4 (also known as LILRB2) are known in the art. Preferred sequences of these receptors of the invention are as follows:

[0073] ILT2: TIFF2025510149000014.tif70135 (SEQ ID NO: 17)

[0074] ILT4: TIFF2025510149000015.tif64135 (SEQ ID NO: 18)

[0075] The sequences of human proinsulin and human insulin, human glutamic acid decarboxylase 65, human islet amyloid polypeptide, and human zinc transporter 8 are known in the art. Preferred sequences for these proteins are as follows:

[0076] Human Proinsulin and Human Insulin Full length human insulin (consisting of a 24 amino acid signal peptide, a 30 amino acid B chain, a 31 amino acid C peptide, and a 21 amino acid A chain): reference sequence >sp|P01308|INS_HUMAN Insulin OS=Homo sapiens OX=9606 GN=INS PE=1 SV=1 TIFF2025510149000016.tif11156 (SEQ ID NO: 19)

[0077] Proinsulin (after signal peptide cleavage) TIFF2025510149000017.tif11156 (SEQ ID NO: 28)

[0078] Mature insulin (B and A chains) FVNQHLCGSHLVEALYLVCGERGFFYTPKT (B chain) (SEQ ID NO: 29) LQKRGIVEQCCTSICSLYQLENYCN (A chain) (SEQ ID NO: 30)

[0079] Human glutamic acid decarboxylase 65 (GAD65): >NP_001127838.1 glutamate decarboxylase 2[Homo sapiens] TIFF2025510149000018.tif57158 (SEQ ID NO: 31; Glutamic acid decarboxylase 2 / GAD2 is synonymous with GAD65.)

[0080] Human Islet Amyloid Polypeptide: >NP_000406.1 islet amyloid polypeptide preproprotein[Homo sapiens] TIFF2025510149000019.tif12145 (SEQ ID NO:32)

[0081] Human zinc transporter 8: >NP_776250.2 zinc transporter 8 isoform a[Homo sapiens] TIFF2025510149000020.tif37155(SEQ ID NO:33)

[0082] The present invention is further illustrated by the following non-limiting examples: (Example) EXAMPLES

[0083] Methods for Producing Recombinant Polypeptides of the Invention Expi-293F cells (Thermo Fisher), grown in Expi-293™ Expression Medium (Thermo Fisher): for DNA complex formation with Expifectamine, Opti-MEM (Thermo Fisher) was used, and 1 μg DNA was transfected at 2.5 × 10 6 Transfect cells / ml, add enhancer according to the protocol after 18-20 hours, and collect supernatant after 4-6 days (37°C, 8% CO 2 , humidified incubator), 19mm 2 Orbital shaker 125 rpm.

[0084] Purification of Spot-Tag proteins: Equilibration of Spot-Cap resin: Transfer desired amount of slurry to appropriate tube, pellet beads by centrifugation (4°C, 4 min, 2500g), remove and discard supernatant, add 10 bed volumes of PBS (chilled) to beads, mix by inversion, pellet beads by centrifugation (4°C, 4 min, 2500g), remove and discard supernatant, repeat twice.

[0085] Add the required volume of beads to the supernatant, incubate overnight at 4° C. on a rotator, wash the beads by repeated centrifugation (4° C., 4 min, 2500 g), and remove the supernatant.

[0086] Prepare 500 μM Spot peptide solution in PBS, remove supernatant, incubate with ⅓ volume of Spot peptide solution for 5-10 min.

[0087] The beads are pelleted by centrifugation. Proteins are concentrated and Spot peptides are removed using an Amicon Ultra-4 centrifugal filter (15 kDa cutoff) over a 15 kDa Amicon cutoff column.

[0088] Amicon Ultra-4 centrifugal filters (15 kDa cutoff) are rinsed with PBS followed by 0.1 N NaOH (centrifugation at 4000 g, 4° C.) to remove traces of glycerol.

[0089] ELISPOT: 1) Cell culture A) PBMC isolation (under laminar flow hood) To isolate peripheral blood mononuclear cells (PBMCs), density gradient centrifugation was performed using leukocytes from the leukapheresis chamber and density gradient medium (e.g. Ficoll, or ROTI Sep 1077). Cells were centrifuged at 1200xg for 20 min with the brake off, after which the interphase ring was collected and washed with 1x PBS (5 min, 300xg). PBMCs were stored frozen until further use.

