Tolerance-inducing constructs and compositions and their use for treating immune disorders

Abstract

The present disclosure relates to constructs and compositions for use in the treatment of conditions involving unwanted immune responses, such as prophylactic or therapeutic treatment of autoimmune diseases, allergic diseases and transplant rejection.

Description

[Technical field]

[0001] The present disclosure relates to constructs and compositions for use in the treatment of conditions involving unwanted immune responses, such as prophylactic or therapeutic treatment of autoimmune diseases, allergic diseases and transplant rejection. [Background technology]

[0002] Immune responses are necessary for defense against diseases, e.g., diseases caused by pathogens such as viruses, bacteria, or parasites. However, unwanted immune activation can cause processes that lead to damage or destruction of one's own tissue. Unwanted immune activation occurs, for example, in autoimmune diseases, where antibodies and / or T lymphocytes react with self-antigens, resulting, for example, in tissue damage and pathology. Unwanted immune activation also occurs in allergic reactions, which are typically characterized by an excessive immune response to harmless substances in the environment and can result in an inflammatory response that leads to tissue destruction. In addition, unwanted immune activation occurs in graft rejection, e.g., rejection of transplanted organs or tissues, which is significantly mediated by alloreactive T cells present in the host, which recognize donor alloantigens or xenoantigens, leading to the destruction of the transplanted organ or tissue. Immune tolerance is the acquired lack of a specific immune response to a substance or tissue that has the ability to induce an immune response in a given organism.

[0003] Typically, to induce tolerance to a particular antigen, the antigen must be presented to other immune cells by antigen-presenting cells (APCs) in the absence of an activation signal, which results in the death or functional inactivation of antigen-specific lymphocytes, or the generation of antigen-specific cells that maintain tolerance. This process generally describes tolerance to self-antigens, or self-tolerance. Immunosuppressants are useful, for example, in the treatment of patients with autoimmune diseases or patients with allogeneic transplants, to prevent or reduce undesirable immune responses.

[0004] Conventional strategies for generating immunosuppression of undesired immune responses are based on broad-acting immunosuppressants. Moreover, to maintain immunosuppression, immunosuppressant therapy is often a lifelong proposition. Unfortunately, the use of broad-acting immunosuppressants is associated with the risk of serious side effects, such as immunodeficiency, since most of them act non-selectively, leading to increased susceptibility to infection and reduced cancer immunosurveillance. Therefore, novel compounds and compositions that induce antigen-specific tolerance would be beneficial.

[0005] APCs such as dendritic cells play a key role in regulating immune responses, providing antigen-specific T cell signals to either combat the presented antigen (putative pathogen) or suppress responses to the presented antigen (putative non-pathogenic antigen) and induce peripheral tolerance, depending on the activation state of the dendritic cell (cytokines and growth factors) and the microenvironment. The challenge in developing tolerogenic immunotherapy is to efficiently deliver antigens to APCs such as dendritic cells in a manner that does not induce an immune response, such as inflammation or a proinflammatory immune response.

[0006] The scientific article "Schjetne KW et al., Eur. J. Immunol. 35(11), 3142-3152, 2005" discloses recombinant antibody constructs called "Troibodies". These Troibodies are recombinant antibodies with V regions specific for APC surface molecules, and T cell epitopes are grafted onto the loops between the β strands in their C domains. Summary of the Invention

[0007] The present disclosure relates to tolerogenic constructs comprising an antigenic unit and a targeting unit that interact with surface molecules on APCs, such as dendritic cells, in a non-inflammatory or tolerogenic manner resulting in antigen presentation in the absence of activation, such as inflammatory activation.

[0008] The inventors have surprisingly found that the Vaccibody platform can deliver disease-associated antigens to antigen-presenting cells (APCs) via binding to and signaling to selected surface receptors on the APCs, which internalize the construct in an optimal manner for the induction of a selected antigen-specific tolerance response and present the antigen in a tolerance-inducing manner, such as the induction of regulatory T cells (Tregs) and suppression of memory and effector T cell responses.

[0009] The tolerance-inducing constructs of the present disclosure may have improved mobilities compared to known constructs such as the "Troibodies" disclosed in Schjetne KW et al., Eur. J. Immunol. 35(11), 3142-3152, 2005. For example, the targeting units of the disclosed constructs are not limited to V regions derived from antibodies, but can be a wide variety of different units.

[0010] A further advantage of the tolerance-inducing constructs of the present disclosure compared to known constructs is that a smaller dose, such as one dose, may be sufficient to achieve the same functional effect, for example, to reduce the level of an immune response, delay the onset or progression of an immune response, and / or reduce the risk of onset or progression of an immune response.

[0011] Vaccibody constructs are multimeric proteins consisting of multiple polypeptides, e.g., dimeric proteins consisting of two polypeptides, each of which comprises a targeting unit, a dimerization unit and an antigenic unit that targets antigen-presenting cells - see, e.g., WO2004 / 076489A1, WO2011 / 161244A1, WO2013 / 092875A1 or WO2017 / 118695A1. These constructs have been shown to be efficient in generating an immune response against an antigen or epitope contained in the antigenic unit.

[0012] The vaccibody or tolerance-inducing construct of the present disclosure may be administered to a subject in the form of a polynucleotide (e.g., a DNA plasmid) that includes a nucleotide sequence encoding a polypeptide. After administration to a host cell, such as a human muscle cell, the polypeptide is expressed and forms a multimeric protein with a multimerization unit; if a dimerization unit is used, the polypeptide forms a dimeric protein when expressed.

[0013] The present disclosure provides tolerance-inducing constructs based on vaccibody structures for use in the prophylactic or therapeutic treatment of immune disorders such as autoimmune diseases, allergic diseases and transplant rejection.

[0014] The tolerogenic construct of the present disclosure comprises an antigenic unit comprising one or more T cell epitopes of an autoantigen, an allergen or an allo- / xeno-antigen, a multimerization unit, e.g., a dimerization unit, and a targeting unit that targets APCs. The targeting unit interacts with a surface molecule on APCs such that the construct is internalized and the epitopes in the antigenic unit are presented in a tolerogenic manner.

[0015] Thus, in a first aspect, the present disclosure provides a method for producing a method for treating a cancer cell comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) multimeric proteins, such as dimeric proteins consisting of multiple polypeptides according to (ii), such as two polypeptides; providing a tolerance-inducing construct comprising The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0016] In another aspect, the present disclosure provides a method for producing a method for manufacturing a pharmaceutical composition comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigenic unit, which target or are capable of targeting antigen-presenting cells; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A multimeric protein consisting of a plurality of polypeptides according to (ii); providing a tolerance-inducing construct comprising The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0017] In another aspect, the present disclosure provides a method for producing a method for manufacturing a pharmaceutical composition comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigenic unit, which targets or is capable of targeting an antigen-presenting cell; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A dimeric protein consisting of two polypeptides according to (ii); providing a tolerance-inducing construct comprising The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0018] In another aspect, the disclosure provides a polynucleotide as described herein.

[0019] In another aspect, the disclosure provides a vector comprising a polynucleotide described herein.

[0020] In another aspect, the disclosure provides a host cell comprising a polynucleotide described herein.

[0021] In another aspect, the present disclosure provides a dimeric protein consisting of two polypeptides described herein.

[0022] In another aspect, the disclosure provides a polypeptide encoded by a nucleic acid described herein.

[0023] In another aspect, the disclosure provides a pharmaceutical composition comprising a tolerance-inducing construct described herein and a pharma- ceutically acceptable carrier.

[0024] In another aspect, the disclosure provides a method for preparing a pharmaceutical composition described herein, the pharmaceutical composition comprising a polypeptide described herein, or a multimer, such as a dimeric protein, described herein, the method comprising: a) transfecting a cell with a polynucleotide described herein; b) culturing the cells; and c) collecting and purifying the multimeric protein, such as a dimeric protein, or the polypeptide expressed from the cell; d) mixing the multimeric protein, such as the dimeric protein, or the polypeptide obtained from step c) with a pharma- ceutically acceptable carrier; Includes.

[0025] In another aspect, the disclosure provides a method for preparing a pharmaceutical composition described herein, the pharmaceutical composition comprising a polynucleotide described herein, the method comprising: a) preparing a polynucleotide; b) optionally, cloning the polynucleotide into an expression vector; c) mixing the polynucleotide obtained from step a) or the vector obtained from step b) with a pharma- ceutically acceptable carrier; Includes.

[0026] In another aspect, the disclosure provides a method for treating a subject suffering from or in need of prevention of a condition involving an unwanted immune response, such as an autoimmune disease, an allergic disease, or transplant rejection, the method comprising administering to the subject a pharmaceutical composition described herein.

[0027] In another aspect, the disclosure provides a pharmaceutical composition as described herein for use in treating a condition involving an unwanted immune response, such as an autoimmune disease, an allergic disease, or transplant rejection. [Brief description of the drawings]

[0028] [Figure 1A] Schematic diagram of an exemplary tolerance-inducing construct. FIG. 1 shows an example of a tolerance-inducing construct of the present disclosure. The tolerance-inducing construct of the present disclosure can be described as a polypeptide with an N-terminal start and a C-terminal end (illustrated in FIG. 1). The elements of the polypeptide--targeting unit (TU), dimerization unit (DimU) and antigenic unit--can be arranged in the polypeptide such that the antigenic unit is located at the C-terminus of the polypeptide (FIG. 1A) or at the N-terminus start of the polypeptide (FIG. 1B). The antigenic unit can include one or more T cell epitopes, and when multiple T cell epitopes are present, can include one or more T cell epitope linkers. A unit linker (UL) can link the dimerization unit and the antigenic unit. FIG. 1 shows an antigenic unit with two T cell epitopes (T1, T2) separated by a T cell epitope linker (TL). The order and orientation of the above units and elements are the same in multimeric proteins, dimeric proteins and polynucleotides. [Figure 1B]Schematic diagram of an exemplary tolerance-inducing construct. FIG. 1 shows an example of a tolerance-inducing construct of the present disclosure. The tolerance-inducing construct of the present disclosure can be described as a polypeptide with an N-terminal start and a C-terminal end (illustrated in FIG. 1). The elements of the polypeptide--targeting unit (TU), dimerization unit (DimU) and antigenic unit--can be arranged in the polypeptide such that the antigenic unit is located at the C-terminus of the polypeptide (FIG. 1A) or at the N-terminus start of the polypeptide (FIG. 1B). The antigenic unit can include one or more T cell epitopes, and when multiple T cell epitopes are present, can include one or more T cell epitope linkers. A unit linker (UL) can link the dimerization unit and the antigenic unit. FIG. 1 shows an antigenic unit with two T cell epitopes (T1, T2) separated by a T cell epitope linker (TL). The order and orientation of the above units and elements are the same in multimeric proteins, dimeric proteins and polynucleotides. [Figure 2A] Expression and secretion of MOG-containing tolerance-inducing constructs. Figures 2A and 2B show protein expression and secretion levels of MOG-containing tolerance-inducing constructs and proinflammatory control constructs VB5002b and VB5052 detected by sandwich ELISA (capture antibody: anti-MOG antibody, detection antibody: anti-hIgG CH3 domain antibody) using (A) supernatants from HEK293 cells transiently transfected with DNA vectors VB5002b, VB5003b, VB5004b, VB5005b, VB5006b, and VB5012b, and (B) supernatants from Expi293F cells transiently transfected with DNA vectors VB5052, VB5046, VB5048, VB5058, VB5059, VB5060, VB5061, and VB5071. All MOG-containing constructs were highly expressed and secreted. Negative controls in (A) are supernatants from HEK293 cells treated with the transfection reagent Lipofectamine alone, and (B) are supernatants from Expi293F cells treated with the transfection reagent ExpiFectamine alone. [Figure 2B]Expression and secretion of MOG-containing tolerance-inducing constructs. Figures 2A and 2B show protein expression and secretion levels of MOG-containing tolerance-inducing constructs and proinflammatory control constructs VB5002b and VB5052 detected by sandwich ELISA (capture antibody: anti-MOG antibody, detection antibody: anti-hIgG CH3 domain antibody) using (A) supernatants from HEK293 cells transiently transfected with DNA vectors VB5002b, VB5003b, VB5004b, VB5005b, VB5006b, and VB5012b, and (B) supernatants from Expi293F cells transiently transfected with DNA vectors VB5052, VB5046, VB5048, VB5058, VB5059, VB5060, VB5061, and VB5071. All MOG-containing constructs were highly expressed and secreted. The negative control in (A) is supernatant from HEK293 cells treated with the transfection reagent Lipofectamine alone, and the negative control in (B) is supernatant from Expi293F cells treated with the transfection reagent ExpiFectamine alone. [Figure 3A] Figure 3: Secretion of full-length MOG-containing tolerance-inducing constructs with different targeting units. Figure 3 shows high-level secretion of full-length tolerance-inducing constructs with different targeting units and the proinflammatory control construct VB5052 detected by sandwich ELISA of supernatants from HEK293 cells or Expi293F cells transiently transfected with vectors VB5005b, VB5006b (HEK293 cells), VB5052, VB5058, VB5059, VB5060 and VB5061 (Expi293F cells). Capture antibody: mouse anti-MOG antibody, 0.25 μg / mL, 100 μL / well, sc-73330, Santa Cruz Biotechnology. Detection antibody: (A) 0.2 μg / mL goat anti-mouse IL-10 biotinylated antibody, 100 μL / well, BAF417, R&D Systems. Negative controls were supernatants from HEK293 cells treated with the transfection reagent Lipofectamine alone or from Expi293F cells treated with the transfection reagent ExpiFectamine alone. [Figure 3B] Figure 3: Secretion of full-length MOG-containing tolerance-inducing constructs with different targeting units. Figure 3 shows high-level secretion of full-length tolerance-inducing constructs with different targeting units and the proinflammatory control construct VB5052 detected by sandwich ELISA of supernatants from HEK293 cells or Expi293F cells transiently transfected with vectors VB5005b, VB5006b (HEK293 cells), VB5052, VB5058, VB5059, VB5060 and VB5061 (Expi293F cells). Capture antibody: mouse anti-MOG antibody, 0.25 μg / mL, 100 μL / well, sc-73330, Santa Cruz Biotechnology. Detection antibody: (B) 0.2 μg / mL goat anti-mouse IL-10 biotinylated antibody, 100 μL / well, BAF417, R&D Systems. Negative controls were supernatants from HEK293 cells treated with the transfection reagent Lipofectamine alone or from Expi293F cells treated with the transfection reagent ExpiFectamine alone. [Figure 3C] Figure 3: Secretion of full-length MOG-containing tolerance-inducing constructs with different targeting units. Figure 3 shows high-level secretion of full-length tolerance-inducing constructs with different targeting units and the proinflammatory control construct VB5052 detected by sandwich ELISA of supernatants from HEK293 cells or Expi293F cells transiently transfected with vectors VB5005b, VB5006b (HEK293 cells), VB5052, VB5058, VB5059, VB5060 and VB5061 (Expi293F cells). Capture antibody: mouse anti-MOG antibody, 0.25 μg / mL, 100 μL / well, sc-73330, Santa Cruz Biotechnology. Detection antibody: (C) 0.8 μg / mL chicken anti-human TGF-β1 biotinylated antibody, 100 μL / well, BAF240, RD Systems. Negative controls were supernatants from HEK293 cells treated with the transfection reagent Lipofectamine alone or from Expi293F cells treated with the transfection reagent ExpiFectamine alone. [Figure 3D] Figure 3: Secretion of full-length MOG-containing tolerance-inducing constructs with different targeting units. Figure 3 shows high-level secretion of full-length tolerance-inducing constructs with different targeting units and the proinflammatory control construct VB5052 detected by sandwich ELISA of supernatants from HEK293 cells or Expi293F cells transiently transfected with vectors VB5005b, VB5006b (HEK293 cells), VB5052, VB5058, VB5059, VB5060 and VB5061 (Expi293F cells). Capture antibody: mouse anti-MOG antibody, 0.25 μg / mL, 100 μL / well, sc-73330, Santa Cruz Biotechnology. Detection antibody: (D) 0.83 μg / mL goat anti-mouse SCGB3A2 biotinylated antibody, 100 μL / well, BAF3465, R&D Systems. Negative controls were supernatants from HEK293 cells treated with only the transfection reagent Lipofectamine or from Expi293F cells treated with only the transfection reagent ExpiFectamine. [Figure 3E] Figure 3: Secretion of full-length MOG-containing tolerance-inducing constructs with different targeting units. Figure 3 shows high-level secretion of full-length tolerance-inducing constructs with different targeting units and proinflammatory control construct VB5052 detected by sandwich ELISA of supernatants from HEK293 cells or Expi293F cells transiently transfected with vectors VB5005b, VB5006b (HEK293 cells), VB5052, VB5058, VB5059, VB5060 and VB5061 (Expi293F cells). Capture antibody: mouse anti-MOG antibody, 0.25 μg / mL, 100 μL / well, sc-73330, Santa Cruz Biotechnology. Detection antibody: (E) 0.8 μg / mL goat anti-mouse CTLA-4 biotinylated antibody, 100 μL / well, BAF476, RD Systems. Negative controls were supernatants from HEK293 cells treated with the transfection reagent Lipofectamine alone or from Expi293F cells treated with the transfection reagent ExpiFectamine alone. [Figure 3F] Figure 3: Secretion of full-length MOG-containing tolerance-inducing constructs with different targeting units. Figure 3 shows high-level secretion of full-length tolerance-inducing constructs with different targeting units and proinflammatory control construct VB5052 detected by sandwich ELISA of supernatants from HEK293 cells or Expi293F cells transiently transfected with vectors VB5005b, VB5006b (HEK293 cells), VB5052, VB5058, VB5059, VB5060 and VB5061 (Expi293F cells). Capture antibody: mouse anti-MOG antibody, 0.25 μg / mL, 100 μL / well, sc-73330, Santa Cruz Biotechnology. Detection antibody: (F) 0.29 μg / mL goat anti-mouse PD-1 biotinylated antibody, 100 μL / well, DY1021, R&D System. Negative controls were supernatants from HEK293 cells treated with the transfection reagent Lipofectamine alone or from Expi293F cells treated with the transfection reagent ExpiFectamine alone. [Figure 3G] Figure 3: Secretion of full-length MOG-containing tolerance-inducing constructs with different targeting units. Figure 3 shows high-level secretion of full-length tolerance-inducing constructs with different targeting units and the proinflammatory control construct VB5052 detected by sandwich ELISA of supernatants from HEK293 cells or Expi293F cells transiently transfected with vectors VB5005b, VB5006b (HEK293 cells), VB5052, VB5058, VB5059, VB5060 and VB5061 (Expi293F cells). Capture antibody: mouse anti-MOG antibody, 0.25 μg / mL, 100 μL / well, sc-73330, Santa Cruz Biotechnology. Detection antibody: (G) 0.2 μg / mL goat anti-human CCL3 biotinylated antibody, 100 μL / well, BAF270, R&D Systems. Negative controls were supernatants from HEK293 cells treated with the transfection reagent Lipofectamine alone or from Expi293F cells treated with the transfection reagent ExpiFectamine alone. [Figure 4] Secretion of MOG(27-63) peptide. Secretion of MOG(27-63) peptide encoded in DNA vector VB5051 was confirmed by direct ELISA (detection antibody: mouse anti-MOG antibody, 3.3 μg / mL, 100 μL / well, sc-73330, Santa Cruz Biotechnology) of supernatant from Expi293F cells transiently transfected with DNA vector VB5051. The negative control was the supernatant from Expi293F cells treated with the transfection reagent ExpiFectamine only. [Diagram 5] Expression and secretion of Met e1-containing tolerance-inducing constructs. Figure 5 shows the protein expression and secretion levels of Met e1-containing tolerance-inducing constructs detected by sandwich ELISA (capture antibody: anti-human IgG3 (CH3 domain) antibody, detection antibody: CaptureSelect™ biotin anti-IgG-Fc (human) conjugate) of supernatants from Expi293F cells transiently transfected with Met e1-containing DNA vectors VB5024, VB5030 and VB5079. All Met e1-containing tolerance-inducing constructs were expressed and secreted. The negative control was supernatant from Expi293F cells treated with the transfection reagent Expifectamine only. [Figure 6] Tolerance-inducing constructs containing scFv anti-DEC205 as targeting units bind to recombinant DEC205 receptor. Figure 6 shows that tolerance-inducing proteins containing scFv anti-DEC205 targeting units bind to recombinant DEC205 receptor by direct ELISA (coating: recombinant DEC205 (216-503), detection antibody: anti-MOG antibody or anti-hIgG CH3 domain antibody) of supernatants from HEK293 cells transiently transfected with scFv anti-DEC205-containing DNA vector VB5004b. Binding to the receptor was confirmed by both antibodies, with the anti-MOG antibody confirming the secretion of full-length protein. [Figure 7]Tolerance-inducing constructs containing IL-10 as a targeting unit bind to recombinant IL-10 receptor. Figure 7 shows that tolerance-inducing proteins containing IL-10 as a targeting unit bind to recombinant IL-10 receptor by direct ELISA (coat: recombinant IL-10 receptor, detection antibody: anti-MOG antibody or anti-hIgG CH3 domain antibody) of supernatants from HEK293 cells transiently transfected with the IL-10-containing DNA vector VB5006b. Binding to the receptor was confirmed by both antibodies, with the anti-MOG antibody confirming the secretion of the full-length protein. [Figure 8A] Characterization of size, protein integrity and dimer formation of secreted tolerance-inducing constructs. Figure 8 shows Western blot (WB) analysis of supernatant from Expi293F cells transiently transfected with MOG-containing DNA vector under reducing and non-reducing conditions. Negative control is supernatant from Expi293F cells treated with the transfection reagent ExpiFectamine (transfection control). Figure 8A: Western blot analysis shows expression and full-length secretion of tolerance-inducing proteins. Reduced supernatant samples (25 μL loading) from transfected Expti293F cells. Primary antibody: mouse anti-MOG (sc-73330). Secondary antibody: Donkey anti-mouse, Dylight800 (SA5-10172). Protein standards were detected in Chemidoc channel Dylight650 (signal not shown) and Chemidoc channel Dylight800. [Figure 8B]Characterization of size, protein integrity and dimerization of secreted tolerance-inducing constructs. Figure 8 shows Western blot (WB) analysis of supernatants from Expi293F cells transiently transfected with MOG-containing DNA vectors under reducing and non-reducing conditions. Negative control is supernatant from Expi293F cells treated with the transfection reagent ExpiFectamine (transfection control). Figure 8B: Western blot analysis shows dimerization of the tolerance-inducing protein (black arrow). Non-reduced supernatant sample (25 μL loading) from transfected Expecti293F cells. Primary antibody: mouse anti-MOG (sc-73330). Secondary antibody: Donkey anti-mouse, Dylight800 (SA5-10172). Chemidoc channels Dylight650 (for protein standards) and 800. [Figure 8C] Characterization of size, protein integrity and dimer formation of secreted tolerance-inducing constructs. Figure 8 shows Western blot (WB) analysis of supernatants from Expi293F cells transiently transfected with MOG-containing DNA vectors under reducing and non-reducing conditions. Negative control is supernatant from Expi293F cells treated with the transfection reagent ExpiFectamine (transfection control). Figure 8C: Western blot analysis shows expression and full-length secretion of tolerance-inducing proteins. Reduced supernatant sample from transfected Expti293F cells (25 μL control). Primary antibody: rat anti-IL10 (MAB417). Secondary antibody: Donkey anti-rat, Dylight488 (SA5-10026). Chemidoc channels Dylight650 (for protein standards) and 488. [Figure 8D]Characterization of size, protein integrity and dimer formation of secreted tolerance-inducing constructs. Figure 8 shows Western blot (WB) analysis of supernatants from Expi293F cells transiently transfected with MOG-containing DNA vectors under reducing and non-reducing conditions. Negative control is supernatant from Expi293F cells treated with the transfection reagent ExpiFectamine (transfection control). Figure 8D: Western blot analysis shows expression and full-length secretion of tolerance-inducing proteins (black arrows). Reduced supernatant samples (35 μL loading) from transfected Expti293F cells. Primary antibody: goat anti-CTLA-4 (AF476). Secondary antibody: Donkey anti-goat, Dylight800 (SA5-10092). Chemidoc channels Dylight650 (for protein standards) and 800. [Figure 9A] Dual color IL-10 / IFNγ Dual color IL-10 / IFNγ FluoroSpot. C57BL / 6 mice were vaccinated once (day 0) with 50 μg of the indicated DNA vectors (VB5004b, VB5002b and VB5001b) and spleens were harvested 7 days post-vaccination. (A) Splenocytes (SFU / 106 splenocytes) and dual color FluoroSpot from mice tested for IFN-γ and IL-10 secretion upon restimulation with MOG(35-55) peptide. n=4 (VB5001b), 5 (VB5004b and VB5002b) or 2 (PBS) individual mice per group are shown. (B) IL-10 / IFN-γ ratios are plotted from the data in (A). Individual mice and means±range are shown, *(p<0.05) **(p<0.01), two-tailed Mann-Whitney test. [Figure 9B]Dual color IL-10 / IFNγ Dual color IL-10 / IFNγ FluoroSpot. C57BL / 6 mice were vaccinated once (day 0) with 50 μg of the indicated DNA vectors (VB5004b, VB5002b and VB5001b) and spleens were harvested 7 days post-vaccination. (A) Splenocytes (SFU / 106 splenocytes) and dual color FluoroSpot from mice tested for IFN-γ and IL-10 secretion upon restimulation with MOG(35-55) peptide. n=4 (VB5001b), 5 (VB5004b and VB5002b) or 2 (PBS) individual mice per group are shown. (B) IL-10 / IFN-γ ratios are plotted from the data in (A). Individual mice and means±range are shown, *(p<0.05) **(p<0.01), two-tailed Mann-Whitney test. [Figure 10A] Detection of % Foxp3+, % IFN-γ and % IL-17 producing CD4+ T cells by flow cytometry. C57BL / 6 mice were vaccinated once (day 0) with 50 μg of the indicated DNA vector (VB5004b, VB5002b, VB5001b) and spleens were harvested 7 days post vaccination. The percentage [%] of (A) Foxp3+, (B) IFN-γ+ and (C) IL-17+ splenocytes in the total CD4+ T cell population upon restimulation with MOG(35-55) peptide is shown. Data are from pools of 4 (VB5001b), 5 (VB5004b and VB5002b) or 2 (PBS) mice / group. Construct ID numbers are indicated on the x-axis. [Figure 10B] Detection of % Foxp3+, % IFN-γ and % IL-17 producing CD4+ T cells by flow cytometry. C57BL / 6 mice were vaccinated once (day 0) with 50 μg of the indicated DNA vector (VB5004b, VB5002b, VB5001b) and spleens were harvested 7 days post vaccination. The percentage [%] of (A) Foxp3+, (B) IFN-γ+ and (C) IL-17+ splenocytes in the total CD4+ T cell population upon restimulation with MOG(35-55) peptide is shown. Data are from pools of 4 (VB5001b), 5 (VB5004b and VB5002b) or 2 (PBS) mice / group. Construct ID numbers are indicated on the x-axis. [Figure 10C] Detection of % Foxp3+, % IFN-γ and % IL-17 producing CD4+ T cells by flow cytometry. C57BL / 6 mice were vaccinated once (day 0) with 50 μg of the indicated DNA vector (VB5004b, VB5002b, VB5001b) and spleens were harvested 7 days post vaccination. The percentage [%] of (A) Foxp3+, (B) IFN-γ+ and (C) IL-17+ splenocytes in the total CD4+ T cell population upon restimulation with MOG(35-55) peptide is shown. Data are from pools of 4 (VB5001b), 5 (VB5004b and VB5002b) or 2 (PBS) mice / group. Construct ID numbers are indicated on the x-axis. [Figure 11A] Dual color IL-10 / IFNγ FluoroSpot. C57BL / 6 mice were vaccinated twice (days 0 and 4) with 50 μg of the indicated DNA vector (VB5012b, VB5052, VB5051) and spleens were harvested 10 days after prime vaccination. (A) Splenocytes (SFU / 106 splenocytes) and dual color FluoroSpot of mice tested for IFN-γ and IL-10 secretion upon restimulation with MOG(35-55) peptide. Individual mice are shown. (B) IL-10 / IFN-γ ratios are plotted from the data in (A). Individual mice and mean ± range are shown, n=5 or n=2 per group (PBS), *(p<0.05)**(p<0.01), two-tailed Mann-Whitney test. [Figure 11B]Dual color IL-10 / IFNγ FluoroSpot. C57BL / 6 mice were vaccinated twice (days 0 and 4) with 50 μg of the indicated DNA vector (VB5012b, VB5052, VB5051) and spleens were harvested 10 days after prime vaccination. (A) Splenocytes (SFU / 106 splenocytes) and dual color FluoroSpot of mice tested for IFN-γ and IL-10 secretion upon restimulation with MOG(35-55) peptide. Individual mice are shown. (B) IL-10 / IFN-γ ratios are plotted from the data in (A). Individual mice and mean ± range are shown, n=5 or n=2 per group (PBS), *(p<0.05)**(p<0.01), two-tailed Mann-Whitney test. [Figure 12] Detection of MOG(38-49)-specific Foxp3+ T cells. C57BL / 6 mice were vaccinated twice (days 0 and 4) with 50 μg of the indicated DNA vector (VB5012b, VB5048, VB5006b, VB5046, VB5051) and spleens were harvested 10 days after prime vaccination. Percentage of splenic Foxp3+ cells detected ex vivo by H-2 Iab / MOG(38-49) tetramer of the total CD4+ population. Data were obtained from pools of 5 mice or 2 mice (PBS) per group. Construct ID numbers are indicated on the x-axis. [Figure 13] Expression and secretion of MOG-containing tolerance-inducing constructs. Figure 13 shows protein expression and secretion levels of MOG-containing tolerance-inducing construct VB5009 with TGFβ1 as the targeting unit, detected by sandwich ELISA (capture antibody: rabbit anti-human TGFβ1 (orb77216, Biorbyte), detection antibody: biotinylated mouse anti-human IgG (05-4240, Invitrogen)) of supernatants from HEK293 cells transiently transfected with VB5009. The negative control is supernatant from HEK293 cells treated with the transfection reagent Lipofectamine only. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Thus, in a first aspect, the present disclosure provides a method for producing a method for treating a cancer cell comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A multimeric protein, such as a dimeric protein consisting of a plurality of polypeptides according to (ii), e.g. two polypeptides; providing a tolerance-inducing construct comprising The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0030] In another aspect, the present disclosure provides a method for producing a method for manufacturing a pharmaceutical composition comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigenic unit, which target or are capable of targeting antigen-presenting cells; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A multimeric protein consisting of a plurality of polypeptides according to (ii); providing a tolerance-inducing construct comprising The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0031] In another aspect, the present disclosure provides a method for producing a method for manufacturing a pharmaceutical composition comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigenic unit, which targets or is capable of targeting an antigen-presenting cell; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A dimeric protein consisting of two polypeptides according to (ii); providing a tolerance-inducing construct comprising The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0032] Such constructs, once administered to a subject, are capable of presenting epitopes in the antigenic unit in a tolerogenic manner and are therefore suitable for use as prophylactic or therapeutic treatments for immune disorders such as autoimmune diseases, allergic diseases and graft rejection.

[0033] The tolerance-inducing constructs do not suppress the general immune system, since they cause the downregulation of disease-specific cells of the immune system that cause the immune disease in question. Thus, treating the immune disease in question with the constructs of the present disclosure does not result in increased susceptibility to infection and reduced cancer immune surveillance. However, bystander suppression of immune cells specific for the relevant disease antigen is expected due to the release of short-range inhibitory cytokines by cell-to-cell contact with the induced antigen-specific regulatory cells.

[0034] The tolerance-inducing constructs of the present disclosure may be administered in the form of a pharmaceutical composition comprising the construct of the present disclosure and a pharma- ceutically acceptable carrier for use in the prophylactic or therapeutic treatment of immune diseases, such as autoimmune diseases, allergic diseases, and transplant rejection.

[0035] A "nucleotide sequence" is a sequence made up of nucleotides. The terms "nucleotide sequence" and "nucleic acid sequence" are used interchangeably herein.

[0036] A "tolerance-inducing construct" is one that, when administered to a subject in a form suitable for administration and in an amount effective to induce tolerance (i.e., an effective amount), does not induce an immune response such as an inflammatory immune response, but rather induces tolerance to a T cell epitope contained in the antigenic unit.

[0037] As used herein, the term "tolerance" refers to a reduced level of an immune response, such as an inflammatory immune response, a delayed onset or progression of an immune response, such as an inflammatory immune response, and / or a reduced risk of onset or progression of an immune response, such as an inflammatory immune response.

[0038] A "subject" is an animal or a human. A subject may be a patient in need of therapeutic treatment, i.e., a human suffering from an immune disorder such as an autoimmune disease, an allergy, or a transplant rejection. The terms "subject" and "individual" are used interchangeably herein.

[0039] A "disease" is an abnormal medical condition typically associated with particular signs and symptoms in a subject suffering from the disease.

[0040] As used herein, "immune disease" refers to a condition, disorder or disease involving an unwanted immune response, including autoimmune disease, allergy or graft rejection, i.e., rejection of an allograft or xenograft, such as rejection by a host of cells, tissues or organs from the same (allo) species or a different (xeno) species that have been transplanted into the host.

[0041] The term "alloantigen" or "allotransplant antigen" as used herein refers to an antigen derived from (shedded from and / or present in) a cell or tissue that, when transferred from a donor to a recipient, can be recognized and bound by the antibodies of the recipient's B-cell receptor or T-cell receptor. Alloantigens are typically the product of polymorphic genes. Alloantigens are proteins or peptides that exhibit slight structural differences when compared between donor and recipient (belonging to the same species). The presence of such donor antigens in the recipient's body can induce an immune response in the recipient. Such an alloreactive immune response is specific to the alloantigen.

[0042] The terms "murine" and "mouse" are used interchangeably to refer to peptides, proteins, nucleic acids and other materials derived from a mouse.

[0043] As used herein, the term "xenoantigen" refers to an antigen derived from an individual of a different species.

[0044] "Treatment" refers to prophylactic or therapeutic treatment.

[0045] A "prophylactic treatment" is a treatment administered to a subject who does not exhibit signs or symptoms of an immune disease or who exhibits only early signs or symptoms of an immune disease, such that the treatment is administered with the intent to prevent the disease or at least reduce the risk of developing the disease. Prophylactic treatment functions as a preventative treatment against an immune disease or as a treatment that inhibits or reduces the further development or enhancement of an immune disease and / or its associated symptoms. The terms "prophylactic treatment", "prophylaxis" and "prevention" are used interchangeably herein.

[0046] A "therapeutic treatment" is a treatment administered to a subject who exhibits symptoms or signs of an immune disease, where the treatment is administered to the subject with the intent of reducing or eliminating those signs or symptoms and / or slowing or halting disease progression.

[0047] "Part" refers to a portion or fragment of an antigen, i.e., a portion or fragment of the amino acid sequence of the antigen, or the nucleotide sequence encoding it, e.g., an epitope; preferably, the portion or fragment of the antigen is immunogenic. These terms are used interchangeably throughout.

[0048] As used herein, "T cell epitope" refers to a single T cell epitope or a portion or region of an antigen that contains multiple T cell epitopes, e.g., multiple minimal epitopes.

[0049] The terms "vaccination" and "administration" are used interchangeably herein.

[0050] The term "minimal epitope" refers to a subsequence of an epitope predicted to bind MHC I or MHC II. In other words, a minimal epitope may be immunogenic, i.e., capable of eliciting an immune response. Thus, the term minimal epitope may refer to a short subsequence of an epitope predicted to bind MHC I or MHC II. Thus, 27-mer epitopes may each have a length shorter than 27 amino acids, each of which may be immunogenic and encompass several minimal epitopes. For example, a minimal epitope may consist of the first 14 amino acids of an epitope if predicted to bind MHC I or MHC II, or may consist of amino acids 9-18, or amino acids 7-22 of an epitope if predicted to bind MHC I or MHC II.

[0051] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0052] Tolerance-inducing constructs Figure 1 shows an example of a tolerance-inducing construct of the present disclosure. The tolerance-inducing construct of the present disclosure can be described as a polypeptide having an N-terminal start and a C-terminus (illustrated in Figure 1). The elements of the polypeptide - targeting unit (TU), dimerization unit (DimU) and antigenic unit - can be arranged in the polypeptide such that the antigenic unit is located at the C-terminus of the polypeptide (Figure 1A) or at the N-terminus of the polypeptide (Figure 1B). Preferably, the antigenic unit is located at the C-terminus of the polypeptide.

[0053] An antigenic unit may contain one or more T cell epitopes, and when multiple T cell epitopes are present, may contain one or more T cell epitope linkers (TL). A unit linker (UL) may link the dimerization unit and the antigenic unit. Figure 1 shows an antigenic unit with two T cell epitopes (T1, T2) separated by a TL. The order and orientation of the above units and elements are the same in the dimeric protein and the polynucleotide.

[0054] In the following, the various units of the construct are described in detail. These units are present in the polynucleotide as nucleic acid sequences encoding the units, while they are present in the polypeptide, multimeric or dimeric protein as amino acid sequences. For ease of reading, in the following, the units of the construct are described mainly with respect to the polypeptide, multimeric or dimeric protein, i.e. based on their amino acid sequences.

[0055] Targeting Unit The tolerogenic constructs of the present disclosure include a targeting unit that targets antigen-presenting cells (APCs).

[0056] The term "targeting unit" as used herein refers to a unit that delivers the construct of the present disclosure to an APC and interacts with a surface molecule on the APC, e.g., binds to a surface receptor on the APC without activating the cell and / or inducing maturation of the cell. The APC internalizes the construct and presents the T cell epitopes contained in the antigenic unit on the MHC on its surface in an anti-inflammatory, tolerogenic manner, e.g., by not upregulating costimulatory signals and / or not upregulating the secretion of inhibitory surface receptors and / or inhibitory cytokines.

[0057] In some embodiments, the targeting unit is a targeting unit for a TGFβR receptor, such as TGFβR1, TGFβR2, or TGFβR3, IL10R, such as IL10RA and IL10RB, IL2R, IL4R, IL6R, IL11R, IL13R, IL27R, IL35R, IL37R, GM-CSFR, FLT3, CCR7, CD11b, CD11c, CD103, CD14, CD36, CD205, CD109, VISTA, MAR The antibody comprises or consists of a portion that binds to a receptor selected from the group consisting of CO, MHCII, CD83, SIGLEC, MGL / Clec10A, ASGR (ASGR1 / ASGR2), CD80, CD86, Clec9A, Clec12A, Clec12B, DCIR2, Langerin, MR, DC-Sign, Treml4, Dectin-1, PDL1, PDL2, HVEM, CD163 and CD141.

[0058] In some embodiments, the targeting unit is an hTGFβ receptor, such as hTGFβR1, hTGFβR2, or hTGFβR3; hIL-10R, such as hIL-10RA and hIL-10RB; hIL2R, hIL4R, hIL6R, hIL11R, hIL13R, hIL27R, hIL35R, hIL37R, hGM-CSFR, hFLT3, hCCR7, hCD11b, hCD11c, hCD103, hCD14, hCD36, hCD205, hCD109, hVISTA, hMARC O, comprising or consisting of a portion that binds to a human (h) receptor selected from the group consisting of hMHCII, hCD83, hSIGLEC, hMGL / hClec10A, hASGR (hASGR1 / hASGR2), hCD80, hCD86, hClec9A, hClec12A, hClec12B, hDCIR2, hLangerin, hMR, hDC-Sign, hTreml4, hDectin-1, hPDL1, hPDL2, hHVEM, hCD163 and hCD141.

[0059] The moiety may be a natural ligand, an antibody or part thereof, such as an scFv, or a synthetic ligand.

[0060] In some embodiments, the portion is an antibody or portion thereof, e.g., an scFv, with specificity for any of the aforementioned receptors, which upon binding to the receptor results in antigens and / or T cell epitopes being presented in an anti-inflammatory tolerogenic manner.

[0061] In other embodiments, the moiety is a synthetic ligand with specificity for any of the aforementioned receptors, which upon binding to the receptor results in antigens and / or T cell epitopes being presented in an anti-inflammatory tolerogenic manner. Protein modeling can be used to design such synthetic ligands.

[0062] In other embodiments, the moiety is a natural ligand.

[0063] In some embodiments the natural ligand is selected from the group consisting of TGFβ, such as TGFβ1, TGFβ2 or TGFβ3, IL-10, IL2, IL4, IL6, IL11, IL13, IL27, IL35, IL37, GM-CSF, FLT3L, CCL19, CCL21, ICAM-1 (intercellular adhesion molecule 1, also known as CD54), keratin, VSIG-3, SCGB3A2, CTLA-4, preferably CTLA-4, the extracellular domain of PD-1, preferably the extracellular domain of PD-1 and BTLA, preferably the extracellular domain of BTLA.

[0064] In another embodiment, the targeting unit is or comprises IL2, preferably human IL2. In another embodiment, the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to the sequence identity of human IL2, such as an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 33. In another embodiment, the targeting unit comprises or consists of a nucleotide sequence encoding human IL2, such as the nucleotide sequence of SEQ ID NO: 36.

[0065] In other embodiments, the targeting unit is or comprises IL-10 or TGFβ, preferably human IL-10, or human TGFβ, including the isoforms TGFβ-1, TGFβ-2 and TGFβ-3.

[0066] In other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to the sequence of human TGFβ, such as an amino acid sequence having at least 80% sequence identity to any of SEQ ID NOs: 205-207.

[0067] In yet other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence of human TGFβ, such as an amino acid sequence having at least 85% sequence identity to any of SEQ ID NOs: 205-207, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, or such as 100% sequence identity.

[0068] In another embodiment, the targeting unit comprises or consists of an amino acid sequence of human TGFβ, such as an amino acid sequence selected from SEQ ID NOs: 205-207, except for up to 22 amino acids, e.g., up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid being substituted, deleted, or inserted.

[0069] In other embodiments, the targeting unit comprises or consists of the amino acid sequence of human TGFβ or a nucleotide sequence encoding human TGFβ.

[0070] In other embodiments, the targeting unit comprises or consists of a nucleotide sequence encoding human TGFβ, such as a nucleotide sequence selected from SEQ ID NOs: 208-210.

[0071] In yet other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to the sequence of mouse TGFβ, such as the mouse TGFβ set forth in SEQ ID NO:177.

[0072] In yet other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to the sequence of human IL-10, such as an amino acid sequence having at least 80% sequence identity to SEQ ID NO:211.

[0073] In yet other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence of human IL-10, such as an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 211, such as an amino acid sequence having at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, or such as 100% sequence identity.

[0074] In other embodiments, the targeting unit comprises or consists of the amino acid sequence of human IL-10, such as the amino acid sequence of SEQ ID NO: 211, except that up to 22 amino acids, e.g. up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid have been substituted, deleted, or inserted.

[0075] In other embodiments, the targeting unit comprises or consists of the amino acid sequence of human IL-10 or a nucleotide sequence encoding human IL-10.

[0076] In other embodiments, the targeting unit comprises or consists of a nucleotide sequence encoding human IL-10, such as the nucleotide sequence of SEQ ID NO:212.

[0077] In yet other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to the sequence of mouse IL-10, such as the mouse IL-10 set forth in SEQ ID NO:169.

[0078] In some embodiments, the targeting unit is or comprises SCGB3A2 or VSIG-3, preferably human VSIG-3 or human SCGB3A2.

[0079] In other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity with the sequence of human SCGB3A2, such as an amino acid sequence having at least 80% sequence identity with SEQ ID NO:213.

[0080] In yet other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence of human SCGB3A2, such as an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 213, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, or such as 100% sequence identity.

[0081] In other embodiments, the targeting unit comprises or consists of the amino acid sequence of human SCGB3A2, such as the amino acid sequence of SEQ ID NO: 213, except that up to 22 amino acids, for example up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, have been substituted, deleted, or inserted.

[0082] In other embodiments, the targeting unit comprises or consists of the amino acid sequence of human SCGB3A2 or a nucleotide sequence encoding human SCGB3A2.

[0083] In other embodiments, the targeting unit comprises or consists of a nucleotide sequence encoding human SCGB3A2, such as the nucleotide sequence of SEQ ID NO:214.

[0084] In yet other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to the sequence of mouse SCGB3A2, such as mouse SCGB3A2 set forth in SEQ ID NO:171.

[0085] In yet other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to that of human VSIG-3, such as an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 215.

[0086] In yet other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence of human VSIG-3, such as an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 215, such as an amino acid sequence having at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, or such as 100% sequence identity.

[0087] In another embodiment, the targeting unit comprises or consists of the amino acid sequence of human VSIG-3, such as the amino acid sequence of SEQ ID NO: 215, except that up to 22 amino acids, e.g., up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, have been substituted, deleted, or inserted.

[0088] In other embodiments, the targeting unit comprises or consists of the amino acid sequence of human VSIG-3 or a nucleotide sequence encoding human VSIG-3.

[0089] In other embodiments, the targeting unit comprises or consists of a nucleotide sequence encoding human VSIG-3, such as the nucleotide sequence of SEQ ID NO:216.

[0090] In yet other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to the sequence of mouse VSIG-3, such as mouse VSIG-3 set forth in SEQ ID NO:173.

[0091] In yet other embodiments, the targeting unit is or comprises an antibody or portion thereof with specificity for CD205, e.g., an scFv, such as an scFv with specificity for human or mouse CD205, or an scFv anti-DEC205. In some embodiments, the scFv with specificity for mouse CD205 comprises or consists of SEQ ID NO:49.

[0092] In other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to human CTLA4, such as an amino acid sequence having at least 80% sequence identity to SEQ ID NO:217.

[0093] In yet other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence of human CTLA4, such as an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 217, such as at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, or such as 100% sequence identity.

[0094] In other embodiments, the targeting unit comprises or consists of the amino acid sequence of human CTLA4, such as the amino acid sequence of SEQ ID NO: 217, except that up to 22 amino acids, e.g., up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, have been substituted, deleted, or inserted.

[0095] In other embodiments, the targeting unit comprises or consists of the amino acid sequence of human CTLA4 or a nucleotide sequence encoding human CTLA4.

[0096] In other embodiments, the targeting unit comprises or consists of a nucleotide sequence encoding human CTLA4, such as the nucleotide sequence of SEQ ID NO:218.

[0097] In yet other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to the sequence of mouse CTLA4, such as the mouse CTLA4 set forth in SEQ ID NO:175.

[0098] In other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to a sequence of human PD-1, such as an amino acid sequence having at least 80% sequence identity to SEQ ID NO:219.

[0099] In yet other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence of human PD-1, such as an amino acid sequence having at least 85% sequence identity to SEQ ID NO:219, such as at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, or such as 100% sequence identity.

[0100] In other embodiments, the targeting unit comprises or consists of the amino acid sequence of human PD-1, such as the amino acid sequence of SEQ ID NO: 219, except that up to 22 amino acids have been substituted, deleted, or inserted, e.g., up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid.

[0101] In other embodiments, the targeting unit comprises or consists of the amino acid sequence of human PD-1 or a nucleotide sequence encoding human PD-1.

[0102] In other embodiments, the targeting unit comprises or consists of a nucleotide sequence encoding human PD-1, such as the nucleotide sequence of SEQ ID NO:220.

[0103] In yet other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to the sequence of mouse PD-1, such as the mouse PD-1 set forth in SEQ ID NO:179.

[0104] In yet other embodiments, the targeting unit comprises or consists of an amino acid sequence having an amino acid sequence having at least 80% sequence identity to the sequence of human IL-10, such as an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 211.

[0105] In yet other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence of human IL-10, such as an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 211, such as an amino acid sequence having at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, or such as 100% sequence identity.

[0106] In other embodiments, the targeting unit comprises or consists of the amino acid sequence of human IL-10, such as the amino acid sequence of SEQ ID NO: 211, except that up to 22 amino acids, e.g. up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid have been substituted, deleted, or inserted.

[0107] In other embodiments, the targeting unit comprises or consists of the amino acid sequence of human IL-10 or a nucleotide sequence encoding human IL-10.

[0108] In other embodiments, the targeting unit comprises or consists of a nucleotide sequence encoding human IL-10, such as the nucleotide sequence of SEQ ID NO:212.

[0109] Antigenic unit The antigenic unit of the tolerogenic construct of the present disclosure comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0110] T cell epitopes suitable for inclusion in an antigenic unit are known in the art, i.e., have been studied, proposed and / or verified to be involved or associated with certain immune diseases and have been published in the literature.

[0111] In some embodiments, the antigenic unit comprises one or more T cell epitopes of an autoantigen, i.e., one T cell epitope of an autoantigen, or two or more T cell epitopes of an autoantigen, i.e., multiple T cell epitopes of an autoantigen. In some embodiments, the multiple T cell epitopes are of the same autoantigen, i.e., are contained in the same autoantigen. In other embodiments, the multiple T cell epitopes are of different autoantigens, i.e., are contained in different autoantigens.

[0112] In some embodiments, the antigenic unit comprises one or more T cell epitopes of an autoantigen, such as a Treg epitope or an inhibitory neoantigen.

[0113] In some embodiments, when the antigenic unit comprises two or more T cell epitopes, the antigenic unit comprises one or more linkers separating the T cell epitopes. In some embodiments, the antigenic unit comprises multiple T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen, and the T cell epitopes are preferably separated by linkers. In yet other embodiments, the antigenic unit comprises multiple T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen, and each T cell epitope is separated from the other T cell epitopes by a linker. An alternative way to describe the separation of each T cell epitope of an autoantigen, allergen, alloantigen or xenoantigen from other T cell epitopes by the linker is that all but the terminal T cell epitopes, i.e. the T cell epitopes at the N-terminus beginning of the polypeptide or at the C-terminus of the polypeptide (i.e. located at the end of the antigenic unit that is not linked to a dimerization unit), are located in a subunit, each subunit comprising or consisting of a T cell epitope and a linker as described herein.

[0114] Thus, an antigenic unit comprising n antigens comprises n-1 subunits, each subunit comprising a T cell epitope of an autoantigen, an allergen, an alloantigen or a xenoantigen, and a linker, and further comprising a terminal T cell epitope. In some embodiments, n is an integer from 1 to 50, e.g., from 3 to 50, or from 15 to 40, or from 10 to 30, or from 10 to 25, or from 10 to 20, or from 15 to 30, or from 15 to 25, or from 15 to 20.

[0115] The linker in the antigenic unit separates the antigens, e.g. epitopes, contained therein. As described above, all T cell epitopes of autoantigens, allergens, alloantigens or xenoantigens can be located in the subunits, separated from each other by linkers.

[0116] In some embodiments, the linker is designed to be non-immunogenic. It may be a rigid linker, meaning that it does not allow the two amino acid sequences that it links to move substantially freely relative to each other. Alternatively, it may be a flexible linker, i.e., a linker that allows the two amino acid sequences that it links to move substantially freely relative to each other.

[0117] Both types of linkers are useful. In one embodiment, the T cell epitope linker is a flexible linker that allows the T cell epitopes to be presented in an optimal manner to T cells, even if the antigenic unit contains multiple T cell epitopes.

[0118] By separating the T cell epitopes by a linker, each T cell epitope of an autoantigen, an allergen, an alloantigen or a xenoantigen is presented in an optimal manner to the immune system.

[0119] By way of example, myelin basic protein (MBP), proteolipid protein (PLP), myelin associated glycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG) and myelin associated basic oligodendrocyte protein (MOBP) have all been studied and proposed as autoantigens involved in multiple sclerosis (MS), and an antigenic unit may, for example, comprise one or more T cell epitopes of MBP, i.e., one T cell epitope of MBP or multiple T cell epitopes of MBP. Furthermore, an antigenic unit may, for example, comprise multiple T cell epitopes of MOG and PLP, e.g., one or more T cell epitopes of MOG and one or more T cell epitopes of PLP.

[0120] In some embodiments, the antigenic unit may comprise one or more T cell epitopes of MOG, such as one or more T cell epitopes of MOG comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 180-182.

[0121] In other embodiments, the antigenic unit comprises one or more T cell epitopes of an allergen, i.e. one T cell epitope of an allergen or more than one T cell epitope of an allergen, i.e. multiple T cell epitopes of an allergen. In some embodiments, the multiple T cell epitopes are of the same allergen, i.e. comprised in the same allergen. In other embodiments, the multiple T cell epitopes are of different allergens, i.e. comprised in different allergens.

[0122] By way of example, Fel d1, Fel d4 and Fel d7 are three of the most prominent cat allergens that account for the majority of human cat allergies, and an antigenic unit may, for example, comprise one or more T cell epitopes of Fel d1, i.e. one T cell epitope of Fel d1 or multiple T cell epitopes of Fel d1. Furthermore, an antigenic unit may, for example, comprise multiple T cell epitopes of Fel d4 and Fel d7, e.g. one or more T cell epitopes of Fel d4 and one or more T cell epitopes of Fel d7.

[0123] In some embodiments, the antigenic unit may comprise one or more T cell epitopes of Met e1, such as one or more T cell epitopes included in SEQ ID NO: 184. In some embodiments, the antigenic unit may comprise one or more T cell epitopes of Met e1, such as Met e1(16-35), Met e1(46-65), Met e1(76-95), Met e1(136-155), Met e1(210-230) and / or Met e1(241-260). In some embodiments, the antigenic unit may comprise one or more T cell epitopes of Met e1, such as one or more T cell epitopes comprising or consisting of a sequence selected from any of SEQ ID NOs: 185-190.

[0124] In other embodiments, the antigenic unit comprises one or more T cell epitopes of an allo / xenoantigen, i.e. one T cell epitope of an allo / xenoantigen, or one or more T cell epitopes of an allo / xenoantigen, i.e. multiple T cell epitopes of an allo / xenoantigen. In some embodiments, the multiple T cell epitopes are of the same allo / xenoantigen, i.e. comprised in the same allo / xenoantigen. In other embodiments, the multiple T cell epitopes are of different allo / xenoantigens, i.e. comprised in different allo / xenoantigens.

[0125] In some embodiments, the antigenic unit comprises one T cell epitope, while in other embodiments, the antigenic unit comprises two or more T cell epitopes, i.e., multiple T cell epitopes.

[0126] The tolerance-inducing constructs of the present disclosure may be individualized treatments, i.e., treatments designed for a particular subject / single patient, hi other embodiments, the tolerance-inducing constructs of the present disclosure are for general use in a patient population or patient, i.e., off-the-shelf treatments.

[0127] Individualized tolerance-inducing constructs For individualized tolerogenic constructs, T cell epitopes are selected for inclusion in the antigenic unit, and the T cell epitopes are optimized for the patient receiving treatment with the construct, which increases the therapeutic effect compared to off-the-shelf treatments that include the tolerogenic construct.

[0128] The antigenic units of the individualized tolerogenic constructs can be designed as follows, as exemplified for patients suffering from MS. 1) Determine the patient's HLA class I and / or HLA class II alleles 2) the T cell epitope is identified as being contained in one or more autoantigens (e.g., autoantigens that have been investigated, proposed, and / or validated as autoantigens involved in MS); 3) T cell epitopes are selected based on predicted binding to the patient's HLA class I and / or class II alleles. 4) One or more tolerogenic test constructs are designed and produced, and T cell epitopes are optionally located in the antigenic units of the constructs as described in this application.

[0129] T cell epitopes are selected in the above method based on their predicted ability to bind to the patient's HLA class I / II alleles, i.e., selected in silico using predictive HLA binding algorithms. After identifying relevant epitopes, the epitopes are ranked according to their ability to bind to the patient's HLA class I / II alleles, and the epitopes predicted to bind best are selected for inclusion in the antigenic unit of the test construct.

[0130] Any suitable HLA binding algorithm may be used, for example one of the following: Available software analysis of peptide-MHC binding (IEDB, NetMHCpan and NetMHCIIpan) can be downloaded or used online from the following websites: www.iedb.org / services.healthtech.dtu.dk / service.php?NetMHCpan-4.0 services.healthtech.dtu.dk / service.php?NetMHCIIpan-3.2

[0131] Off-the-shelf tolerance-inducing constructs The antigenic units of the prefabricated tolerogenic constructs preferably comprise one or more regions of an antigen that contain minimal T cell epitope hotspots, i.e., multiple minimal T cell epitopes (e.g., having a length of 7-15 amino acids) predicted to be presented by various HLA alleles, covering a wide range of subjects, e.g., ethnic groups, or even global populations or world populations.

[0132] By including such hotspots, the construct is maximized in its likelihood of inducing tolerance in a broad range of subjects.

[0133] Further description of antigenic units The T cell epitopes contained in the antigenic units of the constructs of the present disclosure have a length of 7 to about 200 amino acids, and longer T cell epitopes likely contain minimal epitope hotspots.

[0134] In some embodiments, the antigenic unit comprises a T cell epitope having a length of 7 to 150 amino acids, preferably 7 to 100 amino acids, for example, 9 to 100 amino acids, or 15 to 100 amino acids, or 9 to 60 amino acids, or 9 to 30 amino acids, or 15 to 60 amino acids, or 15 to 30 amino acids, or 20 to 75 amino acids, or 25 to 50 amino acids, for example, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids.

[0135] T cell epitopes having a length of about 60 to 200 amino acids may be divided into shorter sequences and may be included in antigenic units separated by linkers as described herein. As an example, a T cell epitope having a length of 150 amino acids may be divided into three sequences of 50 amino acids each and may be included in an antigenic unit with a linker separating the three sequences from each other.