[0090] B) PBMC pulsing (under a laminar flow hood) PBMCs were thawed one day (d-1) prior to PBMC pulsing and placed in wells of a 6-well plate in 5 ml of X-VIVO 15 medium containing 5% human AB serum at 37° C. overnight.

[0091] The next day (d0), cells were counted and cultured at 3 × 10 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). 6 The cells were resuspended at a cell density of 1000 cells / ml.

[0092] For the experiments, 3 × 10 cells were plated in each well of a 12-well plate in a final volume of 1000 μl of X-VIVO complete medium (containing cytokine cocktail and 5 μg / ml of AIM Biomolecules or respective controls). 6 cells were seeded.

[0093] On day 3, 1 ml of complete medium (containing cytokines) was added, and on day 6, a second pulse treatment was performed (after removing the medium) with 5 μg / ml of a recombinant polypeptide of the invention or its surrogate (collectively referred to as "AIM Bio" molecules). On days 7, 10, and 12, 1 ml of complete medium (containing cytokines) was added.

[0094] What you'll need: X-VIVO 15 medium + 5% human AB serum X-VIVO 15 complete medium: X-VIVO 15 medium + 2% human AB serum (containing cytokine cocktail): 10ng / ml TGF-b1, 10ng / ml IL-4, 20ng / ml IL-2, 20ng / ml GM-CSF 6-well plates 12 well plate.

[0095] 2) ELISPOT Laminar Flow Control Hood On day 13, ELISPOT plates were coated with anti-hIL10 (clone 9D-7, 1:500 dilution in PBS, sterile filtered) and aIL10 (10G8-biotin), and on day 14, 200,000 cells were plated per well in duplicate on ELISPOT plates, including a negative control (cells + PBS) and a positive control (e.g., LPS).

[0096] PFDF membranes were activated with 50 μl / well EtOH (35% v / v) for 1 min and then washed 5 times with 200 μl sterile distilled water. Plates were coated overnight at 4 °C with 100 μl / well antibody solution. The next day, unbound coating antibody was removed and after 5 washing steps with 200 μl PBS, 200 μl blocking buffer (X-VIVO 15 5% hAB serum) was added and plates were incubated at room temperature for 30 min to 2 h.

[0097] The respective antigen peptide (e.g. MOG157) in DMSO or DMSO as a control was prepared and a final amount of 5 μg peptide / ml was added to a final volume of 100 μl / well. 150,000 cells were seeded per well in X-VIVO 15 medium containing 5% human AB serum. The blocking buffer (X-VIVO 15 medium + 5% human AB serum) was carefully removed and medium containing PBS as a negative control and stimuli (5 μg / ml total per well) were added to other wells and incubated at 37°C overnight.

[0098] Outside the laminar flow hood Secondary antibodies were prepared: 1 μg / ml aIL-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 μm filter and stored at 4° C. until use.

[0099] The cell supernatant was removed and the cells were washed five times with 100 μl PBS, and the final excess buffer was removed with a paper towel.

[0100] 25 μl of diluted HRP-streptavidin (1:750) was added per well and incubated for 1 hour at room temperature in the dark, followed by 5 washing steps with sterile 1×PBS.

[0101] 100 μl of filtered TMB substrate was added per well and allowed to sit for 15-25 min until blue spots developed. The reaction was stopped by thoroughly washing the wells with tap water.

[0102] The plastic underdrain of the plate was removed and the bottom and sides of the plate were washed with tap water and dried.

[0103] Plates were read using an ImmunoSpot S6 Ultra-V Analyzer (Cellular Technology Limited), analyzed in Excel, and graphs / statistics were performed in Graphad Prism.