[0136] In some embodiments, the length of one T cell epitope is such that the protein is not folded correctly. For example, Fel d1, the most prominent cat allergen, is a protein formed by two heterodimers, each dimer being composed of two chains, chain 1 containing 70 amino acid residues and chain 2 containing 90 or 92 residues. Including long T cell epitopes of both chains in an antigenic unit may induce an allergic reaction if the protein is folded correctly and two or more IgEs on the subject's mast cells and basophils bind to the antigenic unit of the construct.

[0137] If a longer T cell epitope is included in the antigenic unit, protein folding can be tested in vitro, for example by ELISA, using an antibody against the protein (e.g., cat allergen) to determine whether the antibody binds to the T cell epitope.

[0138] In some embodiments, the T cell epitope has a length suitable for presentation by MHC (major histocompatibility complex). There are two major classes of MHC molecules: MHC class I and MHC class II. The terms MHC class I and MHC class II are used interchangeably herein with HLA class I and HLA class II. HLA (human leukocyte antigen) is the major histocompatibility complex in humans. Thus, in one, the antigenic unit comprises a T cell epitope having a length suitable for specific presentation on MHC class I or MHC class II. In some embodiments, the T cell epitope has a length of 7-11 amino acids for MHC class I presentation. In other embodiments, the T cell epitope sequence has a length of 9-60 amino acids, such as 9-30 amino acids, such as 15-60 amino acids, such as 15-30 amino acids, for MHC class II presentation. In other embodiments, the T cell epitope has a length of 15 amino acids for MHC class II presentation.

[0139] The number of T cell epitopes in an antigenic unit may vary and depends on other elements included in the antigenic unit, such as the length and number of T cell epitope linkers as described in this application.

[0140] In some embodiments, the antigenic unit contains up to 3500 amino acids, for example, 60 to 3500 amino acids, for example, about 80 or about 100 or about 150 amino acids to about 3000 amino acids, for example, about 200 to about 2500 amino acids, for example, about 300 to about 2000 amino acids, or about 400 to about 1500 amino acids, or about 500 to about 1000 amino acids.

[0141] In some embodiments, the antigenic unit comprises 1 to 10 T cell epitopes, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 T cell epitopes, or 11 to 20 T cell epitopes, such as 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 T cell epitopes, or 21 to 30 T cell epitopes, such as 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 108, 1 It comprises 24, 25, 26, 27, 28, 29 or 30 T cell epitopes, or 31 to 40 T cell epitopes, for example, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 T cell epitopes, or 41 to 50 T cell epitopes, for example, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 T cell epitopes. In other embodiments, the antigenic unit comprises 1 to 3 T cell epitopes, such as 1, 2, 3 T cell epitopes, or 1 to 5 T cell epitopes, such as 1, 2, 3, 4, 5 T cell epitopes, or 3 to 6 T cell epitopes, such as 3, 4, 5, 6 T cell epitopes, or 5 to 15 T cell epitopes, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, In some embodiments, the antibody comprises 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 T cell epitopes, or 9 to 19 T cell epitopes, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 T cell epitopes.

[0142] In some embodiments, the T cell epitopes are randomly arranged in the antigenic unit. In other embodiments, one or more of the following methods for arranging the T cell epitopes in the antigenic unit can be used.

[0143] In some embodiments, the T cell epitopes are arranged in order from more antigenic to less antigenic in the direction from a multimerization unit, such as a dimerization unit, to the end of the antigenic unit. Alternatively, particularly where hydrophilicity / hydrophobicity varies widely between T cell epitopes, the most hydrophobic T cell epitopes can be located substantially in the center of the antigenic unit and the most hydrophilic T cell epitopes can be located closest to the multimerization unit, such as the dimerization unit or the end of the antigenic unit.

[0144] In some embodiments, the T cell epitopes are arranged in order from more antigenic to less antigenic in the direction from the multimerization unit to the end of the antigenic unit, or, particularly where hydrophilicity / hydrophobicity varies widely between T cell epitopes, the most hydrophobic T cell epitopes may be located substantially in the center of the antigenic unit and the most hydrophilic T cell epitopes may be located closest to the end of the multimerization unit or antigenic unit.

[0145] In some embodiments, the T cell epitopes are arranged in order from more antigenic to less antigenic in the direction from the dimerization unit to the end of the antigenic unit (see FIG. 1). Alternatively, particularly where hydrophilicity / hydrophobicity varies widely between T cell epitopes, the most hydrophobic T cell epitopes may be located substantially in the center of the antigenic unit and the most hydrophilic T cell epitopes may be located closest to the dimerization unit or end of the antigenic unit.

[0146] Since true positioning in the center of the antigenic unit is only possible if the antigenic unit contains an odd number of T cell epitopes, the term "substantially" in this context refers to an antigenic unit that contains an even number of T cell epitopes, with the most hydrophobic T cell epitopes being located as close to the center as possible.

[0147] As an example, an antigenic unit contains five T cell epitopes arranged as follows: 1-2-3 *-4-5;1,2,3 * , 4 and 5 are different T cell epitopes, - is a T cell epitope linker, * indicates the most hydrophobic T cell epitope located in the center of the antigenic unit.

[0148] In another example, the antigenic unit contains six T cell epitopes arranged as follows: 1-2-3 * - 4-5-6, or alternatively arranged as follows: 1-2-4-3 * -5-6;1,2,3 * , 4, 5, and 6 are T cell epitopes, respectively, and - is a T cell epitope linker; * represents the most hydrophobic T cell epitope, located substantially in the center of the antigenic unit.

[0149] Alternatively, the T cell epitopes may alternate between hydrophilic and hydrophobic T cell epitopes. Optionally, the GC-rich T cell epitopes are arranged to avoid GC clusters. In a preferred embodiment, the GC-rich T cell epitopes are arranged such that there is at least one non-GC-rich T cell epitope between them. In some embodiments, the GC-rich sequences encoding the T cell epitopes are arranged such that there is at least one non-GC-rich T cell sequence between them. A GC-rich sequence is a sequence with a GC content of 60% or more, such as 65% or more, such as 70% or more, such as 75% or more, such as 80% or more.

[0150] When an antigenic unit contains multiple T cell epitopes, the epitopes are preferably separated by T cell epitope linkers. This ensures that each T cell epitope is presented in an optimal manner to the immune system. When an antigenic unit contains n T cell epitopes, it preferably contains n-1 T cell epitope linkers, separating each T cell epitope from one or two other T cell epitopes.

[0151] The T cell epitope linker is designed to be non-immunogenic and is preferably also a flexible linker, which allows the T cell epitopes to be presented to the immune system in an optimal manner, even if the antigenic unit contains multiple T cell epitopes.

[0152] Preferably, the T cell epitope linker is a peptide consisting of 4 to 20 amino acids, for example, 5 to 20 amino acids, or 5 to 15 amino acids, or 8 to 20 amino acids, or 8 to 15 amino acids, for example, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, 10 to 15 amino acids, or 8 to 12 amino acids, for example, 8, 9, 10, 11, or 12 amino acids. In a particular preferred embodiment, the T cell epitope linker consists of 10 amino acids.

[0153] All T cell epitope linkers contained in an antigenic unit are preferably identical. However, if one or more T cell epitopes contain a sequence similar to that of a linker, it is advantageous to replace adjacent T cell epitope linkers with linkers of different sequences. Also, if a T cell epitope / linker junction is predicted to constitute an epitope, it is preferable to use T cell epitope linkers of different sequences.

[0154] In one embodiment, T cell epitope linker is designed to be non-immunogenic.It may be a rigid linker, meaning that it does not allow the two amino acid sequences that it links to move substantially freely relative to each other.Alternatively, it may be a flexible linker, i.e., a linker that allows the two amino acid sequences that it binds to move substantially freely relative to each other.

[0155] Both types of linkers are useful. In one embodiment, the T cell epitope linker is a flexible linker that allows the T cell epitopes to be presented in an optimal manner to T cells, even if the antigenic unit contains multiple T cell epitopes.

[0156] Preferably, the T cell epitope linker is a serine (S) and / or glycine (G) rich linker, i.e. a linker containing several serine and / or several glycine residues. Preferred examples are GGGGSGGGSS (SEQ ID NO: 51), GGGSG (SEQ ID NO: 52), GGGGS (SEQ ID NO: 53), SGSSGS (SEQ ID NO: 54), GGSGG (SEQ ID NO: 55) or several variants thereof, such as GGGGSGGGGS (SEQ ID NO: 56), (GGGGS)m (SEQ ID NOs: 53, and 56 to 59), (GGGS)m (SEQ ID NOs: 60 to 64), (GGGSG)m (SEQ ID NOs: 52, and 65 to 68), or (SGSSGS)m (SEQ ID NOs: 54, and 69 to 72), where m is an integer of 1 to 5, for example 1, 2, 3, 4, or 5, and in a preferred embodiment, m is 2. In other preferred embodiments, the serine and / or glycine rich linker further comprises at least one leucine (L) residue, such as at least one or at least two or at least three leucine residues, for example 1, 2, 3 or 4 leucine residues.

[0157] In some embodiments, the T cell epitope linker comprises or consists of LGGGS (SEQ ID NO: 73), GLGGS (SEQ ID NO: 74), GGLGS (SEQ ID NO: 75), GGGLS (SEQ ID NO: 76), or GGGGL (SEQ ID NO: 77). In other embodiments, the T cell epitope linker comprises or consists of LGGSG (SEQ ID NO: 78), GLGSG (SEQ ID NO: 79), GGLSG (SEQ ID NO: 80), GGGLG (SEQ ID NO: 81), or GGGSL (SEQ ID NO: 82). In yet other embodiments, the T cell epitope linker comprises or consists of LGGSS (SEQ ID NO: 83), GLGSS (SEQ ID NO: 84), or GGLSS (SEQ ID NO: 85).

[0158] In yet another embodiment, the T cell epitope linker comprises or consists of LGLGS (SEQ ID NO: 86), GLGLS (SEQ ID NO: 87), GLLGS (SEQ ID NO: 88), LGGLS (SEQ ID NO: 89), GLGGL (SEQ ID NO: 90), or (GLGGL)m (SEQ ID NOs: 90-94). In yet another embodiment, the T cell epitope linker comprises or consists of LGLSG (SEQ ID NO: 95), GLLSG (SEQ ID NO: 96), GGLSL (SEQ ID NO: 97), GGLLG (SEQ ID NO: 98), or GLGSL (SEQ ID NO: 99). In yet another embodiment, the T cell epitope linker comprises or consists of LGLSS (SEQ ID NO: 100), or GGLLS (SEQ ID NO: 101).

[0159] In another embodiment, the T cell epitope linker is a serine-glycine linker having a length of 10 amino acids and containing one or two leucine residues.

[0160] In some embodiments, the T cell epitope linker comprises or consists of LGGGSGGGGS (SEQ ID NO: 102), GLGGSGGGGS (SEQ ID NO: 103), GGLGSGGGGS (SEQ ID NO: 104), GGGLSGGGGS (SEQ ID NO: 105), or GGGGLGGGGS (SEQ ID NO: 106). In other embodiments, the T cell epitope linker comprises or consists of LGGSGGGGSG (SEQ ID NO: 107), GLGSGGGGSG (SEQ ID NO: 108), GGLSGGGGSG (SEQ ID NO: 109), GGGLGGGGSG (SEQ ID NO: 110), or GGGSLGGGSG (SEQ ID NO: 111). In yet other embodiments, the T cell epitope linker comprises or consists of LGGSSGGGSS (SEQ ID NO: 112), GLGSSGGGSS (SEQ ID NO: 113), GGLSSGGGSS (SEQ ID NO: 114), GGGLSGGGSS (SEQ ID NO: 115), or GGGSLGGGSS (SEQ ID NO: 116).

[0161] In further embodiments, the T cell epitope linker comprises or consists of LGGGSLGGGS (SEQ ID NO: 117), GLGGSGLGGS (SEQ ID NO: 118), GGLGSGGLGS (SEQ ID NO: 119), GGGLSGGGLS (SEQ ID NO: 120), or GGGGLGGGGL (SEQ ID NO: 121). In other embodiments, the T cell epitope linker comprises or consists of LGGSGLGGSG (SEQ ID NO: 122), GLGSGGLGSG (SEQ ID NO: 123), GGLSGGGLSG (SEQ ID NO: 124), GGGLGGGGLG (SEQ ID NO: 125), or GGGSLGGGSL (SEQ ID NO: 126). In yet other embodiments, the T cell epitope linker comprises or consists of LGGSSLGGSS (SEQ ID NO: 127), GLGSSGLGSS (SEQ ID NO: 128), or GGLSSGGLSS (SEQ ID NO: 129).

[0162] In yet other embodiments, the T cell epitope linker comprises or consists of GSGGGA (SEQ ID NO: 130), GSGGGAGSGGGA (SEQ ID NO: 131), GSGGGAGSGGGAGSGGGA (SEQ ID NO: 132), GSGGGAGSGGGAGSGGGAGSGGGA (SEQ ID NO: 133), or GENLYFQSGG (SEQ ID NO: 134). In yet other embodiments, the mobile unit comprises or consists of SGGGSSGGGS (SEQ ID NO: 135), GGGGSGGGGS (SEQ ID NO: 56), SSGGGSSGGG (SEQ ID NO: 136), GGSGGGGSGG (SEQ ID NO: 137), GSGSGSGSGS (SEQ ID NO: 138), GGGSSGGGSG (SEQ ID NO: 139), GGGSSS (SEQ ID NO: 140), GGGSSGGGSSGGGSS (SEQ ID NO: 62), or GLGGLAAA (SEQ ID NO: 141).

[0163] In another embodiment, the T cell epitope linker is a rigid linker. Such a rigid linker may be useful to efficiently separate (larger) antigens and prevent their mutual interference. In one embodiment, the T cell epitope linker comprises or consists of KPEPKPAPAPKP (SEQ ID NO: 142), AEAAAKEAAAKA (SEQ ID NO: 143), (EAAAK)m (SEQ ID NOs: 144-148), PSRLEEELRRRLTEP (SEQ ID NO: 149), or SACYCELS (SEQ ID NO: 150).

[0164] In another embodiment, the T cell epitope linker comprises or consists of the sequence TQKSLSLSPGKGLGGL (SEQ ID NO: 151). In another embodiment, the T cell epitope linker comprises or consists of the sequence SLSLSPGKGLGGL (SEQ ID NO: 152). In another embodiment, the T cell epitope linker comprises or consists of AAY or GPGPG (SEQ ID NO: 153).

[0165] In yet other embodiments, the T cell epitope linker is a GSAT linker, i.e., a linker comprising one or more glycine, serine, alanine, and threonine residues, for example, a linker comprising or consisting of the sequence GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 154), or a SEG linker, i.e., a linker comprising one or more serine, glutamic acid, and glycine residues, for example, a linker comprising or consisting of the sequence GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 155) or ELKTPLGDTTHT (SEQ ID NO: 156).

[0166] In other embodiments, the T cell epitope linker is a cleavable linker, e.g., a linker that contains one or more recognition sites for endopeptidases, e.g., furin, caspases, cathepsins, etc. Cleavable linkers can be introduced to release free functional protein domains (e.g., encoded by larger antigens), which can overcome steric hindrance between such domains or other drawbacks due to interference of such domains, such as reduced biological activity, altered biodistribution, etc.

[0167] Examples of T cell epitope linkers are disclosed in paragraphs

[0098] ,

[0099] and the listed sequences of International Publication No. 2020 / 176797A1 (in particular SEQ ID NOs: 37-65 and SEQ ID NOs: 67-76), which are incorporated herein by reference, and in paragraphs

[0135] to

[0139] of U.S. Patent Application Publication No. 2019 / 0022202A1, which is incorporated herein by reference.

[0168] Allergens The tolerance-inducing constructs described herein are useful for inducing tolerance to a variety of protein allergens, e.g., allergens that can be encoded by nucleic acid sequences contained in the polynucleotides of the constructs of the present disclosure, including protein allergens that undergo post-translational modifications.

[0169] In some embodiments, the allergen is a food allergen. In some embodiments, the allergen is a shellfish allergen. In some embodiments, the allergen is tropomyosin, and in other embodiments, the allergen is arginine kinase, myosin light chain, sarcoplasmic calcium binding protein, troponin C, or triosephosphate isomerase, or actin. In some embodiments, the allergen is Pan b1. In some embodiments, the antigenic unit is the Pan b1 T cell epitope (251-270).

[0170] In some embodiments, the allergen is a cow's milk allergen, hi some embodiments, the cow's milk allergen is Bos d4, Bos d5, Bos d6, Bos d7, Bos d8, Bos d9, Bos d10, Bos d11, or Bos d12.

[0171] In some embodiments, the allergen is an egg allergen, hi some embodiments, the egg allergen is ovomucoid, while in other embodiments, the egg allergen is ovalbumin, ovotransferrin, conalbumin, Gal 3 3, egg liaozyme, or ovomucin.

[0172] One T cell epitope known in the art and that has been studied in the context of egg allergy is OVA(257-264), which has the amino acid sequence SIINFEKL (SEQ ID NO:45).

[0173] In some embodiments, the antigenic unit of the construct according to the present disclosure comprises the T cell epitope OVA(257-264). A pharmaceutical composition comprising said T cell epitope can be used for the treatment of egg allergy.

[0174] In some embodiments, the allergen is a fish allergen. In some embodiments, the fish allergen is parvalbumin. In other embodiments, the fish allergen is enolase, aldolase, or vitellogenin. In some embodiments, the allergen is a fruit allergen. In some embodiments, the fruit allergen is pathogenesis-related protein 10, profilin, nsLTP, thaumatin-like protein, gibberellin regulatory protein, isoflavone reductase-related protein, class 1 chitinase, β-1,3 glucanase, germin-like protein, alkaline serine protease, pathogenesis-related protein 1, actinidin, phytocicutin, quiwellin, major latex protein, cupin, or 2S albumin. In some embodiments, the allergen is a plant allergen. In some embodiments, the plant allergen is pathway development-related protein 10, profilin, nsLTP type 1, nsLTP type 2 protein, osmotin-like protein, isoflavone reductase-like protein, β-fructofuranosidase, PR protein TSI-1, cyclophilin, or FAD-containing oxidase.

[0175] In some embodiments, the allergen is a wheat allergen. In some embodiments, the wheat allergen is Tri a12, Tri a14, Tri a15, Tri a18, Tri a19, Tri a20, Tri a21, Tri a25, Tri a26, Tri a27, Tri a28, Tri a29, Tri a30, Tri a31, Tri a32, Tri a33, Tri a34, Tri a35, Tri a36, Tri a37, or Tri a38. In some embodiments, the allergen is a soybean allergen. In some embodiments, the soybean allergen is Gly m1, Gly m2, Gly m3, Gly m4, Gly m5, Gly m6, Gly m7, or Gly m8. In other embodiments, the soybean allergen is Gly m agglutinin, Gly m Bd28K, Gly m30kD, Gly m CPI, or Gly m TI. In some embodiments, the allergen is a peanut allergen. In some embodiments, the peanut allergen is Ara h1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h12, Ara h13, Ara h14, Ara h15, Ara h16, or Ara h17. In some embodiments, the allergen is a nut or seed allergen. In some embodiments, the allergen is 11S globulin, 7S globulin, 2S globulin, PR10, PR-14 nsLTP, oleosin, or profilin.

[0176] In other embodiments, the food allergen is or is derived from buckwheat, celery, color additives, garlic, gluten, oats, legumes, corn, mustard, poultry, meat, rice, sesame.

[0177] In some embodiments, the allergen is a bee venom allergen. In some embodiments, the bee venom allergen is phospholipase A2, hyaluronidase, acid phosphatase, melittin, allergen C / DPP, CRP / lucarapin, or vitellogenin. In some embodiments, the allergen is a vespid allergen. In some embodiments, the vespid allergen is phospholipase A1, hyaluronidase, protease, antigen 5, DPP IV, or vitellogenin.

[0178] In some embodiments, the allergen is a latex allergen. In some embodiments, the latex allergen is Hev b1, Hev b2, Hev b3, Hev b4, Hev b5, Hev b6, Hev b7, Hev b8, Hev b9, Hev b10, Hev b11, Hev b12, Hev b13, Hev b14, Hev b15.

[0179] In some embodiments, the allergen is a dust mite allergen. In some embodiments, the allergen is a Dermatophagoides mite allergen. In some embodiments, the allergen is a storage dust allergen. In some embodiments, the Dermatophagoides mite allergen is Der p1, Der p2, Der p3, Der p4, Der p5, Der p7, Der p8, Der p10, Der p11, Der p21, or Der p 23. In some embodiments, the antigenic unit is a Der p1 T cell epitope (111-139). In some embodiments, the Dermatophagoides mite allergen is Der f1, Der f2, Der f3, Der f7, Der f8, or Der f10. In some embodiments, the Dermatophagoides allergen is Blot t1, Blot t2, Blot t3, Blot t4, Blot t5, Blot t8, Blot t10, Blot t2, or Blot t21.

[0180] In some embodiments, the allergen is a cockroach allergen. In some embodiments, the cockroach allergen is Bla g1, Bla g2, Bla g3, Bla g4, Bla g5, Bla g6, Bla g7, Bla g8, or Bla g11. In some embodiments, the cockroach allergen is Per a1, Per a2, Per a3, Per a6, Per a7, Per a9, or Per a10.

[0181] In some embodiments, the allergen is a mold allergen. In some embodiments, the mold allergen is an Aspergillus fumigatus allergen. In some embodiments, the Aspergillus fumigatus allergen is Asp f1, Asp f2, Asp f3, Asp f4, Asp f5, Asp f6, Asp f7, Asp f8, Asp f9, Asp f 10, Asp f 11, Asp f 12, Asp f13, Asp f14, Asp f15, Asp f16, Asp f17, Asp f18, Asp f22, Asp f23, Asp f27, Asp f28, Asp f29, or Asp f34.

[0182] In some embodiments, the allergen is a fungal allergen. In some embodiments, the fungal allergen is a Malassezia allergen. In some embodiments, the Malassezia allergen is Mala f1, Mala f2, Mala f3, Mala f4, Mala f5, Mala f6, Mala f7, Mala f8, Mala f9, Mala f10, Mala f11, Mala f12, or Mala f13, or MGL_1204.

[0183] In some embodiments, the allergen is a fur animal allergen. In some embodiments, the allergen is a dog allergen. In some embodiments, the dog allergen is Can f1, Can f2, Can f3, Can f4, Can f5, or Can f6. In some embodiments, the allergen is a horse allergen. In some embodiments, the horse allergen is Ecu c1, Ecu c2, Ecu c3, or Ecu c4. In some embodiments, the allergen is a cat allergen. In some embodiments, the cat allergen is Fel d1, Fel d2, Fel d3, Fel d4, Fel d5, Fel d6, Fel d7, or Fel d8. In some embodiments, the allergen is a laboratory animal allergen. In some embodiments, the allergen is lipocalin, urinary prealbumin, secretoglobulin, or serum albumin.

[0184] In some embodiments, the allergen is a pollen allergen. In some embodiments, the allergen is a grass pollen allergen. In some embodiments, the grass pollen allergen is a timothy grass allergen, an orchard grass allergen, a Kentucky blue grass allergen, a perennial lye allergen, an American burdock allergen, a bahia grass allergen, a Johnson grass allergen, or a corn grass allergen. In some embodiments, the grass pollen allergen is Phl p1, Phl p2, Phl p3, Phl p4, Phl p5, Phl p6, Phl p7, Phl p11, Phl p12, or Phl p13.

[0185] In some embodiments, the allergen is a tree pollen allergen. In some embodiments, the tree pollen allergen is an alder pollen allergen, a birch pollen allergen, a rugose bean pollen allergen, a hazel pollen allergen, a European rugose bean pollen allergen, a chestnut pollen allergen, a European beech pollen allergen, a white oak pollen allergen, an ash pollen allergen, a privet pollen allergen, an olive pollen allergen, a lilac pollen allergen, a cypress pollen allergen, or a cypress pollen allergen. In some embodiments, the tree pollen allergen is Aln g1 or Aln g4, Bet v1, Bet v2, Bet v3, Bet v4, Bet v6 or Bet v7, Car b1, Cor a1, Cor a2, Cor a6, Cor a8, Cor a9, Cor a10, Cor a11, Cor a12, Cor a13, Cor a14, Ost c1, Cas 1, Cas 5, Cas 8 or Cas 9, Fag s1, Que a1, Fra e1, Lig v1, Ole e1, Ole e2, 3 Ole e, 4, Ole e5, Ole e6, Ole e7, Ole e8, Ole e9, Ole e10, Ole e11 or Ole e12, Syr v1, Cha o1, Cha o2, Cry j1, Cry j2, Cup s1, Cup s3, Jun a1, Jun a2, Jun a3, Jun o4, Jun v1, Jun v3, Pla a1, Pla a2 or Pla a3 or Pla or 1, Pla or 2 or Pla or 3. In some embodiments the antigenic unit is the Bet v1 T cell epitope (139-152).

[0186] In some embodiments, the allergen is a weed pollen allergen. In some embodiments, the weed allergen is a ragweed pollen allergen, an artemisia pollen allergen, a sunflower pollen allergen, a feverfew pollen allergen, a pyrethrium pollen allergen, a British plantain pollen allergen, an annual mercury pollen allergen, a gooseberry pollen allergen, a Russian thistle pollen allergen, or an amaranth pollen allergen. In some embodiments, the ragweed pollen allergen is Amb a1, Amb a4, Amb a6, Amb a8, Amb a9, Amb a10, or Amb a11. In some embodiments, the artemisia pollen allergen is Art v1, Art v3, Art v4, Art v5, or Art v6. In some embodiments, the sunflower pollen allergen is Hel a1 or Hel a2. In some embodiments, the pyrethrium pollen allergen is Par j1, Par j2, Par j3 or Par j4. In some embodiments, the English plantain pollen allergen is Pla l1. In some embodiments, the common ivy pollen allergen is Mer a1. In some embodiments, the goosefoot pollen allergen is Che a1, Che a2 or Che a3. In some embodiments, the Russian thistle pollen allergen is Sal k1, Sal k4 or Sal k5. In some embodiments, the amaranth pollen allergen is Ama r2.

[0187] In yet other embodiments, the allergen is selected from environmental allergens such as insects, cockroaches, dust mites, or molds.

[0188] In some embodiments, the allergic disease is allergic rhinitis, asthma, atopic dermatitis, allergic gastroenteropathy, contact dermatitis, drug allergy, or a combination thereof.

[0189] Allergies to drugs affect more than 7% of the general population. The constructs of the present disclosure induce tolerance to the immunogenic epitopes present in such drugs, thus allowing affected patients to continue and benefit from drug treatment.

[0190] Thus, in some embodiments, the allergen is included in the drug with undesirable immunogenicity. In some embodiments, the allergen is factor VIII. In some embodiments, the allergen is insulin. In some embodiments, the allergen is one or more monoclonal antibodies used in therapy.

[0191] autoantigen In other embodiments, the tolerogenic construct contains a T cell epitope contained in an autoallergen involved in an autoimmune disease, allowing antigen-specific downregulation of the part of the immune system responsible for the autoimmune disease, without inhibiting the immune system in general.

[0192] In some embodiments, the autoimmune disease is multiple sclerosis (MS). In some embodiments, the autoantigen is myelin oligodendrocyte glycoprotein (MOG). In other embodiments, the autoantigen is MAG, MOBP, CNPase, S100β, or transaldolase. In some embodiments, the autoantigen is myelin basic protein (MBP). In some embodiments, the autoantigen is myelin proteolipid protein (PLP).

[0193] In the examples, we provide constructs for multiple sclerosis that contain either short (35-55 amino acids) or longer (27-63 amino acids) T cell epitopes derived from myelin oligodendrocyte glycoprotein (MOG). MOG is a member of the immunoglobulin superfamily and is expressed exclusively in the central nervous system. MOG(35-55) can induce autoantibody production and relapsing-remitting neurological disease, causing widespread plaque-like demyelination. Autoantibody responses against MOG(35-55) have been observed in MS patients, and MOG(35-55)-induced experimental autoimmune encephalomyelitis (EAE) has been observed in C57 / BL6 mice and Lewis rats.

[0194] Other MS-associated T cell epitopes known in the art and that have been studied include the following: [Table 1]

[0195] In a preferred embodiment, the antigenic unit of the construct of the present disclosure comprises one or more T cell epitopes selected from the group consisting of MOG(35-55), MOG(27-63), PLP(139-151), PLP(131-159), PLP(178-191), PLP(170-199), MBP(84-104) and MBP(76-112). Pharmaceutical compositions comprising such constructs can be used for the treatment of MS.

[0196] In some embodiments, the autoimmune disease is type 1 diabetes. In some embodiments, the autoantigen is glutamic acid decarboxylase 65 kilodalton isoform (GAD65), an autoantigen involved in type 1 diabetes. In some embodiments, the autoantigen is insulin, IA-2, or ZnT8. In further embodiments, the autoantigen is IGRP, ChgA, IAPP, peripherin, tetraspanin-7, GRP78, urocortin-3, or insulin gene enhancer protein isl-1.

[0197] In some embodiments, the autoimmune disease is celiac disease. In some embodiments, the autoantigen is α-gliadin, γ-gliadin, ω-gliadin, low molecular weight glutenin, high molecular weight glutenin, hordein, secalin, or avenin b. In some embodiments, the antigenic unit comprises the T cell epitope α-gliadin (76-95).

[0198] In some embodiments, the autoimmune disease is rheumatoid arthritis. In some embodiments, the autoantigen is collagen. In some embodiments, the autoantigen is heat shock protein 60 (HSP60). In some embodiments, the autoantigen is Band 3. In some embodiments, the autoantigen is small nuclear ribonucleoprotein D1 (SmD1). In some embodiments, the autoantigen is acetylcholine receptor (AChR). In some embodiments, the autoantigen is myelin protein zero (P0).

[0199] In some embodiments, the autoimmune disease is chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and the autoantigen is neurofascin-155. In other embodiments, the autoimmune disease is Hashimoto's thyroiditis (HT) and the autoantigen is thyroid peroxidase and / or thyroglobulin. In other embodiments, the autoimmune disease is pemphigus foliaceus and the autoantigen is desmosome-associated glycoprotein. In other embodiments, the autoimmune disease is pemphigus vulgaris and the autoantigen is desmoglein 3. In other embodiments, the autoimmune disease is thyroid eye disease (TED) and the autoantigen is calcium binding protein (calsequestrin). In other embodiments, the autoimmune disease is Graves' disease and the autoantigen is thyrotropin receptor. In another embodiment, the autoimmune disease is primary biliary cirrhosis (PBC) and the autoantigen is antimitochondrial antibodies (AMA), antinuclear antibodies (ANA), Rim-like / membrane (RL / M) and / or polynuclear dots (MND). In another embodiment, the autoimmune disease is myasthenia gravis and the autoantigen is acetylcholine receptor. In another embodiment, the autoimmune disease is insulin-resistant diabetes and the autoantigen is insulin receptor. In another embodiment, the autoimmune disease is autoimmune hemolytic anemia and the autoantigen is red blood cells. In another embodiment, the autoimmune disease is rheumatoid arthritis and the autoantigen is citrullinated homocitrullinated protein and / or Fc portion of IgG.

[0200] In other embodiments, the autoimmune disease is psoriasis and the autoantigens are cathelicidin (LL-37), a disintegrin-like and metalloprotease domain-containing thrombospondin type 1 motif-like 5 (ADAMTSL5), phospholipase A2 group IVD (PLA2G4D), heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1), and keratin 17.

[0201] Unit Linker The antigenic unit, such as the dimerization unit, and the multimerization unit are preferably linked by a unit linker. The unit linker may include a restriction site to facilitate construction of the polynucleotide. The unit linker is preferably a GLGGL linker (SEQ ID NO: 90) or a GLSGL linker (SEQ ID NO: 163). In some embodiments, the unit linker comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 204.

[0202] The antigenic unit and the multimerization unit are preferably linked by a unit linker. The unit linker may include a restriction site to facilitate construction of the polynucleotide. The unit linker is preferably a GLGGL linker (SEQ ID NO: 90) or a GLSGL linker (SEQ ID NO: 163). In some embodiments, the unit linker comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 204.

[0203] The antigenic unit and the dimerization unit are preferably linked by a unit linker. The unit linker may contain a restriction site to facilitate construction of the polynucleotide. The unit linker is preferably a GLGGL linker (SEQ ID NO: 90) or a GLSGL linker (SEQ ID NO: 163). In some embodiments, the unit linker comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 204.

[0204] In some embodiments, the unit linker comprises or consists of GGGGS (SEQ ID NO:53), GGGGSGGGGS (SEQ ID NO:56), (GGGGS)m (SEQ ID NO:164), EAAAK (SEQ ID NO:144), (EAAAK)m (SEQ ID NO:165), (EAAAK)mGS (SEQ ID NO:166), or (EAAK)mGS (SEQ ID NO:31), where m is an integer of 1 or greater, GPSRLEEELRRRLTEPG (SEQ ID NO:167), AAY, or HEYGAEALERAG (SEQ ID NO:168).

[0205] Multimerization Units and Dimerization Units The constructs of the present disclosure include multimerization units, such as dimerization units.

[0206] In some embodiments, the constructs of the present disclosure comprise a multimerization unit.

[0207] In some embodiments, the constructs of the present disclosure comprise a dimerization unit.

[0208] The term "multimerization unit" as used herein refers to the sequence of nucleotides or amino acids between the antigenic unit and the targeting unit. In addition to linking the antigenic unit and the targeting unit, the multimerization unit facilitates the multimerization / binding of multiple polypeptides, such as two, three, four or more polypeptides, into a multimeric protein, such as a dimeric protein, a trimeric protein or a tetrameric protein. The multimerization unit also provides flexibility in the multimeric protein to allow optimal binding of the targeting unit to surface molecules on the APC, even if they are located at variable distances. The multimerization unit may be any unit that meets one or more of these requirements.

[0209] Multimerization units that promote multimerization / binding of three or more polypeptides In one embodiment, the multimerization unit is a trimerization unit, such as a collagen-derived trimerization unit, such as a human collagen-derived trimerization domain, such as a human collagen-derived XVIII trimerization domain (see, for example, A. Alvarez-Cienfuegos et al., Sci Rep 6, 28643 (2016)) or a human collagen XV trimerization domain. Thus, in one embodiment, the multimerization unit is a trimerization unit that comprises or consists of a nucleotide sequence having SEQ ID NO: 42, or an amino acid sequence encoded by said nucleotide sequence. In another embodiment, the trimerization unit is the C-terminal domain of T4 fibritin. Thus, in one embodiment, the multimerization unit is a trimerization unit that comprises or consists of an amino acid sequence of SEQ ID NO: 43, or a nucleotide sequence encoding said amino acid sequence.

[0210] In another embodiment, the multimerization unit is a tetramerization unit, such as a domain derived from p53, optionally further comprising a hinge region as described below. Thus, in one embodiment, the multimerization unit is a tetramerization unit comprising or consisting of a nucleic acid sequence having SEQ ID NO: 43, or an amino acid sequence encoded by said nucleic acid sequence, optionally further comprising a hinge region as described below.

[0211] The term "hinge region" in relation to a multimerization unit refers to an amino acid sequence contained in the multimerization unit that contributes to the linking of two or more polypeptides, e.g., three or four polypeptides, i.e., contributes to the formation of a multimeric or dimeric protein, and / or functions as a flexible spacer, allowing the targeting unit of the multimeric protein to simultaneously bind to multiple surface molecules on an APC, even if these surface molecules are located at variable distances.

[0212] Dimerization Unit The term "dimerization unit" as used herein refers to the sequence of nucleotides or amino acids between the antigenic unit and the targeting unit. In addition to linking the antigenic unit and the targeting unit, the dimerization unit facilitates the linking of two polypeptides into a dimerization / dimeric protein. The dimerization unit also provides flexibility in the dimeric protein to allow optimal binding of the targeting unit to surface molecules on APCs, even if they are located at variable distances. The dimerization unit may be any unit that meets one or more of these requirements.

[0213] Thus, in some embodiments, constructs of the present disclosure include a dimerization unit that includes a hinge region. In other embodiments, the dimerization unit includes a hinge region and another domain that promotes dimerization. In yet other embodiments, the dimerization unit includes a hinge region, a dimerization unit linker, and another domain that promotes dimerization, where the dimerization unit linker connects the hinge region to the other domain that promotes dimerization. In other embodiments, the dimerization unit includes a hinge region, a dimerization unit linker, and another domain that promotes dimerization, where the dimerization unit linker connects the hinge region to the other domain that promotes dimerization. Dimerization unit linkers are further described below.

[0214] In some embodiments, the dimerization unit linker is a glycine-serine rich linker, preferably GGGSSGGGSG (SEQ ID NO: 139), i.e., the dimerization unit comprises a glycine-serine rich dimerization unit linker, preferably the dimerization unit linker GGGSSGGGSG (SEQ ID NO: 139). In some embodiments, the dimerization unit linker comprises or consists of the nucleotide sequence set forth in SEQ ID NO:201.

[0215] The term "hinge region" refers to an amino acid sequence contained in a dimerization unit that contributes to the joining of two polypeptides, i.e., the formation of a dimeric protein.

[0216] Moreover, the hinge region functions as a flexible spacer, allowing the two targeting units of the dimeric protein to simultaneously bind to two surface molecules on the APC, even if they are located at variable distances. The hinge region may be derived from Ig, such as from IgG, e.g., IgG1, IgG2 or IgG3. In one embodiment, the hinge region is derived from IgM, and comprises or consists of, e.g., a nucleotide sequence having SEQ ID NO: 47, or an amino acid sequence encoded by said nucleic acid sequence. The hinge region may contribute to dimerization (or multimerization) through the formation of a covalent bond, e.g., a disulfide bridge between cysteines. Thus, in some embodiments, the hinge region has the ability to form one or more covalent bonds. Preferably, the covalent bond is a disulfide bridge.

[0217] In some embodiments, the dimerization unit comprises or consists of hinge exon h1 and hinge exon h4 (human hinge region 1 and human hinge region 4) having an amino acid sequence having at least 80% sequence identity to amino acid sequences 1 to 27 of SEQ ID NO:1.

[0218] In a preferred embodiment, the dimerization unit comprises or consists of a hinge exon h1 and a hinge exon h4 having an amino acid sequence having at least 85% sequence identity to the amino acid sequence 1 to 27 of SEQ ID NO: 1, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, or such as at least 99% sequence identity.

[0219] In a preferred embodiment, the dimerization unit comprises or consists of hinge exon h1 and hinge exon h4 having the amino acid sequence 1 to 27 of SEQ ID NO: 1, or a nucleotide sequence encoding the amino acid sequence.

[0220] In a preferred embodiment, the dimerization unit comprises or consists of hinge exon H1 and hinge exon H4 having the amino acid sequence 1-27 of SEQ ID NO: 1, except that up to 10 amino acids, such as up to 9 amino acids, for example up to 8 amino acids, such as up to 7 amino acids, for example up to 6 amino acids, such as up to 5 amino acids, for example up to 4 amino acids, such as up to 3 amino acids, for example up to 2 amino acids, or such as up to 1 amino acid, are substituted, deleted or inserted.

[0221] In some embodiments, the dimerization unit comprises or consists of the amino acid sequence ELKTPLGDTTHT (SEQ ID NO: 156) and / or EPKSCDTPPPCPRCP (SEQ ID NO: 46), or a nucleotide sequence encoding the amino acid sequence. In some embodiments, the dimerization unit comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 200 or SEQ ID NO: 28.

[0222] In another embodiment, the dimerization unit comprises another domain that promotes dimerization, preferably said another domain is an immunoglobulin domain, such as an immunoglobulin constant domain (C domain), such as a CH1 domain, a CH2 domain or a carboxy-terminal C domain (i.e., a CH3 domain), or a sequence substantially identical to such a C domain or a variant thereof. Preferably, the other domain that promotes dimerization is a carboxy-terminal C domain derived from an IgG. More preferably, the other domain that promotes dimerization is a carboxy-terminal C domain derived from an IgG3.

[0223] In some embodiments, the dimerization unit comprises or consists of a carboxy-terminal C domain derived from IgG3 having an amino acid sequence having at least 80% sequence identity to amino acid sequence 39 to 144 of SEQ ID NO:1, or a nucleotide sequence encoding the amino acid sequence.

[0224] In a preferred embodiment, the dimerization unit comprises or consists of a carboxy-terminal C domain derived from IgG3 having an amino acid sequence having at least 85% sequence identity to amino acid sequence 39 to 144 of SEQ ID NO: 1, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, or such as at least 99% sequence identity.

[0225] In a preferred embodiment, the dimerization unit comprises or consists of a carboxy-terminal C domain derived from IgG3 having the amino acid sequence 39 to 144 of SEQ ID NO:1.

[0226] In one preferred embodiment, the dimerization unit comprises or consists of the amino acid sequence 39-144 of SEQ ID NO:1, except that up to 16 amino acids, e.g., up to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid have been substituted, deleted, or inserted.

[0227] Immunoglobulin domains contribute to dimerization through non-covalent interactions, e.g., hydrophobic interactions. Thus, in some embodiments, immunoglobulin domains have the ability to form dimers through non-covalent interactions. Preferably, the non-covalent interactions are hydrophobic interactions.

[0228] It is preferred that if a dimerization unit comprises a CH3 domain, it does not comprise a CH2 domain, and vice versa.

[0229] In a preferred embodiment, the dimerization unit comprises a hinge exon h1, a hinge exon h4, a dimerization unit linker and a CH3 domain of human IgG3. In a further preferred embodiment, the dimerization unit comprises a polypeptide consisting of a hinge exon h1, a hinge exon h4, a dimerization unit linker and a CH3 domain of human IgG3.

[0230] In another preferred embodiment, the dimerization unit consists of a polypeptide consisting of hinge exon h1, hinge exon h4, a dimerization unit linker and the CH3 domain of human IgG3.

[0231] In some embodiments, the dimerization unit comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:1.

[0232] In a preferred embodiment, the dimerization unit comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, or such as at least 99% sequence identity.

[0233] In a more preferred embodiment, the dimerization unit consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:1, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, or such as at least 99% sequence identity.

[0234] In an even more preferred embodiment, the dimerization unit consists of the amino acid sequence of SEQ ID NO:1, or a nucleotide sequence encoding the amino acid sequence.

[0235] In one preferred embodiment, the dimerization unit comprises the amino acid sequence of SEQ ID NO:1, except that up to 22 amino acids, for example up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid have been substituted, deleted or inserted.

[0236] In one preferred embodiment, the dimerization unit consists of the amino acid sequence of SEQ ID NO:1, except that up to 22 amino acids, for example up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid have been substituted, deleted or inserted.

[0237] In some embodiments, the dimerization unit linker is a glycine-serine rich linker, preferably GGGSSGGGSG (SEQ ID NO: 139), i.e., the dimerization unit comprises a glycine-serine rich dimerization unit linker, preferably the dimerization unit linker GGGSSGGGSG (SEQ ID NO: 139).

[0238] Signal peptide In a preferred embodiment, the construct of the present disclosure is a polynucleotide further comprising a nucleotide sequence encoding a signal peptide. The signal peptide is located either at the N-terminus of the targeting unit or at the C-terminus of the targeting unit, depending on the orientation of the targeting unit in the polypeptide (Figure 1). The signal peptide is designed to allow the secretion of the polypeptide encoded by the nucleic acid contained in the polynucleotide in cells transfected with said polynucleotide.

[0239] Any suitable signal peptide can be used. Examples of suitable peptides are the human Ig VH signal peptide or a signal peptide that is naturally present at the N-terminus of any of the targeting units described herein, such as the human signal peptide of human IL-10 or the human signal peptide of human TGFβ.

[0240] Thus, in some embodiments, the polynucleotide comprises a nucleotide sequence encoding a human IL-10 signal peptide, preferably a nucleotide sequence encoding a human IL-10 targeting unit, hi other embodiments, the polynucleotide comprises a nucleotide sequence encoding a human Ig VH signal peptide, preferably a nucleotide sequence encoding an scFv, e.g., human anti-DEC205.

[0241] In some embodiments the polynucleotide comprises a nucleotide sequence encoding a signal peptide comprising an amino acid sequence having at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, or such as at least 99% sequence identity to the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:48.

[0242] In a preferred embodiment, the polynucleotide comprises a nucleotide sequence encoding a signal peptide comprising the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:48.

[0243] In other embodiments, the polynucleotide comprises a signal peptide consisting of an amino acid sequence which has at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, or at least 99%, to the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:48.

[0244] In other preferred embodiments, the polynucleotide comprises a nucleotide sequence encoding a signal peptide having the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:48.

[0245] In other embodiments, the polynucleotide comprises a nucleotide sequence encoding a signal peptide that comprises or consists of the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:48, except that up to 5 amino acids, such as up to 4 amino acids, such as up to 3 amino acids, such as up to 2 amino acids, or up to 1 amino acid have been substituted, deleted or inserted.

[0246] In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a mouse IL-10 signal peptide, such as the IL-10 signal peptide set forth in SEQ ID NO:50, and preferably comprises a nucleotide sequence encoding a mouse IL-10 targeting unit, such as the mouse IL-10 targeting unit set forth in SEQ ID NO:169.

[0247] In some embodiments, the signal peptide is selected from the group consisting of an IL-10 signal peptide, a SCGB3A2 signal peptide, a VSIG-3 signal peptide, a CTLA4 signal peptide, or a PD-1 signal peptide, e.g., a mouse IL-10 signal peptide, a mouse SCGB3A2 signal peptide, a mouse VSIG-3 signal peptide, a mouse CTLA4 signal peptide, or a mouse PD-1 signal peptide. In some embodiments, the signal peptide comprises a sequence having 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 50, 170, 172, 174, 176, and 178.

[0248] Sequence identity Sequence identity can be determined as follows: a high level of sequence identity indicates the likelihood that the second sequence is derived from the first sequence. Amino acid sequence identity requires identical amino acid sequences between two aligned sequences. Thus, a candidate sequence that shares 70% amino acid identity with a reference sequence requires that, after alignment, 70% of the amino acids in the candidate sequence are identical to the corresponding amino acids in the reference sequence. Identity can be determined with the aid of computer analysis, such as, but not limited to, the ClustalW computer alignment program (Higgins D., Thompson J., Gibson JD, Thompson JD, Higgins DG, Gibson TJ, 1994. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignments through sequence weighting, position-specific gap penalties and weight matrix selection. Nucleic Acids Res. 22: 4673-4680), and the default parameters proposed therein. The program is used with default settings to align the mature (biologically active) portion of the query and the reference polypeptide. The number of perfectly conserved residues is counted and divided by the length of the reference polypeptide, so that any tag or fusion protein sequences that form part of the query sequence are disregarded in the alignment and subsequent determination of sequence identity.

[0249] The ClustalW algorithm can also be used to align nucleotide sequences. Sequence identity can be calculated in a similar manner as shown for amino acid sequences.

[0250] Another preferred mathematical algorithm used for comparing sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the FASTA sequence alignment software package (Pearson WR, Methods Mol Biol, 2000, 132:185-219). Align calculates sequence identity based on a global alignment. Align0 does not penalize gaps at the end of the sequence. When utilizing the ALIGN and Align0 programs to compare amino acid sequences, the BLOSUM50 substitution matrix with gap opening / extension penalties of -12 / -2 is preferably used.

[0251] Amino acid sequence variants can be prepared by introducing appropriate changes into the nucleotide sequence encoding the tolerance-inducing construct or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions into and / or substitutions of residues within the amino acid sequence. The terms substituted / substitution, deleted / deletion and inserted / insertion used herein with respect to amino acid sequences and sequence identity are well known and clear to those skilled in the art. Any combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final construct has the desired characteristics. For example, deletion, insertion or substitution of an amino acid residue can produce a silent change and can result in a functionally equivalent peptide / polypeptide.

[0252] Deliberate amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues, so long as the secondary binding activity of the substance is maintained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0253] Included herein are conservative substitutions, i.e., basic, acidic, acidic such as polar, acidic such as polar, and non-conservative substitutions, i.e., basic from one class of residue to another, or similar substitutions including the inclusion of unnatural amino acids such as ornithine, diaminobutyric acid ornithine, norleucine, ornithine, pyrylalanine, thienylalanine, naphthylalanine, and phenylglycine. Conservative substitutions can be made, for example, within the groups of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), aliphatic amino acids (alanine, valine, leucine, isoleucine), polar amino acids (glutamine, asparagine, serine, threonine), aromatic amino acids (phenylalanine, tryptophan, tyrosine), hydroxyl amino acids (serine, threonine), large amino acids (phenylalanine, tryptophan), and small amino acids (glycine, alanine).

[0254] Substitution with unnatural amino acids is also possible. * and α-disubstituted * Amino acids, N-alkyl amino acids * , lactic acid * , trifluorotyrosine * Halide derivatives of natural amino acids such as p-Cl-phenylalanine * , p-Br-phenylalanine * , pI-phenylalanine * , L-allylglycine *, β-alanine * , L-α-aminobutyric acid * , L-γ-aminobutyric acid * , L-α-aminoisobutyric acid * , L-ε-aminocaproic acid * , 7-aminoheptanoic acid * , L-methionine sulfone * , L-norleucine * , L-Norvaline * , p-nitro-L-phenylalanine * , L-hydroxyproline * , L-thioproline * , 4-methylphenylalanine * , Pentamethylphenylalanine * , L-Phenylalanine (4-amino)#, L-Tyr(methyl) * , L-phenylalanine (4-isopropyl) * , L-Tic (l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) * , L-diaminopropionic acid * and L-phenylalanine (4-benzyl) * Examples of methyl derivatives of phenylalanine (Phe) include:

[0255] In the above paragraph: * indicates the hydrophobicity of the substituted residue, # indicates the hydrophilicity of the substituted residue, and # * indicates the amphipathic nature of the substituted residue. The variant amino acid sequence may contain suitable spacer groups that may be inserted between any two amino acid residues of the sequence, including alkyl groups such as methyl, ethyl or propyl groups, in addition to amino acid spacer groups such as glycine or β-alanine residues. A further form of variation involves the presence of one or more amino acid residues in peptoid form.

[0256] Polynucleotides The tolerance-inducing constructs of the present disclosure may be in the form of a polynucleotide.

[0257] A further aspect of the present disclosure is a polynucleotide which is a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen presenting cell, a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen, or a xenoantigen.

[0258] The polynucleotide may be DNA or RNA, including genomic DNA, cDNA and mRNA, and is either double-stranded or single-stranded. In a preferred embodiment, the construct is a DNA plasmid, i.e., the polynucleotide is DNA.

[0259] A polynucleotide is preferably optimized for use in the species to which it is administered. Thus, for administration to humans, the polynucleotide sequence is preferably human codon optimized.

[0260] Polypeptides and multimeric / dimeric proteins The constructs of the present disclosure may be in the form of a polypeptide encoded by a nucleotide sequence contained in the above polynucleotides.

[0261] A further aspect of the present disclosure is a polypeptide comprising a targeting unit that targets or is capable of targeting an antigen presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen, or a xenoantigen.

[0262] A further aspect of the present disclosure is a polypeptide comprising a targeting unit that targets or is capable of targeting an antigen presenting cell, a multimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen, or a xenoantigen.

[0263] A further aspect of the present disclosure is a polypeptide comprising a targeting unit that targets or is capable of targeting an antigen presenting cell, a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen, or a xenoantigen.

[0264] The polypeptides may be expressed in vitro, for example for the production of a pharmaceutical composition comprising the construct, for the production of a tolerance-inducing construct, or the polypeptides may be expressed in vivo as a result of administration of a polynucleotide to a subject, as described above. Due to the presence of the multimerization / dimerization units, a multimerization / dimer protein is formed when the polypeptides are expressed, i.e., by linking multiple polypeptides via their respective multimerization / dimerization units.

[0265] A further aspect of the disclosure is a multimer, such as a dimeric protein, comprising a plurality of polypeptides, such as two polypeptides, each of which comprises a targeting unit that targets or is capable of targeting an antigen presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen, or a xenoantigen.

[0266] A further aspect of the present disclosure is a multimer comprising a plurality of polypeptides, each of which comprises a targeting unit that targets or is capable of targeting an antigen presenting cell, a multimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen, or a xenoantigen.

[0267] Further aspects of the present disclosure include a plurality of polypeptides, each of which comprises a targeting unit that targets or is capable of targeting an antigen presenting cell, a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen, or a xenoantigen.

[0268] The multimeric protein may be a homomultimer, i.e. a multimeric protein in which multiple polypeptide chains are identical and therefore contain identical units and therefore contain identical antigen sequences, or the multimeric protein may be a heteromultimer comprising multiple polypeptide chains, each polypeptide chain may contain a different antigen sequence in its antigenic unit. The dimeric protein may be a homodimer, i.e. a dimeric protein in which two polypeptide chains are identical and therefore contain identical units and therefore contain antigen sequences, or the dimeric protein may be a heterodimer comprising two polypeptide chains, polypeptide chain 1 contains a different T cell epitope in its antigenic unit than polypeptide chain 2. The latter may be relevant when the number of T cell epitopes to be included in an antigenic unit exceeds the upper size limit of the antigenic unit. Preferably, the multimeric / dimeric protein is a homomultimeric / homodimeric protein.

[0269] vector The polynucleotide sequence of the construct may be a DNA polynucleotide contained in a vector suitable for transfecting a host cell and a polypeptide or multimeric / dimeric protein encoded by the nucleic acid sequence contained in the polynucleotide, i.e. an expression vector, preferably a DNA plasmid. In another embodiment, the vector is suitable for transfecting a host cell and expressing the mRNA encoding the polypeptide / multimeric protein.

[0270] A further aspect of the present disclosure is a vector comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen, or a xenoantigen.

[0271] A further aspect of the present disclosure is a vector comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen presenting cell, a multimer, and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen, or a xenoantigen.

[0272] A further aspect of the present disclosure is a vector comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen presenting cell, a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen, or a xenoantigen.

[0273] Preferably, the vector allows easy exchange of the various units mentioned above, in particular the antigenic unit.

[0274] In some embodiments, the vector may be pALD-CV77, or any other vector that does not contain bacterial nucleotide sequences known to induce an immune response in an undesirable manner when introduced into a subject. The antigenic unit may be an antigenic unit cassette restricted by a convenient restriction enzyme, for example, the 5' site may be incorporated into a nucleotide sequence encoding a GLGGL (SEQ ID NO: 90) and / or GLSGL (SEQ ID NO: 163) unit linker, and the 3' site may be replaced with an SfiI restriction enzyme cassette included in the vector after a stop codon.

[0275] A vector of the present disclosure may be any molecule suitable for carrying foreign nucleic acid sequences, such as DNA or RNA, into a cell where they can be expressed, ie, an expression vector.

[0276] In some embodiments, the vector is a DNA vector, such as a DNA plasmid, or a DNA viral vector, such as a DNA viral vector selected from the group consisting of adenovirus, vaccinia virus, adeno-associated virus, cytomegalovirus, and Sendai virus.

[0277] In other embodiments, the vector is an RNA vector, such as an RNA plasmid, or an RNA viral vector, such as a retroviral vector, e.g., a retroviral vector selected from the group consisting of an alphavirus, a lentivirus, a Moloney murine leukemia virus, and a rhabdovirus.

[0278] In a preferred embodiment, the vector is a DNA vector, more preferably a DNA plasmid. In a preferred embodiment, the vector is a DNA plasmid and the polynucleotide is DNA.

[0279] Plasmids Plasmids are small extra-chromosomal DNA molecules within a cell that are physically separated from chromosomal DNA and can replicate independently. Plasmids are mostly found as small circular double-stranded DNA molecules in bacteria, although plasmids can also be found in archaea and eukaryotes. Artificial plasmids are widely used as vectors in molecular cloning, serving to deliver and ensure high expression of recombinant DNA sequences within a host organism. Plasmids contain several important features, including features for the selection of cells containing the plasmid, such as genes for antibiotic resistance, an origin of replication, a multiple cloning site (MCS), and a promoter to drive expression of the inserted gene of interest.

[0280] Generally, a promoter is a sequence that can attract initiation factors and polymerase to the promoter so that the gene is transcribed. A promoter is located upstream on the DNA, near the transcription start site of a gene. A promoter can be from about 100 to about 1000 base pairs long. The nature of a promoter usually depends on the gene and product of transcription, and the type or class of RNA polymerase that is recruited to the site. When the RNA polymerase reads the DNA of the plasmid, an RNA molecule is transcribed. After processing, when the ribosome translates the mRNA into a protein, the mRNA can be translated many times, thus resulting in many copies of the protein encoded by the gene of interest. Generally, the ribosome facilitates decoding by inducing the binding of complementary tRNA anticodon sequences to the mRNA codons. The tRNA carries specific amino acids that are chained together into a polypeptide as the mRNA passes through and is "read" by the ribosome. Translation proceeds in three stages: initiation, elongation, and termination. Following the translation process, the polypeptide is either folded into an active protein and performs its function within the cell, or is exported from the cell and performs its function elsewhere, sometimes after undergoing a significant number of post-translational modifications.

[0281] If the protein is destined for extracellular export, the signal peptide targets the protein to the endoplasmic reticulum, where it is cleaved and the protein is transported to the cell periphery after translation is terminated.

[0282] The DNA plasmids of the present disclosure are not limited to any particular plasmid, and one of skill in the art will understand that any plasmid having an appropriate backbone can be selected and engineered by methods known in the art to contain the elements and units of the present disclosure.