[0104] What you'll need: Capture antibodies: anti-hIL10 (clone: ​​9D-7, Mabtech #3430-3-250; 1:500 dilution), anti-hIL10 biotinylated (Mabtech #3430-6-250), 1xPBS (sterile) 35% ethanol (v / v) Blocking buffer: X-vivo 5% hAB serum (sterile) [Blocking is performed in the same medium as cell culture] Dilution buffer: 0.5% BAS in PBS Washing buffer: 1xPBS Culture medium for T cells: X-VIVO15 medium (Lonza) Filter syringe: Miller GV ELISPOT PVDF plate (#MSIP4510, Millipore) TMB substrate EXAMPLES

[0105] Surrogates of the recombinant polypeptides of the present invention induce IL10-secreting Tregs in mice To induce immune tolerance to the OVA peptide or the viral Gp34 peptide, wild-type black 6 mice were injected with 100 μg of a recombinant polypeptide (also called "AIMBio") having the following sequence: Ova_KbG TIFF2025510149000021.tif44157 (SEQ ID NO: 34), and Gp34_KbG TIFF2025510149000022.tif43158 (sequence number 35). The Gp34 peptide is a well-characterized T cell epitope derived from the Lymphocytic Choriomeningitis Virus (LCMV) glycoprotein. This antigen was previously named Gp33, but it was later found that the epitope presented on H2-Kb contains only amino acids 34-41. (In contrast, the epitope starting at amino acid 33 is presented on H2-Kd.) Therefore, we refer to the H2-Kb epitope as Gp34, which is in line with the latest recommendations. Nevertheless, the use of the nomenclature Gp33 and Gp34 in the literature is ambiguous. The first 8 amino acids of SEQ ID NO: 35 show the correct sequence (AVYNFATM; SEQ ID NO: 58). After 2 weeks, the mice were sacrificed and splenocytes were rechallenged with matched or mismatched peptides. IL-10-secreting cells were quantified by ELIspot. The results are shown in Figure 3. EXAMPLES

[0106] Surrogates of the recombinant polypeptides of the invention selectively prevent CD8+ T cell-induced EAE in mice As described in (Na et al, Brain. 2008 Sep;131(Pt 9):2353-65.), adoptive transfer of CD8+ OT-I T cells that recognize the ovalbumin epitope in H2-Kb into mice expressing ovalbumin in oligodendrocytes induces experimental autoimmune encephalomyelitis that recapitulates many MS and end-stage NMO symptoms. In this animal model, a single injection of 500 μg of a recombinant polypeptide surrogate molecule (also called "AIMBio") that induces tolerance to the targeted ovalbumin epitope almost completely prevented EAE symptoms, whereas a surrogate molecule presenting a control peptide had no significant preventive effect (Figure 4). The sequence of the recombinant polypeptide surrogate molecule is shown below.

[0107] Mog44_DbG TIFF2025510149000023.tif45158 (SEQ ID NO: 36)

[0108] Mog37_DbG TIFF2025510149000024.tif43158 (SEQ ID NO: 37) EXAMPLES

[0109] Some surrogates of the recombinant polypeptides of the invention selectively prevent CD4+ T cell-induced EAE in mice On the day of intraperitoneal injection of 33 μg or 100 μg of a surrogate molecule of a recombinant polypeptide of the invention ("AIM Bio"), 100 μl of MOG35-55 peptide / CFA (complete Freund's adjuvant; final concentration of M. tuberculosis H37RA and peptide 1 mg / ml each) emulsion was injected subcutaneously in the left and right flanks, respectively, and 250 ng pertussis toxin (in 200 μl PBS) was injected intraperitoneally. A second pertussis toxin injection was given 3 days later. In this animal model, a single injection of an AIM Bio surrogate molecule inducing tolerance to a Mog epitope (Mog44_Kb_G) significantly reduced the symptoms of EAE, whereas a surrogate molecule presenting a control peptide (Gp34) or a non-functional Mog peptide (Mog37) showed no significant protective effect (FIG. 5). In this model, Mog44 AIM Bio also prevented inflammation in the spinal cord and infiltration of CD8 T cells (Figure 6). The sequence of the recombinant polypeptide surrogate molecule is shown in Example 3.

[0110] In this model, Mog44 AIM Bio also completely prevented the formation of MOG-specific autoantibodies in serum, as verified by ELISA (Figure 7). This strongly indicates that the recombinant polypeptides of the present invention are effective therapeutic agents for NMO, which is often characterized by an antibody response against human aquaporin 4, and other autoimmune diseases, such as type 1 diabetes, whose pathology is characterized by the presence of autoantibodies. Thus, patient populations are defined by common autoimmune-associated antigens. Some MHC molecules are also associated with NMO.