[0283] host cell A further aspect of the present disclosure is i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen, or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) multimeric proteins, such as dimeric proteins consisting of two polypeptides according to (ii), such as two polypeptides; A host cell comprising:

[0284] A further aspect of the present disclosure is i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen-presenting cell, a multimerization unit and an antigenic unit, the antigenic unit comprising one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A multimeric protein consisting of two polypeptides according to (ii); A host cell comprising:

[0285] A further aspect of the present disclosure is i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen-presenting cell, a dimerization unit and an antigenic unit, the antigenic unit comprising one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A dimeric protein consisting of two polypeptides according to (ii); A host cell comprising:

[0286] Suitable host cells include prokaryotic, yeast, insect or higher eukaryotic cells. In a preferred embodiment, the host cell is a human cell, preferably a cell of a human individual suffering from an immune disorder and in need of prophylactic or therapeutic treatment with a construct of the present disclosure.

[0287] Pharmaceutical Compositions The constructs of the present disclosure may be administered to a subject as a pharmaceutical composition comprising the construct, e.g., a polynucleotide or multimeric / dimeric protein, and a pharma- ceutically acceptable carrier.

[0288] A further aspect of the present disclosure is a pharmaceutical composition comprising a pharma- ceutically acceptable carrier, and i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) multimeric proteins, such as dimeric proteins consisting of multiple polypeptides according to (ii), such as two polypeptides; A pharmaceutical composition comprising: The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0289] A further aspect of the present disclosure is a pharmaceutical composition comprising a pharma- ceutically acceptable carrier, and i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigenic unit, which target or are capable of targeting antigen-presenting cells; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A multimeric protein consisting of a plurality of polypeptides according to (ii); A pharmaceutical composition comprising: The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0290] A further aspect of the present disclosure is a pharmaceutical composition comprising a pharma- ceutically acceptable carrier, and i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigenic unit, which target or are capable of targeting an antigen-presenting cell; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A dimeric protein consisting of two polypeptides according to (ii); A pharmaceutical composition comprising: The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0291] Suitable pharma- ceutically acceptable carriers include, but are not limited to, saline, buffered saline such as PBS, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffer, and combinations thereof.

[0292] In some embodiments, the composition may include one or more adjuvants. Suitable adjuvants include, but are not limited to, dexamethasone, the B subunit of the enterotoxin cholera toxin (CTB), TLR2 ligands, excretory / secretory (ES) products from helminths, rapamycin, or vitamin D3 analogs and aryl hydrocarbon receptor ligands.

[0293] In some particular embodiments, the compositions can include pharma- ceutically acceptable amphiphilic block copolymers comprising blocks of poly(ethylene oxide) and polypropylene oxide.

[0294] As used herein, an "amphiphilic block copolymer" is a linear or branched copolymer that comprises or consists of blocks of poly(ethylene oxide) ("PEO") and blocks of poly(propylene oxide) ("PPO"). Typical examples of useful PEO-PPO amphiphilic block copolymers have the general structures PEO-PPO-PEO (poloxamer), PPO PEO PPO, (PEO PPO-)4ED (poloxamine), and (PPO PEO-)4ED (reverse poloxamine), where "ED" is an ethylenediaminyl group.

[0295] A "poloxamer" is a linear amphiphilic block copolymer composed of one block of poly(propylene oxide) linked to one block of PEO, i.e., one block of poly(ethylene oxide) linked to the formula EOa-POb-EOa, where EO is ethylene oxide, PO is propylene oxide, A is an integer between 2 and 130, and b is an integer between 15 and 67. Poloxamers are conventionally named by using a three-digit identifier, where the first two digits multiplied by 100 provide the approximate molecular weight of the PPO content, and the last digit multiplied by 10 indicates the approximate percentage of PEO content. For example, "poloxamer 188" refers to a polymer containing a PPO block of about 1800 molecular weight (b corresponds to about 31 PPO) and about 80% (w / w) PEO (a corresponds to about 82). However, values ​​are known to vary to some extent, and commercial products such as research grade Lutrol® F68 and clinical grade Kolliphor® P188, both of which are poloxamer 188, have a wide variation in molecular weight (7,680-9,510) according to the manufacturer's datasheets, with the values ​​of a and b provided for these particular products being approximately 79 and 28, respectively. This reflects the heterogeneous nature of the block copolymers, and means that the values ​​of a and b are the averages found in the final formulation.

[0296] "Poloxamine" or "sequential poloxamine" (sold under the name Tetronic®) is an X-shaped block copolymer having four PEO-PPO arms linked to a central ethylenediamine moiety via bonds between the free OH groups in the PEO-PPO-arms and the primary amine groups in the ethylenediamine moiety. Reverse poloxamine is similarly an X-shaped block copolymer having four PPO-PEO arms linked to a central ethylenediamine moiety via bonds between the free OH groups in the PPO-PEO arms and the primary amine groups in the ethylenediamine moiety.

[0297] Preferred amphiphilic block copolymers are poloxamers or poloxamines. Preferred are poloxamers 407 and 188, especially poloxamer 188. Preferred poloxamines are sequential poloxamines of formula (PEO-PPO)4-ED. Particularly preferred poloxamines are those sold under the trademarks Tetronic® 904, 704 and 304, respectively. The characteristics of these poloxamines are as follows: Tetronic® 904 has a total average molecular weight of 6700, a total average weight of PPO units of 4020 and a PEO percentage of about 40%. Tetronic® 704 has a total average molecular weight of 5500, a total average weight of PPO units of 3300 and a PEO percentage of about 40%, and Tetronic® 304 has a total average molecular weight of 1650, a total average weight of PPO units of 990 and a PEO percentage of about 40%.

[0298] In some embodiments, the composition comprises from 0.2% w / v to 20% w / v, e.g., from 0.2% w / v to 18% w / v, from 0.2% w / v to 16% w / v, from 0.2% w / v to 14% w / v, from 0.2% w / v to 12% w / v, from 0.2% w / v to 10% w / v, from 0.2% w / v to 8% w / v, from 0.2% w / v to 6% w / v, The amphiphilic block copolymer may be present in an amount of from 0.2% to 4% w / v, from 0.4% to 18% w / v, from 0.6% to 18% w / v, from 0.8% to 18% w / v, from 1% to 18% w / v, from 2% to 18% w / v, from 1% to 5% w / v, or from 2% to 4% w / v. Particularly preferred are amounts in the range of from 0.5% to 5% w / v. In other embodiments, the composition may comprise an amphiphilic block copolymer in an amount of from 2% to 5% w / v, e.g., about 3% w / v.

[0299] In the case of a pharmaceutical composition comprising a polynucleotide, the composition may further comprise a molecule that facilitates transfection of a cell.

[0300] The pharmaceutical compositions can be formulated in any manner suitable for administration to a subject, such as a patient suffering from or suspected of suffering from an autoimmune disease, an allergic disease, or transplant rejection, such as, for example, a liquid formulation for injection, e.g., intradermal or intramuscular injection.

[0301] In some embodiments, pharmaceutical compositions comprising a polynucleotide described herein, e.g., contained in a vector, may be administered in any manner suitable for administration to a subject, such as by intradermal, intramuscular, or subcutaneous injection, or by mucosal or epithelial applications, such as intranasal or oral administration.

[0302] In a preferred embodiment, the pharmaceutical composition comprises a polynucleotide as described herein, optionally contained in a vector, and is administered by intramuscular or intradermal injection.

[0303] The pharmaceutical composition of the present disclosure typically comprises a polynucleotide in the range of 0.1 μg to 10 mg, for example, about 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.75 μg, 1 μg, 5 μg, 10 μg, 25 μg, 50 μg, 75 μg, or more; for example, 0.1 mg to 10 mg, for example, about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, or 1 mg, or for example, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg. The pharmaceutical composition of the present disclosure typically comprises a polypeptide / dimeric protein in the range of 5 μg to 5 mg.

[0304] The amount of polynucleotide, polypeptide, dimeric protein or multimeric protein may vary depending on whether the pharmaceutical composition is administered for prophylactic or therapeutic treatment, the severity of the immune disease in the affected individual, as well as parameters such as age, weight, sex, medical history and pre-existing conditions.

[0305] Method for producing pharmaceutical composition Suitable methods for preparing a pharmaceutical composition or vaccine according to the present disclosure are disclosed in WO2004 / 076489A1, WO2011 / 161244A1, WO2013 / 092875A1 and WO2017 / 118695A1, which are incorporated herein by reference.

[0306] In one aspect, the present disclosure relates to a method for preparing a pharmaceutical composition comprising a multimeric / dimeric protein or polypeptide as described above by producing the polypeptide in vitro. The in vitro synthesis of polypeptides and proteins can be performed by any suitable method known to the person skilled in the art, such as peptide synthesis or expression of the polypeptide in various expression systems followed by purification.

[0307] In one aspect, the present disclosure relates to a method for preparing a pharmaceutical composition comprising a multimeric protein or polypeptide as described above by producing the polypeptide in vitro. The in vitro synthesis of polypeptides and proteins can be performed by any suitable method known to the person skilled in the art, such as peptide synthesis or expression of the polypeptide in various expression systems followed by purification.

[0308] In one aspect, the present disclosure relates to a method for preparing a pharmaceutical composition comprising the dimeric protein or polypeptide described above by producing the polypeptide in vitro. The in vitro synthesis of polypeptides and proteins can be performed by any suitable method known to the person skilled in the art, such as peptide synthesis or expression of the polypeptide in various expression systems followed by purification.

[0309] Thus, a further aspect of the present disclosure is a method for preparing a multimeric or dimeric protein consisting of a plurality of polypeptides, such as two, three, four or more polypeptides; or a pharmaceutical composition comprising the polypeptides, the method comprising: a) transfecting a cell with a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit; b) culturing the cells; and c) collecting and purifying the multimeric or dimeric protein or the expressed polypeptide from the cell; d) mixing the multimeric or dimeric protein or polypeptide obtained from step c) with a pharma- ceutically acceptable carrier; Includes.

[0310] Thus, a further aspect of the present disclosure is a method for preparing a multimeric protein consisting of a plurality of polypeptides, such as two, three, four or more polypeptides; or a pharmaceutical composition comprising the polypeptides, the method comprising: a) transfecting a cell with a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen-presenting cell, a multimerization unit, and an antigenic unit; b) culturing the cells; and c) collecting and purifying the multimeric or dimeric protein or the expressed polypeptide from the cell; d) mixing the multimeric or dimeric protein or polypeptide obtained from step c) with a pharma- ceutically acceptable carrier; Includes.

[0311] Thus, a further aspect of the present disclosure is a method for preparing a dimeric protein, two polypeptides, or a pharmaceutical composition comprising a polypeptide, the method comprising: a) transfecting a cell with a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit; b) culturing the cells; and c) collecting and purifying the multimeric or dimeric protein or the expressed polypeptide from the cell; d) mixing the multimeric or dimeric protein or polypeptide obtained from step c) with a pharma- ceutically acceptable carrier; Includes.

[0312] In a preferred embodiment, the multimeric protein, dimeric protein or polypeptide obtained from step c) is dissolved in said pharma- ceutically acceptable carrier.

[0313] In a preferred embodiment, the multimeric protein or polypeptide obtained from step c) is dissolved in said pharma- ceutically acceptable carrier.

[0314] In a preferred embodiment, the dimeric protein or polypeptide obtained from step c) is dissolved in said pharma- ceutically acceptable carrier.

[0315] Purification can be carried out according to any appropriate method, such as chromatography, centrifugation, or differential solubility.

[0316] In another aspect, the disclosure provides a method for preparing a pharmaceutical composition comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen, or a xenoantigen, the method comprising: a) preparing a polynucleotide; b) optionally, cloning the polynucleotide into an expression vector; c) mixing the polynucleotide obtained from step a) or the vector obtained from step b) with a pharma- ceutically acceptable carrier; Includes.

[0317] In another aspect, the disclosure provides a method for preparing a pharmaceutical composition comprising a polynucleotide comprising nucleotide sequences encoding a targeting unit that targets or is capable of targeting an antigen presenting cell, a multimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen, or a xenoantigen, the method comprising: a) preparing a polynucleotide; b) optionally, cloning the polynucleotide into an expression vector; c) mixing the polynucleotide obtained from step a) or the vector obtained from step b) with a pharma- ceutically acceptable carrier; Includes.

[0318] In another aspect, the disclosure provides a method for preparing a pharmaceutical composition comprising a polynucleotide comprising nucleotide sequences encoding a targeting unit that targets or is capable of targeting an antigen presenting cell, a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen, or a xenoantigen, the method comprising: a) preparing a polynucleotide; b) optionally, cloning the polynucleotide into an expression vector; c) mixing the polynucleotide obtained from step a) or the vector obtained from step b) with a pharma- ceutically acceptable carrier; Includes.

[0319] The polynucleotides can be prepared by any suitable method known to those skilled in the art. For example, the polynucleotides can be prepared by chemical synthesis using an oligonucleotide synthesizer.

[0320] In particular, nucleotide sequences encoding the targeting unit and / or the dimerization unit may be synthesized individually and then ligated into a vector backbone by ligating a nucleic acid sequence encoding the antigenic unit into the vector to generate the final polynucleotide.

[0321] In one aspect, the present disclosure relates to the use of a construct, polynucleotide, polypeptide, or multimeric protein, such as a dimeric protein, described herein, as a pharmaceutical.

[0322] In one aspect, the disclosure relates to the use of the constructs, polynucleotides, polypeptides, or multimeric proteins described herein as pharmaceuticals.

[0323] In one aspect, the present disclosure relates to the use of the constructs, polynucleotides, polypeptides, or dimeric proteins described herein as pharmaceuticals.

[0324] treatment The constructs or pharmaceutical compositions of the present disclosure may be used to treat autoimmune diseases, allergic diseases or transplant rejection, and treatment may be either prophylactic or therapeutic.

[0325] The constructs / pharmaceutical compositions are administered to induce tolerance in individuals administered with such pharmaceutical compositions. Tolerance is induced by either a single administration or, preferably, multiple administrations appropriately spaced apart.

[0326] In a further aspect, the disclosure provides a method for treating a subject having or suspected of having, or in need of prevention of, an immune disorder selected from the group consisting of an autoimmune disease, an allergic disease, and a transplant rejection, the method comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A multimeric protein, such as a dimeric protein consisting of multiple polypeptides according to (ii); administering to a subject a pharmaceutical composition comprising The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0327] In a further aspect, the disclosure provides a method for treating a subject having or suspected of having, or in need of prevention of, an immune disorder selected from the group consisting of an autoimmune disease, an allergic disease, and a transplant rejection, the method comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A multimeric protein, such as a dimeric protein consisting of multiple polypeptides according to (ii); administering to a subject a pharmaceutical composition comprising The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0328] In a further aspect, the disclosure provides a method for treating a subject having or suspected of having, or in need of prevention of, an immune disorder selected from the group consisting of an autoimmune disease, an allergic disease, and a transplant rejection, the method comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigenic unit, which target or are capable of targeting an antigen-presenting cell; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A dimeric protein, such as a dimeric protein consisting of two polypeptides according to (ii), administering to a subject a pharmaceutical composition comprising The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0329] The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier.

[0330] In some embodiments, a single dose of the pharmaceutical agent is administered to the subject. In some embodiments, multiple doses of the pharmaceutical composition are administered to the subject.

[0331] In yet another aspect, the present disclosure provides a pharmaceutical composition for use in the prophylactic or therapeutic treatment of an immune disease selected from the group consisting of an autoimmune disease, an allergic disease, and a transplant rejection, the pharmaceutical composition comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A multimeric protein, such as a dimeric protein consisting of multiple polypeptides according to (ii); Including, The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0332] In yet another aspect, the present disclosure provides a pharmaceutical composition for use in the prophylactic or therapeutic treatment of an immune disease selected from the group consisting of an autoimmune disease, an allergic disease, and a transplant rejection, the pharmaceutical composition comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigenic unit, which target or are capable of targeting antigen-presenting cells; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A multimeric protein consisting of a plurality of polypeptides according to (ii); Including, The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0333] In yet another aspect, the present disclosure provides a pharmaceutical composition for use in the prophylactic or therapeutic treatment of an immune disease selected from the group consisting of an autoimmune disease, an allergic disease, and a transplant rejection, the pharmaceutical composition comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigenic unit, which targets or is capable of targeting an antigen-presenting cell; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A dimeric protein consisting of two polypeptides according to (ii); Including, The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0334] The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier.

[0335] In some embodiments, a single dose of the pharmaceutical agent is administered to the subject. In some embodiments, multiple doses of the pharmaceutical composition are administered to the subject.

[0336] In a further aspect, the disclosure provides a use of a pharmaceutical composition for the prophylactic or therapeutic treatment of a subject suffering from or suspected of suffering from an immune disease selected from the group consisting of an autoimmune disease, an allergic disease and a transplant rejection, the method comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) multimeric proteins, such as dimeric proteins consisting of multiple polypeptides, such as two polypeptides according to (ii); administering to a subject a pharmaceutical composition comprising The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0337] In a further aspect, the disclosure provides a use of a pharmaceutical composition for the prophylactic or therapeutic treatment of a subject suffering from or suspected of suffering from an immune disease selected from the group consisting of an autoimmune disease, an allergic disease and a transplant rejection, the method comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigenic unit, which target or are capable of targeting antigen-presenting cells; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A multimeric protein consisting of a plurality of polypeptides according to (ii); administering to a subject a pharmaceutical composition comprising The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0338] In a further aspect, the disclosure provides a use of a pharmaceutical composition for the prophylactic or therapeutic treatment of a subject suffering from or suspected of suffering from an immune disease selected from the group consisting of an autoimmune disease, an allergic disease and a transplant rejection, the method comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigenic unit, which targets or is capable of targeting an antigen-presenting cell; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A dimeric protein consisting of two polypeptides according to (ii); administering to a subject a pharmaceutical composition comprising The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0339] The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier.

[0340] In some embodiments, a single dose of the pharmaceutical agent is administered to the subject. In some embodiments, multiple doses of the pharmaceutical composition are administered to the subject.

[0341] In a further aspect, the disclosure provides a use of the pharmaceutical composition for the manufacture of a medicament for the prophylactic or therapeutic treatment of an immune disease selected from the group consisting of an autoimmune disease, an allergic disease and a transplant rejection in a subject suffering from or suspected of suffering from an immune disease or in a subject in need thereof, i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) a multimeric protein, such as a dimeric protein, consisting of multiple polypeptides, such as two polypeptides according to (ii); Including, The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0342] In a further aspect, the disclosure provides a use of the pharmaceutical composition for the manufacture of a medicament for the prophylactic or therapeutic treatment of an immune disease selected from the group consisting of an autoimmune disease, an allergic disease and a transplant rejection in a subject suffering from or suspected of suffering from an immune disease or in a subject in need thereof, i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigenic unit, which target or are capable of targeting antigen-presenting cells; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A multimeric protein consisting of a plurality of polypeptides according to (ii); Including, The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0343] In a further aspect, the disclosure provides a use of the pharmaceutical composition for the manufacture of a medicament for the prophylactic or therapeutic treatment of an immune disease selected from the group consisting of an autoimmune disease, an allergic disease and a transplant rejection in a subject suffering from or suspected of suffering from an immune disease or in a subject in need thereof, i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigenic unit, which targets or is capable of targeting an antigen-presenting cell; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A dimeric protein consisting of two polypeptides according to (ii); Including, The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0344] The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier.

[0345] In some embodiments, a single dose of the pharmaceutical agent is administered to the subject. In some embodiments, multiple doses of the pharmaceutical composition are administered to the subject.

[0346] In a further aspect, the disclosure provides a use of a pharmaceutical composition for the prophylactic or therapeutic treatment of a subject having or suspected of having, or in need of prevention of, an immune disorder selected from the group consisting of an autoimmune disease, an allergic disease, and a transplant rejection, the treatment comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) multimeric proteins, such as dimeric proteins consisting of multiple polypeptides, such as two polypeptides according to (ii); administering to a subject a pharmaceutical composition comprising The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0347] In a further aspect, the disclosure provides a use of a pharmaceutical composition for the prophylactic or therapeutic treatment of a subject having or suspected of having, or in need of prevention of, an immune disorder selected from the group consisting of an autoimmune disease, an allergic disease, and a transplant rejection, the treatment comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigenic unit, which target or are capable of targeting antigen-presenting cells; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A multimeric protein consisting of a plurality of polypeptides according to (ii); administering to a subject a pharmaceutical composition comprising The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0348] In a further aspect, the disclosure provides a use of a pharmaceutical composition for the prophylactic or therapeutic treatment of a subject having or suspected of having, or in need of prevention of, an immune disorder selected from the group consisting of an autoimmune disease, an allergic disease, and a transplant rejection, the treatment comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigenic unit, which targets or is capable of targeting an antigen-presenting cell; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A dimeric protein consisting of two polypeptides according to (ii); administering to a subject a pharmaceutical composition comprising The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0349] The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier.

[0350] In some embodiments, a single dose of the pharmaceutical agent is administered to the subject. In some embodiments, multiple doses of the pharmaceutical composition are administered to the subject.

[0351] In a further aspect, the disclosure provides a medicament for the prophylactic or therapeutic treatment of a subject having or suspected of having, or in need of prevention of, an immune disorder selected from the group consisting of an autoimmune disease, an allergic disease, and a transplant rejection, the medicament comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) a multimeric protein, such as a dimeric protein, consisting of multiple polypeptides, such as two polypeptides according to (ii); Including, The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0352] In a further aspect, the disclosure provides a medicament for the prophylactic or therapeutic treatment of a subject having or suspected of having, or in need of prevention of, an immune disorder selected from the group consisting of an autoimmune disease, an allergic disease, and a transplant rejection, the medicament comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigenic unit, which target or are capable of targeting antigen-presenting cells; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A multimeric protein consisting of a plurality of polypeptides according to (ii); Including, The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0353] In a further aspect, the disclosure provides a medicament for the prophylactic or therapeutic treatment of a subject having or suspected of having, or in need of prevention of, an immune disorder selected from the group consisting of an autoimmune disease, an allergic disease, and a transplant rejection, the medicament comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigenic unit, which targets or is capable of targeting an antigen-presenting cell; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A dimeric protein consisting of two polypeptides according to (ii); Including, The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0354] The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier.

[0355] In some embodiments, a single dose of the pharmaceutical agent is administered to the subject. In some embodiments, multiple doses of the pharmaceutical composition are administered to the subject.

[0356] In a further aspect, the present disclosure provides a method for producing a method for treating a cancer cell comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) a multimeric protein, such as a dimeric protein, consisting of multiple polypeptides, such as two polypeptides according to (ii); and providing a pharmaceutical composition comprising When used in the prophylactic or therapeutic treatment of an immune disease selected from the group consisting of an autoimmune disease, an allergic disease, and a transplant rejection, The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0357] In a further aspect, the present disclosure provides a method for producing a method for treating a cancer cell comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigenic unit, which target or are capable of targeting antigen-presenting cells; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A multimeric protein consisting of a plurality of polypeptides according to (ii); and providing a pharmaceutical composition comprising When used in the prophylactic or therapeutic treatment of an immune disease selected from the group consisting of an autoimmune disease, an allergic disease, and a transplant rejection, The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0358] In a further aspect, the present disclosure provides a method for producing a method for treating a cancer cell comprising: i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigenic unit, which targets or is capable of targeting an antigen-presenting cell; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) A dimeric protein consisting of two polypeptides according to (ii); and providing a pharmaceutical composition comprising When used in the prophylactic or therapeutic treatment of an immune disease selected from the group consisting of an autoimmune disease, an allergic disease, and a transplant rejection, The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen.

[0359] The pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier.

[0360] In some embodiments, a single dose of the pharmaceutical agent is administered to the subject. In some embodiments, multiple doses of the pharmaceutical composition are administered to the subject.

[0361] In a further aspect, the present disclosure provides a method for improving tolerance to an autoantigen, an allergen, an alloantigen or a xenoantigen using a tolerogenic construct according to the present disclosure.

[0362] In a further aspect, the present disclosure provides a method for improving tolerance to an autoantigen, an allergen, an alloantigen or a xenoantigen in a subject, the method comprising administering to the subject a tolerance-inducing construct or pharmaceutical composition according to the present disclosure.

[0363] In some embodiments, a single dose of a tolerance-inducing construct or pharmaceutical composition according to the present disclosure is administered to the subject. In some embodiments, multiple doses of a tolerance-inducing construct or pharmaceutical composition according to the present disclosure are administered to the subject.

[0364] Indicators of successful treatment are known in the art, such as an increase in the level of antigen-specific regulatory T cells, a decrease in the level of antigen-specific effector T cells (and an increase in the level of regulatory T cells), a decrease in the level of effector T cells, a decrease in the level of T cell activation in an ELISPOT when stimulated with the antigenic unit / T cell epitope in the antigenic unit, a decrease in the level of basophil activation in a basophil activation test (BAT), etc.

[0365] Radioallergosorbent testing (RAST) can also be used to compare allergen-specific IgE antibody levels in blood samples from subjects before and after administration of the immunotherapeutic construct, with lower allergen-specific IgE antibody levels indicating successful tolerance induction. EXAMPLES

[0366] Example 1: Design and production of vectors according to the present disclosure All gene sequences described in Examples 1a, 1b and 1c were ordered from GenScript (New Jersey, US) cloned into the expression vector pALD-CV77.

[0367] Example 1a: Design and production of a vector according to the present disclosure for use in the treatment of multiple sclerosis Myelin oligodendrocyte glycoprotein (MOG) is a protein located in the central nervous system. The immunodominant 35-55 epitope of MOG (MOG35-55) is a major target of both cellular and humoral immune responses during multiple sclerosis. MOG(35-55)-induced experimental autoimmune encephalomyelitis (EAE) is the most commonly used animal model of multiple sclerosis (Hunterman, H. et al., 2022).

[0368] DNA vectors were designed containing nucleotide sequences encoding the following units / portions, as described in Table 1: [Table 2]

[0369] DNA vectors VB5003b, VB5004b, VB5005b, VB5006b, VB5012b, VB5046, VB5048, VB5058, VB5059, VB5060, VB5061 and VB5071 encode tolerogenic constructs comprising a targeting unit, a dimerization unit and an antigenic unit as specified in Table 1. The mouse MOG(27-63) antigenic unit contains the T cell epitope MOG(35-55).

[0370] DNA vectors VB5002b and VB5052 encode constructs ("Vaccibodies") containing a human CCL3L1 targeting unit, which is known to target APCs in a pro-inflammatory manner, i.e., constructs containing such targeting units induce enhanced immune responses in subjects to which they are administered, and the compounds are expected to induce activated immune responses accompanied by increased IFN-γ production (see, e.g., WO2011161244A1).

[0371] The DNA vectors VB5001b and VB5051 do not encode a targeting unit or a dimerization unit, but only MOG(27-63) as the antigenic unit, i.e. a single protein / peptide.

[0372] Example 1b: Design and production of vectors according to the present disclosure for use in treating diabetes mellitus Glutamic acid decarboxylase 65 (GAD65) is considered the major autoantigen in diabetes. The peptide GAD65(201-220) gave the greatest T cell response in transgenic mice expressing the major histocompatibility complex class II allele HLA-DQ8 after immunization with GAD65. GAD65(206-220) is the immunodominant T cell epitope of GAD65 in NOD mice (Liu, J. et al., 1999).

[0373] DNA vectors were designed containing nucleotide sequences encoding the following units / portions, as described in Table 2: [Table 3]

[0374] The DNA vector VB5016b encodes a tolerogenic construct comprising a targeting unit, a dimerization unit and an antigenic unit, as described in Table 2.

[0375] The DNA vector VB5015b encodes a construct ("Vaccibody") containing a human CCL3L1 targeting unit, which is known to target APCs in a pro-inflammatory manner, i.e., constructs containing such targeting units induce an inflammatory immune response in subjects to which they are administered, and the compound is expected to induce IFN-γ production (see, e.g., WO2011161244 A1).

[0376] The DNA vector VB5014b does not encode a targeting unit or a dimerization unit, and encodes only GAD65(202-221) as the antigenic unit, i.e., a single protein / peptide.

[0377] The mouse glutamic acid decarboxylase 65 (GAD65) 202-221 (SEQ ID NO: 183) antigenic unit contains the known T cell epitopes GAD65(206-220) and GAD65(202-221).

[0378] Example 1c: Design and production of a vector according to the present disclosure for use in treating shrimp allergy Tropomyosin is a major shellfish allergen. Six major T cell epitopes were identified for tropomyosin from the species Metapenaeus ensis (Met e 1) in a Balb / c mouse model of Met e 1 hypersensitivity. Oral immunotherapy with peptides of the six T cell epitopes effectively reduced the allergic response to shrimp tropomyosin (Wai, CYY et al., 2015).

[0379] DNA vectors were designed containing nucleotide sequences encoding the following units / portions, as described in Table 3: [Table 4]

[0380] The sequences of the antigenic units are further specified in Table 3a below. [Table 5]

[0381] The DNA vectors VB5024, VB5030 and VB5079 encode tolerance-inducing constructs containing the targeting unit, dimerization unit and antigenic unit described in Table 3.

[0382] The Met e 1 (241-260), (210-230), (136-155), (76-95), (46-65), (16-35) antigenic units (SEQ ID NO: 29) contain the GGGGSGGGGS (SEQ ID NO: 56) linker between the T cell epitopes. The Met e 1 (1-274) antigenic unit (SEQ ID NO: 30) contains the complete Met e 1 allergen.

[0383] Example 1d: Design and production of a DNA construct of the present disclosure for use in the treatment of multiple sclerosis All gene sequences of the tested constructs described in Examples 1d and 1e were ordered from Genscript (860 Centennial Ave., Piscataway, NJ 08854, USA) and cloned into the expression vectors pUMVC4a and / or pALD-CV77, except for construct VB5017, which was cloned only into the expression vector pALD-CV77.

[0384] Eight constructs (DNA plasmids) were designed for use in mice, containing different mouse signal peptides and targeting units, identical mouse dimerization units, and antigenic units containing either a short T cell epitope of the mouse multiple sclerosis (MS) autoantigen myelin oligodendrocyte glycoprotein (MOG), i.e., MOG(35-55), or a longer T cell epitope of mouse MOG, i.e., MOG(27-63). [Table 6]

[0385] Example 1e: Design and production of a DNA construct of the present disclosure for use in treating diabetes mellitus One construct (DNA plasmid) was designed for use in mice containing a T cell epitope derived from mouse glutamic acid decarboxylase GAD65 (202-221). GAD65 is an important diabetic autoantigen. The construct further contains the elements (all mouse protein) shown in the table below. [Table 7]

[0386] Example 1f: Design and production of a DNA construct of the present disclosure for use in treating shrimp allergy The construct (DNA plasmid) is designed for use in mice, containing a T cell epitope derived from tropomyosin (Pan b 1 epitope), which is an important shellfish allergen. The construct further contains the elements (all mouse proteins) shown in the table below. [Table 8]

[0387] Example 2a: In vitro characterization of protein expression and secretion of MOG-containing constructs The objective of this study was to characterize the expression and secretion of proteins encoded by MOG-containing DNA vectors following transient transfection of mammalian cells.

[0388] HEK293 cells were obtained from ATCC and transiently transfected with MOG containing DNA vectors (VB5002b, VB5003b, VB5004b, VB5005b, VB5006b and VB5012b). 5 Cells / well were seeded into 24-well tissue culture plates containing 10% FBS growth medium and transfected with 1 μg of each DNA vector using Lipofectamine® 2000 reagent under conditions suggested by the manufacturer (Thermo Fischer Scientific). Transfected cells were maintained at 37° C., 5% CO2 for 5 days and cell supernatants were harvested.

[0389] Expi293F cells were obtained from Thermo Fisher and transiently transfected with MOG(27-63) containing DNA vectors (VB5052, VB5046, VB5048, VB5058, VB5059, VB5060, VB5061 and VB5071). Briefly, Expi293F cells (1.7 × 10 6 Cells / mL, 1 mL) were seeded into 96-well culture plates. Cells were transfected with 0.64 μg / mL plasmid DNA using ExpiFectamine 293 reagent (Thermo Fisher Sci.) and plates were incubated in a humidified CO2 cell incubator (8% CO2, 37 °C) on an orbital shaker (3 mm diameter, 900 rpm). Supernatants were harvested 72 h after transfection.

[0390] Expression and secretion of proteins encoded by MOG-containing vectors was characterized by sandwich ELISA of supernatants using antibodies against MOG (capture antibody, mouse anti-MOG antibody, 0.25 μg / mL, 100 μL / well, sc-73330, Santa Cruz Biotechnology) and hIgG CH3 domain (detection antibody, mouse anti-human IgG Fc secondary antibody, biotin, 0.1 μg / mL, 100 μL / well, 05-4240, Invitrogen). Figures 2A and 2B show that all 14 MOG-containing constructs were expressed and secreted at high levels.

[0391] Secretion of full-length tolerogenic proteins with the MOG antigenic unit and six different targeting units was confirmed by sandwich ELISA of supernatants with antibodies against MOG and targeting units with mouse sequences of IL-10, TGFβ1, SCGB3A2, CTLA-4, PD-1, and CCL1L3, respectively. The results of tolerogenic proteins are shown in Figures 3A-3F, and the proinflammatory control is shown in Figure 3G.

[0392] Detection of IL-10 (VB5005b, VB5006b and VB5058), TGFβ1 (VB5059), SCGB3A2 (VB5060), CTLA-4 (VB5061), PD-1 (VB5071) and CCL3L1 (VB5052) respectively in combination with detection of MOG indicates secretion of high levels of full-length tolerance-inducing proteins and pro-inflammatory controls.

[0393] To evaluate the secretion and expression of the MOG(27-63) antigen-only control peptide encoded by vector VB5051, Expi293F cells were transiently transfected with VB5051 and supernatants were harvested after 3 days. Secretion and expression of VB5051 was evaluated by direct ELISA using the supernatant as coating and detection using an antibody against MOG (mouse anti-MOG antibody, 3.3 μg / mL, 100 μL / well, sc-73330, Santa Cruz Biotechnology). Figure 4 shows that the MOG(27-63) peptide is secreted upon transfection of mammalian cells with VB5051.

[0394] Example 2b: In vitro characterization of protein expression and secretion of constructs of the present disclosure The purpose of this study was to characterize protein expression levels after transient transfection of mammalian cells with DNA plasmids by measuring the presence of protein in cell supernatants by ELISA assay using binding of specific antibodies to the targeting, dimerization, and antigenic units of the protein. HEK293 cells were obtained from ATCC. HEK293 cells were transiently transfected with the constructs. Briefly, 2×10 5Cells / well were seeded into 24-well tissue culture plates containing 10% FBS growth medium and transfected with 1 μg of DNA plasmid using Lipofectamine® 2000 reagent under conditions suggested by the manufacturer (Invitrogen, Thermo Fischer Scientific). The transfected cells were then maintained at 37° C. with 5% CO for 6 days and cell supernatants were harvested for characterization of protein expression.

[0395] ELISA was performed to confirm the amount of protein produced by HEK293 cells and secreted into the cell supernatant. MaxiSorp Nunc-immuno plates were coated with 0.06 μg / mL rabbit anti-human TGFβ1 (orb77216, Biorbyte) as capture antibody in 1×PBS with 100 μL / well and the plates were incubated overnight at 4° C. Microtiter wells were blocked by the addition of 200 μL / well 4% BSA in 1×PBS. 100 μL of cell supernatant from transfected HEK293 cells containing protein expressed from DNA plasmid was added to the plate.

[0396] For the detection antibody, 1 μg / mL of biotinylated mouse anti-human IgG (HP6017, Invitrogen, binds to the CH3 domain of the dimerization unit contained in the construct) was added and incubated. Then, SA-HRP (streptavidin horseradish peroxidase, S2438-250UG, Sigma-Aldrich, 1:3000) was added and incubated. All incubations were performed at 37 °C for 1 h, unless otherwise specified, followed by three washes with PBS-Tween®. Then, 100 μL / well of TMB solution was added, and color development was stopped after 5–15 min by adding 100 μL / well of 1 M HCl. The optical density at 450 nm was measured with an automated plate reader (Thermo Scientific Multiscan GO).

[0397] FIG. 13 shows that construct VB5009 was expressed and secreted as a protein.

[0398] Example 3: In vitro characterization of protein expression and secretion of Met e1-containing constructs The aim of this study was to characterize the protein expression and secretion of the tolerance-inducing protein encoded by the Met e 1-containing DNA vector (see Table 3) following transient transfection of mammalian cells.

[0399] Briefly, Expi293F cells (1.7 × 10 6 Cells / mL, 1 mL, Thermo Fisher Sci.) were seeded into 96-well culture plates. Cells were transfected with 0.64 μg / mL plasmid DNA using ExpiFectamine 293 reagent (Thermo Fisher Sci.) and plates were incubated in a humidified CO2 cell incubator (8% CO2, 37 °C) on an orbital shaker (3 mm diameter, 900 rpm). Supernatants were harvested 72 h after transfection.

[0400] Secreted proteins encoded by Met e 1-containing vectors were characterized by sandwich ELISA of the supernatants using an antibody against the hIgG CH3 domain (capture antibody, 0.5 μg / mL mouse anti-human IgG3 (CH3 domain) antibody, 100 μL / well, MCA878G, BioRad) and an antibody against the hIgG Fc domain (detection antibody, 0.250 μg / mL CaptureSelect™ Biotin Anti-IgG-Fc (Human) Conjugate, 100 μL / well, 7103262100, Invitrogen). The results are shown in FIG. 5.

[0401] As is clear from FIG. 5, all Met e 1 containing tolerance-inducing proteins (VB5024, VB5030 and VB5079) were expressed and secreted.

[0402] Example 4: In vitro characterization of the binding of tolerance-inducing constructs to the DEC205 receptor The aim of this study was to characterize the functional binding of the scFv anti-DEC205 targeting unit of VB5004b (see Table 1) to the recombinant DEC205 receptor. Functional binding of the targeting unit was assessed in an ELISA with supernatants of HEK293 cells transiently transfected with a tolerogenic DNA vector encoding scFv anti-DEC205 as the targeting unit by coating the recombinant DEC205 receptor on an ELISA-plate and using an antibody against the antigenic unit or the dimerization unit as the detection antibody.

[0403] HEK293 cells were obtained from ATCC and transiently transfected with DNA vector VB5004b encoding scFv anti-DEC205. 5 HEK293 cells / well were seeded in 24-well tissue culture plates containing 10% FBS growth medium and transfected with 1 μg of each DNA vector using Lipofectamine® 2000 reagent under conditions suggested by the manufacturer (Invitrogen, Thermo Fischer Scientific). Transfected cells were maintained at 37° C., 5% CO2 for 5 days and cell supernatants were harvested. Secreted proteins encoded by scFv anti-DEC205-containing vectors were characterized by direct ELISA of the supernatants. ELISA plates were coated with 100 μL / well of 5 μg / mL recombinant DEC205 (216-503) (OPCD05072, Aviva Systems Biology) and blocked before adding the supernatants. Binding to the recombinant receptor was detected by antibodies against MOG (100 μL / well, 1 μg / mL mouse anti-MOG antibody, sc-73330, Santa Cruz Biotechnology) or hIgG CH3 domain (100 μL / well, 0.1 μg / mL mouse anti-human IgG Fc secondary antibody, biotin, 05-4240, Invitrogen). The results are shown in Figure 6.

[0404] FIG. 6 confirms binding of scFv anti-DEC205 containing the tolerance-inducing protein VB5004b to the DEC205 receptor and secretion of the full-length tolerance-inducing protein.

[0405] Example 5: In vitro characterization of the binding of tolerogenic constructs to the IL-10 receptor The aim of this study was to characterize the functional binding of the IL-10 targeting unit of VB5006b (see Table 1) to recombinant IL-10 receptor (IL-10R). Functional binding of the targeting unit was assessed in an ELISA with supernatants of HEK293 cells transiently transfected with a DNA vector encoding IL-10 as the targeting unit by coating recombinant IL-10 receptor on an ELISA-plate and using an antibody against the antigenic unit or the dimerization unit as the detection antibody.

[0406] Briefly, HEK293 cells were obtained from ATCC and transiently transfected with IL-10-containing DNA vector VB5006b. 5Cells / well were seeded in 24-well tissue culture plates containing 10% FBS growth medium and transfected with 1 μg of each DNA vector using Lipofectamine® 2000 reagent under conditions suggested by the manufacturer (Invitrogen, Thermo Fischer Scientific). Transfected cells were maintained at 37° C., 5% CO2 for 5 days and cell supernatants were harvested. Secreted proteins encoded by IL-10-containing vectors were characterized by direct ELISA of the supernatants. ELISA plates were coated with 100 μL / well of 2.5 μg / mL recombinant IL-10 receptor and blocked before adding the supernatants. Binding to the recombinant receptor was detected by antibodies against MOG (100 μL / well, 1 μg / mL mouse anti-MOG antibody, sc-73330, Santa Cruz Biotechnology) or hIgG CH3 domain (100 μL / well, 0.1 μg / mL mouse anti-human IgG Fc secondary antibody, biotin, 05-4240, Invitrogen). Results for VB5006b are shown in Figure 7.

[0407] FIG. 7 confirms binding of the IL-10-containing tolerogenic protein VB5006b to the IL-10 receptor and secretion of the full-length tolerogenic protein.

[0408] Example 6: In vitro characterization of size and protein integrity of tolerance-inducing constructs To further characterize the proteins encoded by VB5046, VB5052, VB5058, VB5059, VB5061 and VB5071, Western blot analysis was performed on supernatant samples from transfected i293F cells.

[0409] Briefly, Expi293F cells (1.7 × 10 6Cells / mL, 1 mL) were seeded into 96-well culture plates. Cells were transfected with 0.64 μg / mL plasmid DNA using ExpiFectamine 293 reagent (Thermo Fisher Sci.) and plates were incubated in a humidified CO2 cell incubator (8% CO2, 37 °C) on an orbital shaker (3 mm diameter, 900 rpm). Supernatants were harvested 72 h after transfection.

[0410] Samples were prepared by mixing 14 μL of supernatant from transfected Expi293F cells with 5 μL of 4× Laemmli sample buffer (Bio-Rad) and 1 μL of DTT (Cayman Chemical) or 1 μL of ultrapure water for reducing and non-reducing conditions, respectively (scaling up the total sample volume at the given ratio). Samples (reduced or non-reduced) were heated at 70 °C for 10 min and then loaded onto 4%-20% Criterion TGX Stain-Free precast gels (Bio-Rad). SDS-PAGE was performed using 1× Tris / Glycine / SDS running buffer (Bio-Rad) with Precision Plus Protein All Blue prestained protein standard (Bio-Rad). Proteins were transferred from the gel to EtOH-activated low fluorescence (LF) 0.45 μm PVDF membranes (Bio-Rad) by using a Trans-Blot Turbo semi-dry transfer system (Bio-Rad). PVDF membranes were blocked in EveryBlot buffer (Bio-Rad) for 5 min and probed with mouse anti-MOG (sc-73330, Santa Cruz Biotechnology), rat anti-mouse IL-10 (MAB417, R&D Systems) or goat anti-mouse CTLA-4 (AF467, R&D Systems) to detect MOG, IL-10 or CTLA-4, respectively. Membranes were incubated with fluorochrome-conjugated species-specific secondary antibodies for 1 h at room temperature, then washed and dried. For IL-10 detection in the Dylight 488 channel, membranes were reprobed with Dylight-488 secondary antibody. Membranes were reactivated in ethanol and TBST. Membranes were blocked and incubated with Dylight 488-conjugated secondary antibodies for 1 h at room temperature, then washed and dried. Images were acquired using a ChemiDoc™ MP Imaging System.

[0411] Western blot analysis using anti-MOG antibody shows full-length secretion of the tolerance-inducing proteins and VB5052 (Figure 8A). All proteins except VB5059 appear to have a higher molecular weight than expected based on the protein sequence, which is likely due to post-translational modifications. VB5059 has TGFβ1 as a targeting unit, which is known to reduce its size by 28 kDa upon cleavage into latency-associated peptide and mature TGFβ1. Figure 8B shows that the protein forms a dimer under non-reducing conditions. Figure 8B also shows the presence of monomeric formation of the protein under non-reducing conditions. Membranes probed with anti-IL-10 and anti-CTLA-4 (Figures 8C and 8D, respectively) showed bands corresponding to the same molecular weight as the bands detected in membranes probed with anti-MOG (Figure 8A). Thus, Figures 8C and 8D confirm that MOG and IL-10 or CTLA-4, respectively, are part of the same fusion protein. In Figures 8C and 8D, VB5048 was included as a control to demonstrate that anti-IL-10 and anti-CTLA-4, respectively, do not bind proteins nonspecifically.

[0412] Example 7: Evaluation of tolerance-inducing ability of the DNA vector of the present disclosure The tolerance-inducing potential of VB5004b (listed in Table 1) was assessed in spleens from mice vaccinated with VB5004b and determined by calculating the induced IL-10 / IFN-γ ratio. IL-10 (a non-inflammatory cytokine associated with immune tolerance) and IFN-γ (a marker for the induction of inflammatory immune responses) signals were determined in a two-color FluoroSpot assay after restimulation of splenocytes harvested from mice vaccinated with MOG(35-55) peptide. The IL-10 / IFN-γ ratio indicates the extent to which the immune response induced by the DNA vector is skewed towards a tolerogenic response. The tolerogenic profile was determined by the frequency of induced Foxp3+ cells and the CD4+ expression level after VB5004b vaccination. +This was further assessed by the lack of IFN-γ+ and IL-17+ production from T cells. The results obtained were compared with the responses induced by the proinflammatory control vaccine VB5002b (listed in Table 1) and the tolerance-inducing potential of VB5001b (listed in Table 1).

[0413] Murine vaccination and FluoroSpot The following study design was applied: Female 6-week-old C57BL / 6 mice were obtained from Janvier Labs (France). All animals were housed in the animal facility at Radium Hospital (Oslo, Norway). All animal protocols were approved by the Norwegian Food Safety Authority (Oslo, Norway). Four to five mice / group were used for testing VB5004b, VB5002b and VB5001b, and two mice / group for the negative control (PBS only). VB5002b is included as a proinflammatory version of the MOG(27-63) construct, containing a human CCL3L1 targeting unit known to target APCs and induce inflammatory immune responses, i.e. constructs containing such targeting units will induce a proinflammatory immune response in subjects to which they are administered, and it is anticipated that the compound will induce IFN-γ production in T cells specific for the encoded antigen. The MOG(27-63) peptide alone, a DNA vector encoding VB5001b, was included as a comparison to VB5004b.

[0414] A single dose of 50 μg of VB5004b or control DNA vectors VB5001b and VB5002b dissolved in sterile PBS was administered by intramuscular needle injection into each tibialis anterior muscle (2 × 25 μL, 1000 μg / mL) followed by electroporation with an AgilePulse in vivo electroporation system (BTX, USA).

[0415] Spleens were harvested 7 days after vaccination and mashed in a cell strainer to obtain a single cell suspension. Red blood cells were lysed using ammonium chloride potassium (ACK) lysis buffer. After washing, splenocytes were counted using a NucleoCounter NC-202 (ChemoMetec, Denmark) and 6 × 10 6 Resuspend to a final concentration of 6 x 10 cells / mL and plate on a 96-well IFN-γ / IL-10 dual-color FluoroSpot plate. 5 Splenocytes were then restimulated with 16.67 μg / mL of MOG(35-55) peptide for 44 h before being tested for IFN-γ and IL-10 cytokine production in a dual-color FluoroSpot assay according to the manufacturer's protocol (Mabtech AB, Sweden). Spot-forming cells were measured with an IRIS Fluorospot and ELISpot plate reader (Mabtech AB) and analyzed using Apex software (Mabtech AB). Results were reported as IL-10+ or ​​IFN-γ+ spots / 10 6 Shown are the average numbers of splenocytes in triplicates.

[0416] As can be seen from Figure 9A, VB5004b induced higher levels of IL-10 compared to the levels induced by VB5001b. Furthermore, in contrast to VB5002b, which induces high levels of IFN-γ, low background levels of IFN-γ were detected in response to VB5004b vaccination. Figure 9B shows the ratio of IL-10 / IFN-γ calculated from the values ​​shown in Figure 9A. The ratio of IL-10 / IFN-γ was higher for VB5004b, indicating that VB5004b induced significantly higher levels of the immunosuppressive cytokine IL-10 than the proinflammatory cytokine IFN-γ. In contrast, splenocytes from mice administered VB5002b showed an IL-10 / IFN-γ ratio of approximately 1, indicating that both cytokines were produced at comparable levels after restimulation with MOG(35-55) peptide. To avoid excessive inflammation and ensure the final resolution of inflammation, it is important that the production of pro-inflammatory cytokines such as IFN-γ is regulated by a negative feedback mechanism including the production of anti-inflammatory cytokines such as IL-10 (Sugimoto MA et al. 2016). Therefore, the increased levels of IL-10 observed in response to VB5002b may be explained by such a feedback mechanism to control the induced inflammatory response. A significantly increased IL-10 / IFN-γ ratio was also detected for VB5004b compared to VB5001b, indicating a higher tolerance-inducing potential of VB5004b compared to both VB5002b and VB5001b.

[0417] Flow cytometry Splenocytes were further analyzed by flow cytometry for expression of Foxp3, IFN-γ and IL-17. Foxp3 acts as a master regulator of suppressive pathways in regulatory T cell (Treg) development and function.

[0418] Briefly, after 16 h restimulation with MOG(35-55) peptide, cells were harvested, counted, washed and then incubated with fixable viability dye (FVD) for 10 min in the dark. Cells were then centrifuged and washed before adding a surface staining mixture: anti-CD3 (BUV395), anti-CD4 (BV785) and anti-CD8 and incubated for 30 min at 4°C in the dark. After incubation, cells were centrifuged, cell pellets were resuspended and washed in flow buffer (PBS, 10% FBS and 2 mM EDTA). Foxp3 fixation / permeabilization solution was added to cells and incubated for 60 min at 4°C in the dark. After the centrifugation step, cells were resuspended and washed in permeabilization buffer, then centrifuged and added with an intracellular antibody mixture: anti-Foxp3 (Alexa fluor 700), anti-IFN-γ (APC), anti-IL-17 (Alexa fluor 488) and incubated for 30 min at 4°C in the dark. Cells were then washed and resuspended in flow buffer until acquisition. Correction for fixable viability dyes was performed using single-stained Ultra comp eBeads and ArC-reactive beads. To assess the quality of staining, a gating strategy was devised to exclude fluid misalignment, cell debris, doublets, and dead cells. We further defined T cells based on CD3 expression. CD3+ T cells were examined for CD4 expression, and cells were then analyzed for Foxp3 and cytokine expression. Unstimulated cells from each group were used to assess background levels of cytokine production in the assay. Flow cytometry files (FCS) were exported from FACSDiva™ software and analyzed using FlowJo. Data obtained from flow cytometry analysis was analyzed using GraphPad prism 9.

[0419] As shown in Figure 10A, the percentage of Foxp3+ cells among CD4+ T cells was highly increased in mice vaccinated with VB5004b compared to the levels detected in mice vaccinated with VB5002b, VB5001b, or PBS. Both IFN-γ and IL-17 are proinflammatory cytokines that contribute to the pathogenesis of chronic inflammatory and autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (Gobel K et al. 2018). Thus, a tolerogenic vaccine must reliably induce tolerance without inadvertently sensitizing autoantigen immune responses, for example by inducing proinflammatory cytokines, that could exacerbate autoimmunity. As seen in Figures 10B and 10C, elevated IFN-γ and IL-17 expression was detected in response to VB5002b, whereas the lack of these proinflammatory cytokines was confirmed in mice vaccinated with VB5004b.

[0420] Thus, Example 7 shows that VB5004b, encoding the scFv anti-DEC205 targeting unit and the MOG(27-63) antigenic unit, induces a higher non-inflammatory to inflammatory cytokine ratio (IL-10 / IFN-γ) and shows a lack of inflammatory cytokine production compared to the proinflammatory version VB5002b. Example 7 further shows that VB5004b induces a higher frequency of Foxp3+CD4+ T cells in PBS vaccinated mice compared to VB5001b, VB5002b and background levels, indicating a higher presence of Tregs induced by VB5004b compared to controls.

[0421] Example 8: Evaluation of tolerance-inducing ability of DNA vectors according to the present disclosure The tolerance-inducing potential of VB5012b (listed in Table 1) was determined by calculating the IL-10 / IFN-γ ratio as described in Example 7. The tolerogenic profile of VB5012b, VB5048, VB5058 and VB5046 (all constructs listed in Table 1) was further assessed by the percentage of MOG(38-49)-specific Foxp3+ T cells induced in response to vaccination.

[0422] Murine vaccination and FluoroSpot The following study design was applied: Female 6-week-old C57BL / 6 mice were obtained from Janvier Labs (France). All animals were housed in the animal facility at Radium Hospital (Oslo, Norway). All animal protocols were approved by the Norwegian Food Safety Authority (Oslo, Norway). Five mice / group were used for testing VB5012b, VB5048, VB5006b, VB5046, VB5052 and VB5051, whereas two mice / group were used for the negative control (PBS-vaccinated mice only). As in Example 7, VB5052 was included as a pro-inflammatory MOG(27-63)-encoded construct and VB5051 was included as a MOG(27-63)-encoded antigen alone for comparison with VB5012b, VB5048, VB5006b and VB5046.

[0423] A 50 μg dose of DNA vector VB5012b, VB5048, VB5006b, VB5046, VB5051 or VB5052 was administered intramuscularly twice (days 0 and 4) followed by electroporation, and spleens were harvested 10 days after the first vaccination and mashed in a cell strainer to obtain a single cell suspension as described in Example 7. Splenocytes were restimulated with MOG(35-55) peptide for 44 hours and tested for IFN-γ and IL-10 cytokine production in a dual-color FluoroSpot assay as described in Example 7.

[0424] Figure 11A shows increased levels of IL-10 produced in the spleens of mice vaccinated with VB5012b, along with a lack of IFN-γ production upon restimulation with MOG(35-55) peptide. This is in contrast to VB5052, which shows increased IFN-γ levels in splenocytes from vaccinated mice. As shown in Figure 11B, a significantly higher IL-10 / IFN-γ ratio was detected for VB5012b compared to VB5052, and a trend toward a higher IL-10 / IFN-γ ratio was detected compared to VB5051.

[0425] MOG(38-49)-specific CD4 in splenocytes from mice vaccinated with tetramer (H-2IAb / GWYRSPFSRVVH) + Flow cytometric analysis of T cells The generation of MOG-specific Foxp3+ cells, which represent Treg cells that act to suppress and control MOG-specific inflammatory immune responses, thereby maintaining self-tolerance, was demonstrated by MOG-specific tetramer staining and flow cytometry (CD4 + Foxp3+MOG(38-49)-tet+ cells).

[0426] Briefly, 2 × 10 pooled from each group 6Splenocytes were transferred to 96-well V-bottom plates. Tetramers and Abs were diluted in PBS with 5% FBS before use and protected from light. All steps requiring cell washing were performed with PBS with 5% FBS unless otherwise stated. First, cells were stained with ProT2® MHC class II tetramer specific for MOG(38-49) (1 μg / ml, H-2 IAb-GWYRSPFSRVVH-ProT2® Tetramer PE, 2958, Proimmune) and plates were incubated for 2 hours in a humidified CO2 cell incubator (5% CO2, 37°C). Without washing the cells, Fc receptors were blocked for 5 minutes on ice to prevent non-specific binding of flow cytometry antibodies (Abs) to Fc receptors (0.25 μg / ml, TruStain FcX™ PLUS (anti-mouse CD16 / 32) antibody, 156604, Biolegend). Without washing the cells, the cells were stained with a surface Ab cocktail containing anti-mouse CD8 PE-Cy7 (0.25 μg / ml, clone: ​​53-6.7, 100721, BD Biosciences), anti-mouse CD4 eFluor450 (0.25 μg / ml, clone: ​​GK1.5, 48-0041-82, Thermofischer / eBioscience), and anti-mouse CD25 PerCP-Cy5.5 (0.25 μg / ml, clone: ​​PC61, 102030, Biolegend) for 30 min on ice. The cells were washed twice with PBS. The cells were then stained with fixable viability stain (150 μL per well, 1:8000 dilution in PBS, Fixable Viability Stain 780, 565388, BD biosciences) for 10 min on ice. Cells were washed twice with PBS only and fixed and permeabilized using Foxp3 / transcription factor staining buffer set according to the manufacturer's instructions (200 μL per well, 00-5523-00, Thermofischer / eBioscience).Cells were washed and stained with an intracellular Ab cocktail containing anti-mouse FOXP3 eFluor 660 (0.25 μg / mL, clone: ​​FJK-16s, 50-5773-82, Thermofischer / eBioscience), anti-mouse Ki-67 Alexa Fluor 488 (0.25 μg / mL, clone: ​​11F6, 151204, Biolegend) for 30 min on ice. Cells were washed, resuspended in 150 μL PBS with 5% FBS, and analyzed on a BD FACSymphony™ A3 Cell Analyzer. The following controls were used as a guide to gating the desired populations using FlowJo™ v10.8 Software (BD Life Sciences), an unstained control (=cells did not receive any Abs), and a Fluorescent Minus One (FMO) control (=all but one of the samples stained with fluorophore-labeled Abs to accurately discriminate positive vs. negative signals).

[0427] As shown in Figure 12, a higher percentage of MOG(38-49)-specific Foxp3+ cells can be observed after a two-dose vaccination regimen (day 0 + day 4) with the tolerance-inducing constructs VB5012b, VB5048, VB5006b and VB5046 compared to vaccination with VB5051 or PBS.