[0111] Mog-reactive antibodies in serum from mice treated with AIM Bio (33 or 100 μg) were detected by 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 loaded with mouse serum diluted 1:25 in PBS 1% BSA for 1 hour. Detection was performed with anti-mouse IgG-HRP or anti-mouse heavy and light chain HRP antibodies diluted 1:5000. EXAMPLES

[0112] Human recombinant polypeptide candidates of the present invention for T1D 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 believed to enable the embryo to influence the maternal immune system to tolerate embryonic antigens but to better antagonize pathogen-derived antigens. The recombinant polypeptide of the present invention containing variable peptides could selectively eliminate peptide-specific cytotoxic effector T cells and induce peptide-specific Treg in vitro. T1D autoantigens according to the invention include (pro)insulin (INS), glutamic acid decarboxylase 65 (GAD65), islet amyloid polypeptide (IAPP) or zinc transporter 8 (ZNT8).

[0113] FIG. 8 shows a list of candidate human T1D recombinant polypeptides.

[0114] According to our findings, single-chain proteins containing INS, GAD65, IAPP or ZNT8 peptide antigens and HLA-Gα3 domains can induce tolerogenic T cells in healthy donors, thus upregulating CD8 Tregs by at least 30% in 75% of all healthy blood donors (Figure 9). Correlating with the in vivo experiments presented here, it is believed that these constructs suppress CD8 T cell- and antibody-mediated responses to islet cell antigens in patients. EXAMPLES

[0115] Further proof of principle regarding the stability and efficacy of recombinant polypeptides of the invention Furthermore, the inventors set out to obtain and test recombinant polypeptides having the general structure of the recombinant polypeptides of the present invention but containing a variety of different peptide antigens to further provide proof of principle that the recombinant polypeptides of the present invention and their surrogates are stable and effective. As shown in Figures 10 and 11, respectively, the tested recombinant polypeptides are stable during freeze-thaw and storage, and are heat stable. Furthermore, these recombinant polypeptides induce Tregs in a dose-dependent manner (Figure 12) and inhibit T cell lysis in a dose-dependent manner (Figure 13). The effect of the recombinant polypeptides on serum cytokine profile in EAE-ODC Ova mice is shown in Figure 14. There is an induction of IL-10, and possibly IL-4, both of which are known immunosuppressive cytokines that downregulate immune responses in an inflammatory environment. This requires the HLA-Gα3 domain and the cognate peptide. IL-2 appears to be induced in response to presentation of the cognate peptide to cells, independent of the α3 domain. IL-2 is required for T cell activation and survival.

[0116] In addition, the high melting temperature shown in Figure 16 confirms good protein stability of the recombinant polypeptide of the invention for therapeutic applications. Furthermore, the data in Figure 17 show that the T1D single chain MHC1b molecule (recombinant polypeptide of the invention) can be purified and stored, and is resistant to freeze-thaw cycles. EXAMPLES

[0117] As shown in FIG. 15, upregulation of CD8 Tregs occurs in PBMCs in healthy blood donors by the recombinant polypeptides of the invention. In vitro Treg induction mediated by peptide-HLA-G containing constructs (AIM Biologicals) was performed as follows: PBMCs from healthy donors were isolated from leukocytes obtained from a leukapheresis chamber by density gradient centrifugation on Ficoll. Cells were centrifuged for 20 min at 1200 g without brake, followed by collection of the interphase ring and washing with 1x PBS (5 min, 300 g). PBMCs were frozen for further use.

[0118] PBMCs were thawed 1 day before PBMC pulsing (d-1) and maintained overnight at 37°C in 5 ml of X-VIVO 15 medium containing 5% human AB serum in wells of a 6-well plate. The next day (d0), cells were counted and plated at 3x10 in X-Vivo15 complete medium (5% hAB serum and cytokine cocktail: 20ng / ml hIL-2, 20ng / ml hGM-CSF, 10ng / ml hIL-4 and 10ng / ml hTGF-b1). 6 The cells were resuspended at a cell concentration of 100 x 10 cells / ml. In the experiment, 3 x 10 cells were placed in each well of a 12-well plate. 6 Cells were plated in a final volume of 1000 μl of X-VIVO complete medium and cytokine cocktail, as well as 5 μg / ml of each control AIM Bio molecule.