[0428] Thus, Example 8 shows that VB5012b, encoding the MARCO ligand SCGB3A2 as the targeting unit and MOG(27-63) as the antigenic unit, induced a higher non-inflammatory to inflammatory cytokine ratio (IL-10 / IFN-γ) compared to the proinflammatory version VB5052. Example 8 further shows that vaccination with the tolerogenic constructs VB5012b, VB5048, VB5006b or VB5046 induced a higher percentage of MOG(38-49)-specific Foxp3+ cells and showed higher levels of induced Tregs compared to vaccination with VB5051 and background levels in PBS vaccinated mice. These results indicate that the DNA vectors VB5048, VB5012b, VB5006b and VB5046 can induce a tolerogenic response.

[0429] array SEQ ID NO:1 [ka]

[0430] SEQ ID NO:2 VB5003 Mouse IgVH signal peptide (1-19), mouse anti-DEC205 scFv (20-265), dimerization unit (266-442), unit linker (443-447), mouse MOG 35-55 (448-468) [ka]

[0431] SEQ ID NO:3 VB5004 Mouse IgVH signal peptide (1-19), mouse anti-DEC205 scFv (20-265), dimerization unit (266-442), unit linker (443-447), mouse MOG 27-63 (448-484) [ka]

[0432] SEQ ID NO:4 VB5012 Mouse SCGB3A2 signal peptide (1-21), mouse SCGB3A2 (22-91), dimerization unit (92-268), unit linker (269-273), mouse MOG 35-55 (274-294) [ka]

[0433] SEQ ID NO:5 VB5013 Mouse VSIG-3 signal peptide (1-22), mouse VSIG-3 (23-428), dimerization unit (429-605), unit linker (606-610), mouse MOG 35-55 (611-631) [ka]

[0434] SEQ ID NO:6 Mouse IgVH signal peptide (1-19) MNFGLRLIFLVLTLKGVQC

[0435] SEQ ID NO:7 VB5016 Mouse IgVH signal peptide (1-19), mouse anti-DEC205 scFv (20-265), dimerization unit (266-442), unit linker (443-447), mouse GAD65 202-221 (448-467) [ka]

[0436] SEQ ID NO:8 VB5005 Mouse IL10 signal peptide (1-18), mouse IL10 (2-178), dimerization unit (179-355), unit linker (356-360), mouse MOG 35-55 (361-381) [ka]

[0437] SEQ ID NO:9 VB5006 Mouse IL10 signal peptide (1-18), mouse IL10 (2-178), dimerization unit (179-355), unit linker (356-360), mouse MOG 27-63 (361-397) [ka]

[0438] SEQ ID NO:10 VB5009 Mouse TGFβ1 signal peptide (1-29), mouse TGFβ1 (30-390), dimerization unit (391-567), unit linker (568-572), mouse MOG 35-55 (573-593) [ka]

[0439] SEQ ID NO:11 VB5017 Mouse CTLA4 signal peptide (1-35), mouse CTLA4 (36-161), dimerization unit (162-338), unit linker (339-343), mouse MOG 35-55 (344-364) [ka]

[0440] SEQ ID NO:12 VB5022 Mouse IgVH signal peptide (1-19), mouse anti-DEC205 scFv (20-265), dimerization unit (266-442), unit linker (443-447), mouse Pan b 1 epitope (448-468) [ka]

[0441] Embodiment 1. i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen-presenting cell (APC), a multimerization unit, such as a dimerization unit, and an antigenic unit; or ii) a polypeptide encoded by the nucleic acid sequence according to (i); or iii) multimeric proteins, such as dimeric proteins consisting of multiple polypeptides according to (ii), such as two polypeptides; A tolerance-inducing construct comprising: The antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen, Tolerance-inducing constructs.

[0442] 2. The tolerance-inducing construct of embodiment 1, wherein said construct comprises a multimerization unit.

[0443] 3. The tolerance-inducing construct of embodiment 1, wherein the construct comprises a dimerization unit and / or the multimeric protein is a dimeric protein.

[0444] 4. A tolerogenic construct according to any of the previous embodiments, wherein said targeting unit is capable of delivering the construct to an antigen presenting cell and interacts with a surface molecule on an APC without activating the APC.

[0445] 5. A tolerance-inducing construct according to any of the previous embodiments, wherein said targeting unit is capable of binding to a surface receptor on an APC.

[0446] 6. The tolerance-inducing construct according to any of the previous embodiments, wherein said targeting unit comprises or consists of a moiety that binds to a receptor selected from the group consisting of TGFβ receptors, such as TGFβR1, TGFβR2, or TGFβR3, IL10R, such as IL-10RA and IL10-RB, IL2R, IL4R, IL6R, IL11R and IL13R, IL27R, IL35R, IL37R, CCR7, CD11b, CD11c, CD103, CD14, CD36, CD205, CD109, VISTA, MARCO, MHCII, MHCII, CD83, SIGLEC, MGL, CD80, CD86, Clec9A, Clec12A, Clec12B, DCIR2, Langerin, MR, DC-Sign, Treml4, Dectin-1, PDL1, PDL2, and HVEM.

[0447] 7. A tolerance-inducing construct according to any of the previous embodiments, wherein said moiety is selected from a natural ligand, a synthetic ligand and an antibody or part thereof, such as an scFv.

[0448] 8. A tolerogenic construct according to any of the previous embodiments, wherein said moiety is an antibody or part thereof, e.g. an scFv, having specificity for said receptor, and binding to said receptor results in the antigen being presented in an anti-inflammatory tolerogenic manner.

[0449] 9. A tolerogenic construct according to any of the previous embodiments, wherein said moiety is a synthetic ligand having specificity for said receptor, and binding to said receptor results in the antigen being presented in an anti-inflammatory tolerogenic manner.

[0450] 10. A tolerance-inducing construct according to any of the previous embodiments, wherein said moiety is a natural ligand.

[0451] 11. The tolerance-inducing construct of any of the previous embodiments, wherein said natural ligand is selected from the group consisting of TGFβ, such as TGFβ1, TGFβ2 or TGFβ3, IL-10, IL2, IL4, IL6, IL11, IL13, IL27, IL35, IL37, GM-CSF, FLT3L, CCL19, CCL21, ICAM-1 (intercellular adhesion molecule 1, also known as CD54), keratin, VSIG-3, SCGB3A2, CTLA-4, such as the extracellular domain of CTLA-4, PD-1, such as the extracellular domain of PD-1, and BTLA.

[0452] 12. A tolerance-inducing construct according to any of the previous embodiments, wherein said natural ligand is selected from the group consisting of TGFβ, IL-10, SCGB3A2, CTLA-4, such as the extracellular domain of CTLA-4.

[0453] 13. A tolerance-inducing construct according to any of the previous embodiments, wherein said natural ligand is the extracellular domain of BTLA.

[0454] 14. A tolerance-inducing construct according to any of the previous embodiments, wherein said targeting unit consists of or comprises IL-10 or TGFβ, preferably human IL-10 or human TGFβ.

[0455] 15. A tolerance-inducing construct according to any of the previous embodiments, wherein the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity with a sequence of human TGFβ, such as an amino acid sequence selected from SEQ ID NOs: 205-207.

[0456] 16. A tolerance-inducing construct according to any of the previous embodiments, wherein said targeting unit comprises or consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence of human TGFβ, such as an amino acid sequence selected from SEQ ID NOs: 205-207, such as at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, or 100% sequence identity.

[0457] 17. The tolerance-inducing construct according to any of the preceding embodiments, wherein the targeting unit comprises or consists of an amino acid sequence of human TGFβ, such as an amino acid sequence selected from SEQ ID NOs: 205-207, except for up to 22 amino acids, e.g. up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid being substituted, deleted, or inserted.

[0458] 18. A tolerance-inducing construct according to any of the previous embodiments, wherein the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to that of human IL-10, such as the amino acid sequence of SEQ ID NO: 211.

[0459] 19. A tolerance-inducing construct according to any of the previous embodiments, wherein said targeting unit comprises or consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence of human IL-10, such as the amino acid sequence of SEQ ID NO: 211, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, such as at least 99%, or 100% sequence identity.

[0460] 20. The tolerance-inducing construct according to any of the previous embodiments, wherein the targeting unit comprises or consists of the amino acid sequence of human IL-10, such as the amino acid sequence of SEQ ID NO: 211, except that up to 22 amino acids, e.g. up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid have been substituted, deleted, or inserted.

[0461] 21. A tolerance-inducing construct according to any of the previous embodiments, wherein said targeting unit is or comprises SCGB3A2 or VSIG-3, such as human VSIG-3 or human SCGB3A2.

[0462] 22. A tolerance-inducing construct according to any of the previous embodiments, wherein the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to that of human VSIG-3, such as the amino acid sequence of SEQ ID NO: 215.

[0463] 23. A tolerance-inducing construct according to any of the previous embodiments, wherein the targeting unit comprises or consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence of human VSIG-3, such as the amino acid sequence of SEQ ID NO: 215, such as an amino acid sequence having at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99%, or such as 100% sequence identity.

[0464] 24. A tolerance-inducing construct according to any of the previous embodiments, wherein the targeting unit comprises or consists of an amino acid sequence of human VSIG-3, such as an amino acid sequence selected from SEQ ID NO: 215, except that up to 22 amino acids, e.g. up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid have been substituted, deleted, or inserted.

[0465] 25. A tolerance-inducing construct according to any of the previous embodiments, wherein the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to that of human SCGB3A2, such as the amino acid sequence of SEQ ID NO: 213.

[0466] 26. A tolerance-inducing construct according to any of the previous embodiments, wherein the targeting unit comprises or consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence of human SCGB3A2, such as the amino acid sequence of SEQ ID NO: 213, such as an amino acid sequence having at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, or such as 100% sequence identity.

[0467] 27. A tolerance-inducing construct according to any of the previous embodiments, wherein the targeting unit comprises or consists of an amino acid sequence of human SCGB3A2, such as an amino acid sequence selected from SEQ ID NO: 213, except that up to 22 amino acids, e.g. up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid have been substituted, deleted, or inserted.

[0468] 28. A tolerance-inducing construct according to any of the previous embodiments, wherein said targeting unit consists of or comprises an antibody or part thereof having specificity for CD205.

[0469] 29. A tolerance-inducing construct according to any of the previous embodiments, wherein said targeting unit consists of or comprises an scFv with specificity for CD205, such as an anti-DEC205 scFv, such as an scFv comprising or consisting of SEQ ID NO: 49.

[0470] 30. A tolerance-inducing construct according to any of the previous embodiments, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, such as one T cell epitope of an autoantigen or multiple T cell epitopes of an autoantigen.

[0471] 31. A tolerogenic construct according to any of the previous embodiments, wherein the multiple T cell epitopes are the same autoantigen or multiple different autoantigens.

[0472] 32. A tolerogenic construct according to any of the previous embodiments, wherein the antigenic unit comprises one or more linkers separating the T cell epitopes.

[0473] 33. A tolerogenic construct according to any of the previous embodiments, wherein the antigenic unit comprises multiple T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen, and the T cell epitopes are separated by a linker.

[0474] 34. A tolerogenic construct according to any of the previous embodiments, wherein all T cell epitopes except the terminal T cell epitope are located in the subunits, and each subunit comprises or consists of a T cell epitope and a linker.

[0475] 35. A tolerogenic construct according to any of the previous embodiments, wherein all T cell epitopes except the N-terminal T cell epitope are located in the subunits, and each subunit comprises or consists of a T cell epitope and a linker.

[0476] 36. A tolerogenic construct according to any of the previous embodiments, wherein all T cell epitopes except the C-terminal T cell epitope are located in the subunits, and each subunit comprises or consists of a T cell epitope and a linker.

[0477] 37. A tolerance-inducing construct according to any of the previous embodiments, wherein the antigenic unit comprises n antigens and n-1 subunits, each subunit comprising a T cell epitope of an autoantigen, an allergen, an alloantigen or a xenoantigen, and a linker, and further comprising a terminal T cell epitope.

[0478] 38. The tolerance-inducing construct according to any of the preceding embodiments, wherein n is an integer between 1 and 50, for example, between 3 and 50, between 15 and 40, between 10 and 30, between 10 and 25, between 10 and 20, between 15 and 30, between 15 and 25, between 15 and 20.

[0479] 39. A tolerance-inducing construct according to any of the previous embodiments, wherein the linker is non-immunogenic.

[0480] 40. A tolerance-inducing construct according to any of the previous embodiments, wherein the linker is a rigid linker or a flexible linker.

[0481] 41. The tolerance-inducing construct according to any of the previous embodiments, wherein the antigenic unit comprises one or more T cell epitopes of myelin basic protein (MBP), such as one T cell epitope of MBP or multiple T cell epitopes of MBP, myelin oligodendrocyte glycoprotein (MOG), such as one T cell epitope of MOG or multiple T cell epitopes of MOG, or proteolipid protein (PLP), such as one T cell epitope of PLP or multiple T cell epitopes of PLP.

[0482] 42. A tolerance-inducing construct according to any of the preceding embodiments, wherein the antigenic unit comprises one or more T cell epitopes of MOG, for example comprising or consisting of a sequence selected from the group consisting of SEQ ID NOs: 180-182.

[0483] 43. A tolerance-inducing construct according to any of the previous embodiments, wherein the antigenic unit comprises one or more T cell epitopes of an allergen, such as one T cell epitope of an allergen or multiple T cell epitopes of an allergen.

[0484] 44. A tolerance-inducing construct according to any of the previous embodiments, wherein the multiple T cell epitopes are the same allergen or multiple different allergens.

[0485] 45. A tolerogenic construct according to any of the previous embodiments, wherein the antigenic unit comprises one or more T cell epitopes of Fel d1, such as one T cell epitope of Fel d1 or multiple T cell epitopes of Fel d1.

[0486] 46. ​​A tolerogenic construct according to any of the previous embodiments, wherein the antigenic unit comprises one or more T cell epitopes of Fel d4 and / or Fel d7, such as one or more T cell epitopes of Fel d4 and / or one or more T cell epitopes of Fel d7.

[0487] 47. A tolerance-inducing construct according to any of the previous embodiments, wherein the antigenic unit comprises one or more T cell epitopes of an alloantigen / xenoantigen, such as one T cell epitope of an alloantigen / xenoantigen or multiple T cell epitopes of an alloantigen / xenoantigen.

[0488] 48. A tolerogenic construct according to any of the previous embodiments, wherein the multiple T cell epitopes are the same allo / xenoantigen or multiple different allo / xenoantigens.

[0489] 49. A tolerance-inducing construct according to any of the preceding embodiments, wherein the or T cell epitopes have a length of between 7 and about 200 amino acids.

[0490] 50. The tolerance-inducing construct according to any of the preceding embodiments, wherein the one or T cell epitopes have a length of 7 to 150 amino acids, preferably 7 to 100 amino acids, for example 9 to 100 amino acids, or 15 to 100 amino acids, or 9 to 60 amino acids, or 9 to 30 amino acids, or 15 to 60 amino acids, or 15 to 30 amino acids, or 20 to 75 amino acids, or 25 to 50 amino acids.

[0491] 51. The tolerance-inducing construct of any of the previous embodiments, wherein the one or the T cell epitopes have a length of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids.

[0492] 52. A tolerogenic construct according to any of the previous embodiments, wherein the antigenic unit comprises one or more T cell epitopes having a length of 7 to 11 amino acids.

[0493] 53. A tolerance-inducing construct according to any of the previous embodiments, wherein the antigenic unit comprises one or more T cell epitopes having a length of 9 to 60 amino acids, such as 9 to 30 amino acids, for example 15 to 60 amino acids, such as 15 to 30 amino acids.

[0494] 54. A tolerogenic construct according to any of the previous embodiments, wherein the antigenic unit comprises one or more T cell epitopes having a length of 15 amino acids.

[0495] 55. The tolerance-inducing construct according to any of the preceding embodiments, wherein the antigenic unit comprises up to 3500 amino acids, such as 60 to 3500 amino acids, such as about 80 or about 100 or about 150 amino acids to about 3000 amino acids, such as about 200 to about 2500 amino acids, such as about 300 to about 2000 amino acids, or about 400 to about 1500 amino acids, or about 500 to about 1000 amino acids.

[0496] 56. The antigenic unit comprises 1 to 10 T cell epitopes, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 T cell epitopes, or 11 to 20 T cell epitopes, for example 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 T cell epitopes, or 21 to 30 T cell epitopes, for example 21, 22, 23, 24, 25, 26, 27, 2. The tolerance-inducing construct according to any of the preceding embodiments, comprising 28, 29 or 30 T cell epitopes, or 31-40 T cell epitopes, such as 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 T cell epitopes, or 41-50 T cell epitopes, such as 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 T cell epitopes.

[0497] 57. The antigenic unit comprises 1 to 3 T cell epitopes, for example 1, 2, 3, or 1 to 5 T cell epitopes, for example 1, 2, 3, 4, 5, or 3 to 6 T cell epitopes, for example 3, 4, 5, 6, or 5 to 15 T cell epitopes, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 T cell epitopes. or 7 to 17 T cell epitopes, for example 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 T cell epitopes, for example 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 T cell epitopes.

[0498] 58. A tolerogenic construct according to any of the previous embodiments, wherein the one or more T cell epitopes are randomly arranged in the antigenic unit.

[0499] 59. A tolerogenic construct according to any of the previous embodiments, wherein the one or more T cell epitopes are arranged in order from more antigenic to less antigenic in the direction from the multimerization unit to the end of the antigenic unit.

[0500] 60. A tolerogenic construct according to any of the previous embodiments, wherein the most hydrophobic T cell epitope is located substantially in the center of the antigenic unit and the most hydrophilic T cell epitope is located closest to the end of the multimerization unit or antigenic unit.

[0501] 61. A tolerogenic construct according to any of the previous embodiments, wherein the T cell epitopes are arranged in order from more antigenic to less antigenic in the direction from the dimerization unit to the end of the antigenic unit.

[0502] 62. A tolerogenic construct according to any of the previous embodiments, wherein the most hydrophobic T cell epitope is located substantially in the center of the antigenic unit and the most hydrophilic T cell epitope is located closest to the end of the dimerization unit or antigenic unit.

[0503] 63. A tolerance-inducing construct according to any of the previous embodiments, wherein the one or more T cell epitopes alternate between hydrophilic and hydrophobic T cell epitopes.

[0504] 64. A tolerogenic construct according to any of the previous embodiments, wherein the GC-rich T cell epitopes are positioned such that there is at least one non-GC-rich T cell epitope between them.

[0505] 65. A tolerance-inducing construct according to any of the previous embodiments, wherein the multiple T cell epitopes are separated by a T cell epitope linker.

[0506] 66. A tolerance-inducing construct according to any of the previous embodiments, wherein the antigenic unit comprises n T cell epitopes and n-1 T cell epitope linkers.

[0507] 67. The tolerance-inducing construct of any of the previous embodiments, wherein the T cell epitope linker is non-immunogenic.

[0508] 68. The tolerance-inducing construct of any of the previous embodiments, wherein the T cell epitope linker is a flexible linker.

[0509] 69. The tolerance-inducing construct according to any of the previous embodiments, wherein the T cell epitope linker is a peptide consisting of 4 to 20 amino acids, such as 5 to 20 amino acids, or 5 to 15 amino acids, or 8 to 20 amino acids, or 8 to 15 amino acids, such as 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, 10 to 15 amino acids, or 8 to 12 amino acids, such as 8, 9, 10, 11, or 12 amino acids.

[0510] 70. The tolerance-inducing construct of any of the previous embodiments, wherein the T cell epitope linker consists of 10 amino acids.

[0511] 71. A tolerance-inducing construct according to any of the previous embodiments, wherein all T cell epitope linkers contained in the antigenic unit are preferably identical.

[0512] 72. A tolerance-inducing construct according to any of the previous embodiments, wherein the T cell epitope linker is a serine (S) and / or glycine (G) rich linker.

[0513] 73. The tolerance-inducing construct of any of the previous embodiments, wherein the serine and / or glycine-rich T cell epitope linker further comprises at least one leucine (L) residue, such as at least 1 or at least 2 or at least 3 or at least 4 leucine residues.

[0514] 74. A tolerance-inducing construct according to any of the previous embodiments, wherein the T cell epitope linker is a serine-glycine linker having a length of 10 amino acids and containing one or two leucine residues.

[0515] 75. A tolerance-inducing construct according to any of the previous embodiments, wherein the T cell epitope linker is a GSAT linker or a SEG linker.

[0516] 76. The tolerance-inducing construct of any of the previous embodiments, wherein the T cell epitope linker comprises or consists of GLGGL (SEQ ID NO: 90).

[0517] 77. The tolerance-inducing construct of any of the previous embodiments, wherein the T cell epitope linker is a cleavable linker.

[0518] 78. A tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is a food allergen, such as a shellfish allergen, such as tropomyosin, arginine kinase, myosin light chain, sarcoplasmic calcium binding protein, troponin C, triosephosphate isomerase or actin.

[0519] 79. A tolerogenic construct according to any of the previous embodiments, wherein said allergen is Pan b 1 and optionally said antigenic unit consists of or comprises the Pan b 1 T-cell epitope (251-270).

[0520] 80. The tolerance-inducing construct of any of the previous embodiments, wherein the cow's milk allergen is such as Bos d 4, Bos d 5, Bos d 6, Bos d 7, Bos d 8, Bos d 9, Bos d 10, Bos d 11 or Bos d 12.

[0521] 81. A tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is an egg allergen, such as ovomucoid, ovalbumin, ovotransferrin, conalbumin, Gal 3, egg liaozyme or ovomucin.

[0522] 82. The tolerance-inducing construct of any of the previous embodiments, wherein the allergen is OVA(257-264) comprising the amino acid sequence SIINFEKL (sequence number 45).

[0523] 83. A tolerogenic construct according to any of the previous embodiments, wherein the antigenic unit comprises the T cell epitope OVA(257-264).

[0524] 84. A tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is a fish allergen, such as parvalbumin, enolase, aldolase or vitellogenin.

[0525] 85. A tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is a fruit allergen, such as pathogenesis-related protein 10, profilin, nsLTP, thaumatin-like protein, gibberellin regulatory protein, isoflavone reductase-related protein, class 1 chitinase, β-1,3 glucanase, germin-like protein, alkaline serine protease, pathogenesis-related protein 1, actinidin, phytocictatin, quiwelin, major latex protein, cupin or 2S albumin.

[0526] 86. A tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is a plant allergen, such as pathway formation-related protein 10, profilin, nsLTP type 1, nsLTP type 2 protein, osmolyte-like protein, isoflavone reductase-like protein, β-fructofuranosidase, PR protein TSI-1, cyclophilin or FAD-containing oxidase.

[0527] 87. The tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is a wheat allergen, such as Tri a 12, Tri a 14, Tri a 15, Tri a 18, Tri a 19, Tri a 20, Tri a 21, Tri a 25, Tri a 26, Tri a 27, Tri a 28, Tri a 29, Tri a 30, Tri a 31, Tri a 32, Tri a 33, Tri a 34, Tri a 35, Tri a 36, ​​Tri a 37 or Tri a 38.

[0528] 88. A tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is a soy allergen, such as Gly ml, Gly m2, Gly m3, Gly m4, Gly m5, Gly m6, Gly m7 or Gly m8, Gly m agglutinin, Gly m Bd28K, Gly m 30 kD, Gly m CPI or Gly m TI.

[0529] 89. The tolerance-inducing construct of any of the previous embodiments, wherein the allergen is a peanut allergen, such as Ara h1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h 10, Ara h 11, Ara h 12, Ara h 13, Ara h 14, Ara h 15, Ara h 16, or Ara h 17.

[0530] 90. A tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is a nut or seed allergen.

[0531] 91. A tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is an 11S globulin, a 7S globulin, a 2S globulin, PR10, PR-14 nsLTP, oleosin or profilin.

[0532] 92. A tolerance-inducing construct according to any of the previous embodiments, wherein the food allergen is buckwheat, celery, colour additives, garlic, gluten, oats, legumes, corn, mustard, poultry, meat, rice or sesame.

[0533] 93. A tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is a bee venom allergen, such as phospholipase A2, hyaluronidase, acid phosphatase, melittin, allergen C / DPP, CRP / lucarapin or vitellogenin.

[0534] 94. A tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is a vesicoallergen, such as phospholipase A1, hyaluronidase, protease, antigen 5, DPP IV or vitellogenin.

[0535] 95. The tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is a latex allergen, such as Hev b 1, Hev b 2, Hev b 3, Hev b 4, Hev b 5, Hev b 6, Hev b 7, Hev b 8, Hev b 9, Hev b 10, Hev b 11, Hev b 12, Hev b 13, Hev b 14 or Hev b 15.

[0536] 96. A tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is a dust mite allergen, such as a Dermatophagoides mite allergen or a reservoir dust mite allergen, such as Der p1, Der p2, Der p3, Der p4, Der p5, Der p7, Der p8, Der p10, Der p11, Der p21, Der p 23, Der f1, Der f2, Der f3, Der f7, Der f8, Der f10, Blot t1, Blot t2, Blot t3, Blot t4, Blot t5, Blot t8, Blot t10, Blot t2, Blot t21.

[0537] 97. A tolerogenic construct according to any of the previous embodiments, wherein the antigenic unit consists of or comprises the Der p 1 T-cell epitope (111-139).

[0538] 98. The tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is a cockroach allergen, such as Bla g 1, Bla g 2, Bla g 3, Bla g 4, Bla g 5, Bla g 6, Bla g 7, Bla g 8, Bla g 11, Per a 1, Per a 2, Per a 3, Per a 6, Per a 7, Per a 9 or Per a 10.

[0539] 99. The tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is a mold allergen, such as an Aspergillus fumigatus allergen, such as Asp f1, Asp f2, Asp f3, Asp f4, Asp f5, Asp f6, Asp f7, Asp f8, Asp f9, Asp f 10, Asp f 11, Asp f 12, Asp f13, Asp f14, Asp f15, Asp f16, Asp f17, Asp f18, Asp f22, Asp f23, Asp f27, Asp f28, Asp f29 or Asp f34.

[0540] 100. The tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is a fungal allergen, such as a Malassezia allergen, such as Mala f1, Mala f2, Mala f3, Mala f4, Mala f5, Mala f6, Mala f7, Mala f8, Mala f9, Mala f10, Mala f11, Mala f12 or Mala f13 or MGL_1204.

[0541] 101. A tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is a dog allergen, such as Can f1, Can f2, Can f3, Can f4, Can f5 or Can f6, or the allergen is a horse allergen, such as Ecu c1, Ecu c2, Ecu c3 or Ecu c4, or the allergen is a cat allergen, such as Fel d1, Fel d2, Fel d3, Fel d4, Fel d5, Fel d6, Fel d7, or Fel d8, or the allergen is an experimental animal allergen, such as lipocalin, urinary prealbumin, secretoglobulin or serum albumin.

[0542] 102. The tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is a pollen allergen, such as a grass pollen allergen, such as timothy grass allergen, orchard grass allergen, Kentucky blue grass allergen, perennial rye allergen, Japanese holly allergen, bahia grass allergen, johnson grass allergen, oak leaf allergen, Phl p1, Phl p2, Phl p3, Phl p4, Phl p5, Phl p6, Phl p7, Phl p11, Phl p12 or Phl p13.

[0543] 103. The allergen is a tree pollen allergen, an alder pollen allergen, a birch pollen allergen, a humb pollen allergen, a hazel pollen allergen, a European humb pollen allergen, a chestnut pollen allergen, a European beech pollen allergen, a white oak pollen allergen, an ash pollen allergen, a privet pollen allergen, an olive pollen allergen, a lilac pollen allergen, a cypress pollen allergen or a cypress pollen allergen, such as Aln g1 or Aln g4, Bet v1, Bet v2, Bet v3, Bet v4, Bet v6 or Bet v7, Car b1, Cor a1, Cor a2, Cor a6, Cor a8, Cor a9, Cor a10, Cor a11, Cor a12, Cor a13, Cor a14, Ost c1, Cas 1, Cas 5, Cas 8 or Cas 9, Fag s1, Que a1, Fra e1, Lig v1, Ole e1, Ole e2, 3 Ole e, 4, Ole e5, Ole e6, Ole e7, Ole e8, Ole e9, Ole e10, Ole e11 or Ole e12, Syr v1, Cha o1, Cha o2, Cry j1, Cry j2, Cup s1, Cup s3, Jun a1, Jun a2, Jun a3, Jun o4, Jun v1, Jun v3, Pla a1, Pla a2 or Pla a3 or Pla or 1, Pla or2 or Pla or3.

[0544] 104. A tolerogenic construct according to any of the previous embodiments, wherein the antigenic unit consists of or comprises the Bet v1 T-cell epitope (139-152).