[0119] On day 3, 1 ml of complete medium (with cytokines) was added, and a second pulse (after removing the medium) with 5 μg / ml AIM Biomolecule was performed on day 6. On days 7, 10, and 12, 1 ml of complete medium (with cytokines) was added.

[0120] On day 13, the PVDF membrane of the ELISpot plate was activated by adding 50 μl / well EtOH (35% v / v) for 1 min, followed by washing 5 times with 200 μl sterile distilled water. The plate was coated overnight at 4°C with 100 μl / well anti-hIL10 (clone 9D-7, diluted 1:500 in PBS, sterile filtered). The next day, unbound coating antibody was removed, 5 washing steps with 200 μl PBS were performed, and 200 μl blocking buffer (X-VIVO 15 5% hAB serum) was added and incubated for 30 min to 2 h at room temperature. On day 14, 200,000 cells per well were plated in duplicate on the ELISpot plate for 48 h, including a negative control (cells plus PBS) and a positive control (e.g. LPS). Secondary antibodies were prepared: 1 μg / ml aIL-10 biotinylated antibody (1:1000 dilution) in 0.5% BSA / 1xPBS, and horseradish peroxidase-conjugated streptavidin (1:750 in 0.5% BSA / PBS). Tetramethylbenzidine was filtered through a 0.45 μm filter and stored at 4°C until use. Cell supernatant was removed and washed 5 times with 100 μl PBS. The final excess buffer was removed using a paper towel. 25 μl of diluted HRP-streptavidin (1:750) was added to each well and incubated for 1 hour at room temperature in the dark, followed by washing 5 times with sterile 1xPBS. 100 μl of filtered TMB substrate was added to each well for 15-25 minutes until a blue spot was formed. 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 allowed to dry. Some recombinant polypeptides of the invention induced at least 30% more IL-10 secreting Tregs in PBMCs of about 75% of all healthy blood donors.

[0121] Industrial Applicability The pharmaceutical compositions, polypeptides, nucleic acids, cells and products used in the present invention can be industrially applicable, for example, they can be used in the manufacture of medicines or as medicines.

Claims

1. A recombinant polypeptide capable of presenting a peptide antigen, the recombinant polypeptide comprising, in order from N-terminus to C-terminus: i) a peptide antigen presented by the recombinant polypeptide, the peptide antigen being a peptide of human proinsulin or human insulin, human glutamic acid decarboxylase 65, human islet amyloid polypeptide, or human zinc transporter 8; ii) optionally, a linker sequence; iii) optionally a sequence of a human polypeptide domain comprising 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 set forth in SEQ ID NO: 5; iv) optionally, a linker sequence; v) optionally, the [α]1 domain of an MHC molecule; vi) optionally, the [α]2 domain of an MHC molecule; vii) an [α]3 domain of an MHC class Ib molecule, or a derivative of an [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 comprising:

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

3. 2. The recombinant polypeptide of claim 1, 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: 2, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO:

27.

4. 2. The recombinant polypeptide of claim 1, 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.

5. 2. The recombinant polypeptide of claim 1, 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.

6. The [α]3 domain or derivative according to (vii) is identical to or has at least 80% amino acid sequence identity, preferably at least 90% amino acid sequence identity with the [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 21, or the [α]3 domain or derivative according to (vii) is identical to or has at least 92% amino acid sequence identity with the [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 21, or the [α]3 domain or derivative according to (vii) is identical to or has at least 94% amino acid sequence identity with the [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 21, or the [α]3 domain or derivative according to (vii) is identical to or has at least 94% amino acid sequence identity with the [α]3 domain having the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 21, or 9 or SEQ ID NO:21, or the [α]3 domain or derivative described in (vii) is identical to or has at least 96% amino acid sequence identity with the [α]3 domain having the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:21, or the [α]3 domain or derivative described in (vii) is identical to or has at least 98% amino acid sequence identity with the [α]3 domain having the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:21, or the [α]3 domain or derivative described in (vii) is identical to or has at least 99% amino acid sequence identity with the [α]3 domain having the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:21, or the [α]3 domain or derivative described in (vii) is identical to or has at least 99% amino acid sequence identity with the [α]3 domain having the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:

21.