[0545] 105. The allergen is a weed pollen allergen, such as ragweed pollen allergen, artemisia pollen allergen, sunflower pollen allergen, feverfew pollen allergen, pyrethrium pollen allergen, English plantain pollen allergen, common ivy pollen allergen, goosefoot pollen allergen, Russian thistle pollen allergen or amaranth pollen allergen, such as Amb a1, Amb a4, Amb a6, Amb a8, Amb a9, Amb a10 or Amb a11, Art v1, Art v3, Art v4, Art v5 or Art v6, Hel a1 or Hel a2, Par j1, Par j2, Par j3 or Par j4, Pla l1, Mer a1, Che a1, Che a2 or Che a3, Sal k1, Sal k4 or Sal k5, or Amaranth pollen allergen. The tolerance-inducing construct of any of the preceding embodiments, which is r2.

[0546] 106. A tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is selected from environmental allergens such as insects, cockroaches, dust mites or molds.

[0547] 107. A tolerance-inducing construct according to any of the previous embodiments, wherein the allergen causes an allergic disease selected from allergic rhinitis, asthma, atopic dermatitis, allergic gastroenteropathy, contact dermatitis, drug allergy, or a combination thereof.

[0548] 108. A tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is comprised in a drug having undesirable immunogenicity.

[0549] 109. A tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is Factor VIII.

[0550] 110. A tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is insulin.

[0551] 111. A tolerance-inducing construct according to any of the previous embodiments, wherein the allergen is one or more monoclonal antibodies used in therapy.

[0552] 112. A tolerance-inducing construct according to any of the previous embodiments, wherein the construct comprises a T cell epitope contained in an autoallergen whose autoantigen is involved in an autoimmune disease.

[0553] 113. The tolerance-inducing construct of any of the previous embodiments, wherein the autoantigen is involved in multiple sclerosis (MS).

[0554] 114. The tolerance-inducing construct of any of the previous embodiments, wherein the autoantigen is myelin oligodendrocyte glycoprotein (MOG), MAG, MOBP, CNPase, S100β, transaldolase, myelin basic protein (MBP), myelin proteolipid protein (PLP).

[0555] 115. The tolerance-inducing construct of any of the previous embodiments, wherein the antigenic unit comprises one or more T-cell epitopes selected from the group consisting of MOG(35-55), MOG(27-63), PLP(139-151), PLP(131-159), PLP(178-191), PLP(170-199), MBP(84-104) and MBP(76-112).

[0556] 116. The tolerance-inducing construct according to any of the previous embodiments, wherein the antigenic unit comprises one or more T cell epitopes selected from the group consisting of SEQ ID NOs: 185-190 or 192-197.

[0557] 117. The tolerance-inducing construct of any of the previous embodiments, wherein the autoantigen is involved in type 1 diabetes.

[0558] 118. A tolerance-inducing construct according to any of the previous embodiments, wherein the autoantigen is glutamic acid decarboxylase 65-kilodalton isoform (GAD65), insulin, IA-2 or ZnT8, IGRP, ChgA, IAPP, peripherin, tetraspanin-7, GRP78, urocortin-3 or insulin gene enhancer protein isl-1.

[0559] 119. A tolerance-inducing construct according to any of the previous embodiments, wherein the autoantigen is involved in celiac disease.

[0560] 120. A tolerance-inducing construct according to any of the previous embodiments, wherein the autoantigen is α-gliadin, γ-gliadin, ω-gliadin, low molecular weight glutenin, high molecular weight glutenin, hordein, secalin or avenin b.

[0561] 121. A tolerance-inducing construct according to any of the previous embodiments, wherein the antigenic unit comprises the T cell epitope alpha-gliadin (76-95).

[0562] 122. The tolerance-inducing construct of any of the previous embodiments, wherein the autoantigen is involved in rheumatoid arthritis.

[0563] 123. A tolerance-inducing construct according to any of the previous embodiments, wherein the autoantigen is collagen, heat shock protein 60 (HSP60), band 3, small nuclear ribonucleoprotein D1 (SmD1), acetylcholine receptor (AChR) or myelin protein zero (P0).

[0564] 124. A tolerance-inducing construct according to any of the previous embodiments, wherein the autoantigen is involved in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), and the autoantigen is neurofascin 155.

[0565] 125. The tolerance-inducing construct of any of the previous embodiments, wherein the autoantigen is involved in Hashimoto's thyroiditis (HT), and the autoantigen is thyroid peroxidase and / or thyroglobulin.

[0566] 126. A tolerance-inducing construct according to any of the previous embodiments, wherein the autoantigen is involved in pemphigus foliaceus, and the autoantigen is desmosome-associated glycoprotein.

[0567] 127. A tolerance-inducing construct according to any of the previous embodiments, wherein the autoantigen is involved in pemphigus vulgaris and the autoantigen is desmoglein 3.

[0568] 128. The tolerance-inducing construct of any of the previous embodiments, wherein the autoantigen is involved in thyroid eye disease (TED), and the autoantigen is calcium-binding protein (calsequestrin).

[0569] 129. A tolerance-inducing construct according to any of the previous embodiments, wherein the autoantigen is involved in Graves' disease, and the autoantigen is the thyroid-stimulating hormone receptor.

[0570] 130. A tolerance-inducing construct according to any of the previous embodiments, wherein the autoantigen is involved in primary biliary cirrhosis (PBC) and the autoantigen is antimitochondrial antibody (AMA), antinuclear antibody (ANA), Rim-like / membrane (RL / M) and / or multiple nuclear dots (MND).

[0571] 131. A tolerance-inducing construct according to any of the previous embodiments, wherein the autoantigen is involved in myasthenia gravis, and the autoantigen is an acetylcholine receptor.

[0572] 132. A tolerance-inducing construct according to any of the previous embodiments, wherein the autoantigen is involved in insulin-resistant diabetes, and wherein the autoantigen is the insulin receptor.

[0573] 133. A tolerance-inducing construct according to any of the previous embodiments, wherein the autoantigen is involved in autoimmune hemolytic anemia, and the autoantigen is an erythrocyte.

[0574] 134. A tolerance-inducing construct according to any of the previous embodiments, wherein the autoantigen is involved in rheumatoid arthritis, the autoantigen being a citrullinated homocitrullinated protein and the Fc portion of IgG.

[0575] 135. A tolerogenic construct according to any of the previous embodiments, wherein the antigenic unit and the multimerization unit are linked by a unit linker.

[0576] 136. A tolerogenic construct according to any of the previous embodiments, wherein the antigenic unit and the dimerization unit are linked by a unit linker.

[0577] 137. A tolerance-inducing construct according to any of the previous embodiments, wherein the unit linker comprises a restriction site.

[0578] 138. A tolerance-inducing construct according to any of the previous embodiments, wherein the unit linker is a GLGGL linker (SEQ ID NO: 90) or a GLSGL linker (SEQ ID NO: 163).

[0579] 139. The tolerance-inducing construct according to any of the previous embodiments, wherein the unit linker comprises or consists of GGGGS (SEQ ID NO: 53), GGGGSGGGGS (SEQ ID NO: 56), (GGGGS)m (SEQ ID NO: 164), EAAAK (SEQ ID NO: 144), (EAAAK)m (SEQ ID NO: 165), (EAAAK)mGS (SEQ ID NO: 166), or (EAAK)mGS (SEQ ID NO: 31), where m is an integer equal to or greater than 1, GPSRLEEELRRRLTEPG (SEQ ID NO: 167), AAY or HEYGAEALERAG (SEQ ID NO: 168).

[0580] 140. A tolerance-inducing construct according to any of the previous embodiments, wherein the construct comprises a multimerization unit.

[0581] 141. A tolerance-inducing construct according to any of the previous embodiments, wherein the construct comprises a dimerization unit.

[0582] 142. A tolerance-inducing construct according to any of the previous embodiments, wherein the multimerization unit is a trimerization unit or a tetramerization unit.

[0583] 143. A tolerance-inducing construct according to any of the previous embodiments, wherein the multimerization unit is a trimerization unit, such as a collagen-derived trimerization unit, such as a human collagen-derived trimerization domain, such as a human collagen-derived XVIII trimerization domain or a human collagen XV trimerization domain.

[0584] 144. A tolerance-inducing construct according to any of the previous embodiments, wherein the multimerization unit is a trimerization unit comprising or consisting of a nucleotide sequence having SEQ ID NO: 42, or an amino acid sequence encoded by the nucleotide sequence.

[0585] 145. A tolerance-inducing construct according to any of the previous embodiments, wherein the multimerization unit is the C-terminal domain of T4 fibritin.

[0586] 146. A tolerance-inducing construct according to any of the previous embodiments, wherein the multimerization unit is a trimerization unit comprising or consisting of the amino acid sequence of SEQ ID NO: 43, or a nucleotide sequence encoding said amino acid sequence.

[0587] 147. A tolerance-inducing construct according to any of the previous embodiments, wherein the multimerization unit is a tetramerization unit, such as a domain derived from p53.

[0588] 148. A tolerance-inducing construct according to any of the previous embodiments, wherein the multimerization unit is a tetramerization unit comprising or consisting of a nucleic acid sequence having SEQ ID NO: 44, or an amino acid sequence encoded by said nucleic acid sequence.

[0589] 149. A tolerance-inducing construct according to any of the previous embodiments, wherein the dimerization unit comprises a hinge region.

[0590] 150. A tolerance-inducing construct according to any of the previous embodiments, wherein the dimerization unit comprises a hinge region and another domain that promotes dimerization.

[0591] 151. A tolerance-inducing construct according to any of the previous embodiments, wherein the dimerization unit comprises a hinge region, a dimerization unit linker, and another domain that promotes dimerization, and the dimerization unit linker connects the hinge region to the other domain that promotes dimerization.

[0592] 152. A tolerance-inducing construct according to any of the previous embodiments, wherein the dimerization unit comprises a hinge region, a dimerization unit linker, and another domain that promotes dimerization, and the dimerization unit linker connects the hinge region to the other domain that promotes dimerization.

[0593] 153. The tolerance-inducing construct according to any of the previous embodiments, wherein the dimerization unit linker is a glycine-serine rich linker, such as GGGSSGGGSG (sequence number 139).

[0594] 154. A tolerance-inducing construct according to any of the previous embodiments, wherein the hinge region is from Ig, such as from IgG, e.g. IgG1, IgG2 or IgG3.

[0595] 155. A tolerance-inducing construct according to any of the previous embodiments, wherein the hinge region is derived from IgM and optionally comprises or consists of a nucleotide sequence having SEQ ID NO: 47, or an amino acid sequence encoded by said nucleic acid sequence.

[0596] 156. A tolerance-inducing construct according to any of the previous embodiments, wherein the hinge region has the ability to form one or more covalent bonds, such as one or more disulfide bridges.

[0597] 157. A tolerance-inducing construct according to any of the previous embodiments, wherein the dimerization unit comprises or consists of hinge exon h1 and hinge exon h4 (human hinge region 1 and human hinge region 4) having an amino acid sequence having at least 80% sequence identity to amino acid sequence 1 to 27 of SEQ ID NO:1.

[0598] 158. A tolerance-inducing construct according to any of the previous embodiments, wherein the dimerization unit comprises or consists of a hinge exon h1 and a hinge exon h4 having an amino acid sequence having at least 85% sequence identity to the amino acid sequence 1 to 27 of SEQ ID NO: 1, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, or such as at least 99% sequence identity.

[0599] 159. A tolerance-inducing construct according to any of the previous embodiments, wherein the dimerization unit comprises or consists of hinge exon H1 and hinge exon H4 having amino acid sequences 1 to 27 of SEQ ID NO:1, or a nucleotide sequence encoding the amino acid sequence.

[0600] 160. In a preferred embodiment, the tolerance-inducing construct according to any of the previous embodiments, wherein the dimerization unit comprises or consists of hinge exon H1 and hinge exon H4 having the amino acid sequence 1 to 27 of SEQ ID NO: 1, except that at most 10 amino acids, such as at most 9 amino acids, for example at most 8 amino acids, such as at most 7 amino acids, for example at most 6 amino acids, such as at most 5 amino acids, for example at most 4 amino acids, such as at most 3 amino acids, for example at most 2 amino acids, or such as at most 1 amino acid, etc. are substituted, deleted or inserted.

[0601] 161. A tolerance-inducing construct according to any of the preceding embodiments, wherein the dimerization unit comprises another domain that promotes dimerization, such as an immunoglobulin domain, e.g., an immunoglobulin constant domain (C domain), e.g., a CH1 domain, a CH2 domain or a carboxy-terminal C domain (i.e., a CH3 domain), or a sequence substantially identical to such a C domain or a variant thereof.

[0602] 162. A tolerance-inducing construct according to any of the previous embodiments, wherein the other domain that promotes dimerization is a carboxy-terminal C domain derived from IgG, such as a carboxy-terminal C domain derived from IgG3.

[0603] 163. The tolerance-inducing construct according to any of the previous embodiments, wherein the dimerization unit comprises or consists of a carboxy-terminal C domain derived from IgG3 having an amino acid sequence having at least 80% sequence identity to amino acid sequence 39 to 144 of SEQ ID NO: 1, or a nucleotide sequence encoding said amino acid sequence.

[0604] 164. A tolerance-inducing construct according to any of the previous embodiments, wherein the dimerization unit comprises or consists of a carboxy-terminal C-domain derived from IgG3 having an amino acid sequence having at least 85% sequence identity to amino acid sequence 39 to 144 of SEQ ID NO: 1, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, or such as at least 99% sequence identity.

[0605] 165. A tolerance-inducing construct according to any of the preceding embodiments, wherein the dimerization unit comprises or consists of a carboxy-terminal C domain derived from an IgG3 having amino acid sequence 39 to 144 of SEQ ID NO:1.

[0606] 166. A tolerance-inducing construct according to any of the preceding embodiments, wherein the dimerization unit comprises or consists of the amino acid sequence 39 to 144 of SEQ ID NO:1, except that up to 16 amino acids, e.g. up to 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, have been substituted, deleted, or inserted.

[0607] 167. A tolerance-inducing construct according to any of the previous embodiments, wherein the immunoglobulin domain has the ability to form dimers via non-covalent interactions, such as hydrophobic interactions.

[0608] 168. A tolerance-inducing construct according to any of the previous embodiments, wherein the dimerization unit comprises a CH3 domain and does not comprise a CH2 domain, or wherein the dimerization unit comprises a CH2 domain and does not comprise a CH3 domain.

[0609] 169. A tolerance-inducing construct according to any of the previous embodiments, wherein the dimerization unit comprises hinge exon h1, hinge exon h4, dimerization unit linker and CH3 domain of human IgG3.

[0610] 170. A tolerance-inducing construct according to any of the previous embodiments, wherein the dimerization unit comprises a polypeptide consisting of hinge exon h1, hinge exon h4, a dimerization unit linker and the CH3 domain of human IgG3.

[0611] 171. A tolerance-inducing construct according to any of the previous embodiments, wherein the dimerization unit consists of a polypeptide consisting of hinge exon h1, hinge exon h4, a dimerization unit linker and the CH3 domain of human IgG3.

[0612] 172. The tolerance-inducing construct of any of the previous embodiments, wherein the dimerization unit comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:1.

[0613] 173. A tolerance-inducing construct according to any of the previous embodiments, wherein the dimerization unit comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, or such as at least 99% sequence identity.

[0614] 174. A tolerance-inducing construct according to any of the previous embodiments, wherein the dimerization unit consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:1, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, for example at least 97%, such as at least 98%, or such as at least 99%.

[0615] 175. A tolerance-inducing construct according to any of the previous embodiments, wherein the dimerization unit consists of the amino acid sequence of SEQ ID NO:1, or a nucleotide sequence encoding the amino acid sequence.

[0616] 176. A tolerance-inducing construct according to any of the previous embodiments, wherein the dimerization unit comprises the amino acid sequence of SEQ ID NO: 1, except that up to 22 amino acids, such as up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid have been substituted, deleted or inserted.

[0617] 177. A tolerance-inducing construct according to any of the previous embodiments, wherein the dimerization unit consists of the amino acid sequence of SEQ ID NO: 1, except that up to 22 amino acids, such as up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid have been substituted, deleted or inserted.

[0618] 178. The tolerance-inducing construct of any of the previous embodiments, wherein the dimerization unit linker is a glycine-serine rich linker, such as GGGSSGGGSG (sequence number 139).

[0619] 179. The tolerance-inducing construct of any of the previous embodiments, wherein the polynucleotide further comprises a nucleotide sequence encoding a signal peptide.

[0620] 180. The tolerance-inducing construct according to any of the previous embodiments, wherein the signal peptide is located at the N-terminus of the targeting unit or the C-terminus of the targeting unit.

[0621] 181. A tolerance-inducing construct according to any of the previous embodiments, wherein the signal peptide is a human Ig VH signal peptide or a signal peptide naturally occurring at the N-terminus of any of the targeting units described herein, such as the human signal peptide of human IL-10 or the human signal peptide of human TGFβ.

[0622] 182. The tolerance-inducing construct according to any of the previous embodiments, wherein the polynucleotide comprises a nucleotide sequence encoding a human IL-10 signal peptide, preferably a nucleotide sequence encoding a human IL-10 targeting unit.

[0623] 183. A tolerance-inducing construct according to any of the previous embodiments, wherein the polynucleotide comprises a nucleotide sequence encoding a human Ig VH signal peptide, preferably encoding an scFv, such as human anti-DEC205.

[0624] 184. A tolerance-inducing construct according to any of the previous embodiments, wherein the polynucleotide comprises a nucleotide sequence encoding a signal peptide comprising an amino acid sequence having at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 48.

[0625] 185. The tolerance-inducing construct of any of the previous embodiments, wherein the polynucleotide comprises a nucleotide sequence encoding a signal peptide comprising the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:48.

[0626] 186. A tolerance-inducing construct according to any of the previous embodiments, wherein the polynucleotide comprises a nucleotide sequence encoding a signal peptide consisting of an amino acid sequence having at least 80%, preferably at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, or at least 99%, to the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 48.

[0627] 187. The tolerance-inducing construct of any of the previous embodiments, wherein the polynucleotide comprises a nucleotide sequence encoding a signal peptide having the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:48.

[0628] 188. A tolerance-inducing construct according to any of the previous embodiments, wherein the polynucleotide comprises a nucleotide sequence encoding a signal peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 48, except that up to 5 amino acids, such as up to 4 amino acids, such as up to 3 amino acids, such as up to 2 amino acids, or up to 1 amino acid, etc., have been substituted, deleted or inserted.

[0629] 189. A tolerance-inducing construct according to any of the previous embodiments, wherein the polynucleotide is a DNA sequence or an RNA sequence.

[0630] 190. A polynucleotide according to any of the preceding embodiments.

[0631] 191. A vector comprising a polynucleotide according to embodiment 190.

[0632] 192. A host cell comprising a polynucleotide according to embodiment 190 and / or a vector according to embodiment 191.

[0633] 193. A polypeptide encoded by a nucleotide sequence according to any one of embodiments 1 to 189.

[0634] 194. A dimeric protein according to any one of embodiments 1 to 189, consisting of two polypeptides.

[0635] 195. A multimeric protein according to any one of embodiments 1 to 189, consisting of two or more polypeptides.

[0636] 196. A pharmaceutical composition comprising a tolerance-inducing construct according to any of embodiments 1 to 189 and a pharma- ceutically acceptable carrier.

[0637] 197. A pharmaceutical composition comprising a polynucleotide according to any one of embodiments 1 to 189, a vector according to embodiment 191, a polypeptide according to embodiment 193, a dimeric protein according to embodiment 194, or a multimeric protein according to embodiment 195, and a pharma- ceutically acceptable carrier.

[0638] 198. The pharmaceutical composition according to any of embodiments 196-197, further comprising one or more pharma- ceutically acceptable excipients and / or diluents.

[0639] 199. The pharmaceutical composition of any of embodiments 196-198, wherein the pharma- ceutically acceptable carrier is selected from the group consisting of saline, buffered saline, PBS, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffer, and combinations thereof.

[0640] 200. A method for preparing a pharmaceutical composition according to any of embodiments 196 to 199, comprising a multimeric protein, a dimeric protein or a polypeptide, the method comprising: i) transfecting a cell with a polynucleotide according to embodiment 190; ii) culturing the cells; iii) collecting and purifying the multimeric protein, the dimeric protein, or the expressed polypeptide from the cell; iv) mixing the dimeric protein or polypeptide obtained from step iii) with a pharma- ceutically acceptable carrier; A method comprising:

[0641] 201. A method for preparing a pharmaceutical composition according to any one of embodiments 196 to 199, wherein the pharmaceutical composition comprises a polynucleotide according to embodiment 190, the method comprising: i) preparing a polynucleotide; ii) optionally, cloning the polynucleotide into an expression vector; iii) mixing the polynucleotide obtained from step i) or the vector obtained from step ii) with a pharma- ceutically acceptable carrier; A method comprising:

[0642] 202. A method for treating a subject suffering from or in need of prevention of a condition involving an unwanted immune response, such as an autoimmune disease, an allergic disease, or a transplant rejection, comprising administering to the subject a pharmaceutical composition described in any of embodiments 196 to 199.

[0643] 203. A pharmaceutical composition according to any of embodiments 196 to 199, for use in the treatment of a condition involving an unwanted immune response, such as an autoimmune disease, an allergic disease, or transplant rejection.

[0644] 204. Use of a pharmaceutical composition according to any of embodiments 196 to 199 for the treatment of conditions involving unwanted immune responses, such as autoimmune diseases, allergic diseases or graft rejection.

[0645] 205. Use of a pharmaceutical composition according to any of embodiments 196 to 199 for the manufacture of a medicament for the treatment of a condition involving an unwanted immune response, such as an autoimmune disease, an allergic disease, or transplant rejection.

[0646] 206. Use of a pharmaceutical composition according to any of embodiments 196 to 199 for treating a subject having a condition involving an unwanted immune response, such as an autoimmune disease, an allergic disease, or transplant rejection.

[0647] 207. A pharmaceutical comprising a tolerance-inducing construct described in any of embodiments 1 to 189 for treating a condition involving an unwanted immune response, such as an autoimmune disease, an allergic disease, or transplant rejection.

[0648] 208. Use of a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a tolerance-inducing construct described in any one of embodiments 1 to 189 for the manufacture of a medicament for treating a subject having a condition involving an undesired immune response, such as an autoimmune disease, an allergic disease, or transplant rejection.

[0649] 209. Use of a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a tolerance-inducing construct described in any of embodiments 1 to 189 for the treatment of a subject having a condition involving an unwanted immune response, such as an autoimmune disease, an allergic disease, or transplant rejection.

[0650] 210. A pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a tolerance-inducing construct described in any of embodiments 1 to 189 when used to treat a condition involving an unwanted immune response, such as an autoimmune disease, an allergic disease, or transplant rejection.

[0651] 211. A method for improving tolerance to an autoantigen, an allergen, an alloantigen or a xenoantigen using a tolerance-inducing construct described in any of embodiments 1 to 189.

[0652] 212. A method for improving tolerance to an autoantigen, an allergen, an alloantigen or a xenoantigen in a subject, comprising administering to the subject a tolerance-inducing construct according to any one of embodiments 1 to 189 or a pharmaceutical composition according to any one of embodiments 196 to 199.

Claims

1. i) a polynucleotide comprising a nucleotide sequence encoding a targeting unit that targets or is capable of targeting an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit; or ii) a polypeptide encoded by a nucleotide sequence according to (i); or iii) A multimeric protein, such as a dimeric protein consisting of a plurality of polypeptides according to (ii), e.g. two polypeptides; A tolerance-inducing construct comprising: the antigenic unit comprises one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen, the targeting unit interacts with a surface molecule on the antigen-presenting cell without activating the cell; Tolerance-inducing constructs.

2. The tolerance-inducing construct of claim 1 , wherein the construct comprises a dimerization unit and the multimeric protein is a dimeric protein.

3. 3. The tolerance-inducing construct of claim 1 or 2, wherein the targeting unit comprises or consists of a moiety that binds to a receptor selected from the group consisting of TGFβR1, TGFβR2, TGFβR3, IL10R, IL10RA and IL10RB, IL2R, IL4R, IL6R, IL11R and IL13R, IL27R, IL35R, IL37R, CCR7, CD11b, CD11c, CD103, CD14, CD36, CD205, CD109, VISTA, MARCO, MHCII, MHCII, CD83, SIGLEC, MGL, CD80, CD86, Clec9A, Clec12A, Clec12B, DCIR2, Langerin, MR, DC-Sign, Treml4, Dectin-1, PDL1, PDL2 and HVEM.

4. The tolerance-inducing construct of claim 3, wherein the portion is an antibody or a portion thereof, such as an scFv, preferably the portion is an antibody having specificity for CD205 or a portion of an antibody having specificity for CD205, such as an scFv.

5. 4. The tolerance-inducing construct of claim 3, wherein said moiety is a synthetic or natural ligand, preferably said moiety is selected from the group consisting of TGFβ, IL-10, IL2, IL4, IL6, IL11, IL13, IL27, IL35, IL37, CCL19, CCL21, ICAM-1, keratin, VSIG-3, SCGB3A2, CTLA-4, preferably the extracellular domain of CTLA-4, PD-1, the extracellular domain of PD-1, and BTLA, preferably the extracellular domain of BTLA, more preferably said natural ligand is selected from the group consisting of IL-10, TGFβ, SCGB3A2 and VSIG-3.

6. 3. The tolerance-inducing construct of claim 1 or 2, wherein the antigenic unit comprises one T cell epitope of one autoantigen or one allergen or one alloantigen or one xenoantigen, or the antigenic unit comprises multiple T cell epitopes of one autoantigen or one allergen or one alloantigen or one xenoantigen, or the antigenic unit comprises multiple T cell epitopes of multiple different autoantigens or multiple different allergens or multiple different alloantigens or multiple different xenoantigens.

7. 3. The tolerance-inducing construct of claim 1 or 2, wherein the antigenic unit comprises one or more T cell epitopes of an allergen, such as an allergen selected from the group consisting of a food allergen, a bee venom allergen, a latex allergen, a dust mite allergen, a cockroach allergen, a mold allergen, a fungal allergen, a fur animal allergen, a pollen allergen and an allergen contained in a drug, and optionally, when the antigenic unit comprises two or more T cell epitopes, the T cell epitopes are separated by a T cell epitope linker.

8. 3. The tolerance-inducing construct of claim 1 or 2, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, such as an autoantigen selected from the group consisting of multiple sclerosis autoantigens, type 1 diabetes autoantigens, celiac disease autoantigens, rheumatoid arthritis autoantigens, chronic inflammatory demyelinating polyradiculoneuropathy autoantigens, Hashimoto's thyroiditis autoantigens, pemphigus foliaceus autoantigens, pemphigus vulgaris autoantigens, thyroid eye disease autoantigens, Graves' disease autoantigens, primary biliary cirrhosis autoantigens, myasthenia gravis autoantigens, insulin-resistant diabetes autoantigens and hemolytic anemia autoantigens, and optionally, when the antigenic unit comprises two or more T cell epitopes, the T cell epitopes are separated by a T cell epitope linker.

9. A tolerance-inducing construct as described in claim 1 or 2, comprising a dimerization unit, the dimerization unit comprising hinge exon h1 and hinge exon h4 of human IgG3, a dimerization unit linker and a CH3 domain.

10. 3. The tolerance-inducing construct of claim 1 or 2, wherein the construct is a polynucleotide (i), such as RNA or DNA, and optionally the polynucleotide further comprises a nucleotide sequence encoding a signal peptide.

11. A polynucleotide described in claim 1 or 2, such as a polynucleotide contained in a vector.

12. A host cell comprising a polynucleotide according to claim 11, such as a polynucleotide contained in a vector.

13. 3. A polypeptide encoded by the polynucleotide of claim 1 or 2, or a multimeric protein consisting of a plurality of such polypeptides, such as a dimeric protein consisting of two such polypeptides.

14. A tolerance-inducing construct according to claim 1 or 2, or a polynucleotide according to claim 11, or a polypeptide according to claim 13, or a multimeric protein, such as a dimeric protein, for use as a medicament.

15. 13. A pharmaceutical composition comprising a tolerance-inducing construct according to claim 1 or 2, or a polynucleotide according to claim 11, or a multimeric protein, such as a polypeptide or dimeric protein, according to claim 13, and a pharma- ceutically acceptable carrier, optionally wherein the pharma-ceutically acceptable carrier is selected from the group consisting of saline, buffered saline, PBS, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffer, and combinations thereof.

16. A pharmaceutical composition for use in the prophylactic or therapeutic treatment of a condition involving an unwanted immune response, such as an autoimmune disease, an allergic disease, or a transplant rejection, comprising a tolerance-inducing construct according to claim 1 or 2, or a polynucleotide according to claim 11, or a multimeric protein, such as a polypeptide or dimeric protein, according to claim 13, and a pharma- ceutically acceptable carrier.

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