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

8. 2. The recombinant polypeptide of claim 1, 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.

9. The recombinant polypeptide of claim 1, wherein the polypeptide is a dimer or a multimer.

10. 2. The recombinant polypeptide of claim 1, wherein the polypeptide comprises or consists of all of (i) to (vii) and / or the polypeptide does not comprise (viii) to (x).

11. 2. The recombinant polypeptide of claim 1, wherein the polypeptide comprises or consists of all of (i) through (x).

12. The recombinant polypeptide of claim 1, further comprising an N-terminal secretory signal peptide sequence.

13. The recombinant polypeptide comprises, in order from N-terminus to C-terminus, the following ((a) and (b)): (a) a peptide antigen selected from the group consisting of the amino acid sequences of SEQ ID NO:2, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:2; and (b) the amino acid sequence of SEQ ID NO: 16 2. The recombinant polypeptide of claim 1, comprising an amino acid sequence consisting of:

14. The recombinant polypeptide of claim 1 , wherein the recombinant polypeptide is soluble.

15. 2. A nucleic acid encoding one or more polypeptides according to claim 1, wherein the nucleic acid is preferably a vector.

16. A pharmaceutical composition comprising at least one nucleic acid according to claim 15.

17. A pharmaceutical composition or kit comprising at least one recombinant polypeptide according to claim 1.

18. 18. The pharmaceutical composition or kit of claim 17, comprising at least two different recombinant polypeptides according to claim 1, each of the different polypeptides comprising a different peptide antigen as defined in claim 3, and / or the pharmaceutical composition or kit preferably comprises at least the following (A) to (C): (A) a recombinant polypeptide in which the peptide antigen is a peptide antigen of human proinsulin or human insulin; (B) a recombinant polypeptide in which the peptide antigen is a peptide antigen of human glutamic acid decarboxylase 65; (C) a recombinant polypeptide in which the peptide antigen is a peptide antigen of human zinc transporter 8, and optionally (D) a recombinant polypeptide in which the peptide antigen is a peptide antigen of human islet amyloid polypeptide.

19. 18. The pharmaceutical composition or kit of claim 17, comprising at least three different recombinant polypeptides according to claim 1, wherein the peptide antigen of a first recombinant polypeptide of the at least three different recombinant polypeptides consists of the amino acid sequence of SEQ ID NO: 2, the peptide antigen of a second recombinant polypeptide of the at least three different recombinant polypeptides consists of the amino acid sequence of SEQ ID NO: 22, and the peptide antigen of a third recombinant polypeptide of the at least three different recombinant polypeptides consists of the amino acid sequence of SEQ ID NO:

23.

20. 18. The pharmaceutical composition or kit according to claim 16 or claim 17 for use in the treatment of type 1 diabetes in a human patient, wherein the treatment is preferably an immunotherapeutic treatment, and / or the treatment is preferably by inducing immune tolerance to human proinsulin and / or human insulin, human glutamic acid decarboxylase 65, human islet amyloid polypeptide, and / or human zinc transporter 8, and / or the treatment is for reducing plasma levels of autoantibodies against insulin (insulin autoantibody IAA), glutamic acid decarboxylase (GAD-65), islet antigen-2A (IA-2A), or zinc transporter ZnT8, preferably as assessed by a radioactive binding assay or a non-radioactive electrochemiluminescence antigen binding assay, and / or the human patient is preferably a patient who has plasma autoantibodies against insulin (insulin autoantibody IAA), glutamic acid decarboxylase (GAD-65), islet antigen-2A (IA-2A), or zinc transporter ZnT8 before initiation of treatment.

21. A recombinant host cell comprising the nucleic acid of claim 15 and expressing the recombinant polypeptide of claim 1.

22. A method for obtaining a pharmaceutical composition comprising a recombinant polypeptide of claim 1, comprising the steps of: (a) culturing a recombinant host cell comprising the nucleic acid of claim 15 and expressing the recombinant polypeptide of claim 1 under conditions that allow expression of the recombinant polypeptide from the nucleic acid molecule; (b) recovering the recombinant polypeptide; (c) purifying the recombinant polypeptide; and (d) formulating the recombinant polypeptide into a pharmaceutical composition.

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