Co-expression of constructs and immunoinhibitory compounds

Abstract

The present invention relates to vectors, such as DNA plasmids, that contain multiple nucleic acid sequences engineered to be co-expressed as separate molecules, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit comprising one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen, and one or more immune inhibitory compounds.

Description

[Technical field]

[0001] The present invention relates to vectors, such as DNA plasmids, which contain multiple nucleic acid sequences of interest engineered to be co-expressed as separate molecules, pharmaceutical compositions comprising such vectors, and the use of such vectors and such pharmaceutical compositions in the treatment or prevention of disease. [Background technology]

[0002] Immune responses are necessary to protect against diseases caused by pathogens such as viruses, bacteria, and parasites. However, unwanted immune activation can lead to processes that damage or destroy one's own tissues. Unwanted immune activation occurs, for example, in autoimmune diseases, where antibodies and / or T lymphocytes react with self-antigens, resulting in, for example, tissue damage and lesions. Unwanted immune activation also occurs in allergic reactions, which are characterized by an excessive immune response to typically harmless substances in the environment and can cause an inflammatory response that leads to tissue destruction. Furthermore, in graft rejection, for example, rejection of transplanted organs or tissues, unwanted immune activation occurs, which is significantly mediated by alloreactive T cells present in the host, which recognize donor allo- or xenoantigens and lead to the destruction of the transplanted organ or tissue.

[0003] Immune tolerance is the acquired lack of a specific immune response to substances or tissues capable of eliciting an immune response in a particular organism.

[0004] Typically, to induce tolerance to a specific antigen, the antigen must be presented to other immune cells by antigen-presenting cells (APCs) in the absence of activation signals, resulting in either 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, i.e., self-tolerance. Immunosuppressants are useful for preventing or reducing unwanted immune responses, for example, in the treatment of autoimmune disease patients or allogeneic transplant patients. Traditional strategies for immunosuppressing unwanted immune responses are based on broad-acting immunosuppressants. Furthermore, lifelong immunosuppressant therapy is often required to maintain immunosuppression. Unfortunately, the use of broad-acting immunosuppressants carries the risk of severe side effects, such as immune deficiency, because most of them act nonselectively, increasing susceptibility to infections and reducing immune surveillance of cancer. Therefore, new compounds and compositions that induce antigen-specific tolerance would be beneficial.

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

[0006] The present invention relates to a construct comprising an antigen unit comprising one or more T cell epitopes of an autoantigen, an allergen, an alloantigen or a xenoantigen, and a targeting unit which interacts with a surface molecule on an APC in a non-inflammatory or tolerogenic manner, resulting in the presentation of the antigen in the absence of inflammatory activation conditions.

[0007] Vaccibody constructs are dimeric fusion proteins consisting of two polypeptides, each of which comprises a targeting unit for targeting antigen-presenting cells, a dimerization unit, and an antigenic unit comprising one or more disease-associated antigens or portions thereof. In another embodiment, the Vaccibody constructs are multimeric fusion proteins consisting of multiple polypeptides, each of which comprises a targeting unit for targeting APCs, a multimerization unit, and an antigenic unit comprising one or more disease-associated antigens or portions thereof - see, for example, WO 2004 / 076489 (A1), WO 2011 / 161244 (A1), WO 2013 / 092875 (A1) or WO 2017 / 118695 (A1). These constructs have been shown to be efficient in generating an immune response against an antigen or a portion thereof, e.g., an epitope, contained in the antigenic unit.

[0008] The vaccibody construct can be administered to a subject in the form of a polynucleotide encoding the polypeptide, e.g., a polynucleotide contained in a vector, such as a DNA plasmid. After administration to a host cell, e.g., administration to a muscle cell of a subject, e.g., a human, the polypeptide is expressed and forms a multimeric fusion protein, such as a dimeric protein, with a multimerization unit, such as a dimerization unit.

[0009] The inventors have surprisingly found that the modified vaccibody platform can be used to deliver disease-associated antigens to APCs in an optimal manner to induce a selected antigen-specific tolerance response by binding to and signaling through selected surface receptors on APCs that internalize the construct and present the antigen contained therein in a tolerogenic manner. Summary of the Invention

[0010] The present invention provides vectors, e.g., DNA plasmids, for co-expressing the constructs and one or more immunosuppressive compounds. The vectors and pharmaceutical compositions comprising such vectors are 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.

[0011] Such constructs and immunoinhibitory compounds enable the epitopes in the antigenic units to be presented in a tolerogenic manner once the vector is administered to a subject, making the vectors of the invention suitable for use in the prophylactic or therapeutic treatment of immune disorders, such as autoimmune diseases, allergic diseases and transplant rejection.

[0012] The constructs and immunoinhibitory compounds cause the downregulation of disease-specific cells of the immune system that cause the immune disease in question, and do not suppress the general immune system, therefore treating the immune disease in question with the vectors of the invention does not increase susceptibility to infections or decrease immune surveillance of cancer.

[0013] The one or more immunoinhibitory compounds serve to generate or promote an environment favorable for the presentation of epitopes in an antigenic unit in a tolerance-inducing manner or, for example, by promoting the induction of tolerance-maintaining cells or favoring the maintenance of such cells.

[0014] In a first aspect, the present invention provides a method for producing a composition comprising the steps of: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as 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; (b) one or more additional nucleic acid sequences encoding one or more immunosuppressant compounds; and Including, The present invention relates to a vector which allows for the co-expression of the first polypeptide and one or more immunosuppressive compounds as separate molecules.

[0015] In one embodiment, a vector of the present invention comprises a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell without activating it.

[0016] The vectors of the present invention can be used, for example, in the form of a pharmaceutical composition, i.e., a composition comprising the vector and a pharma- ceutically acceptable carrier or diluent, for the prophylactic or therapeutic treatment of immune disorders by administering the vector / pharmaceutical composition to a subject in need of such prophylactic or therapeutic treatment. [Brief description of the drawings]

[0017] [Figure 1] 1 shows an IRES co-expression element for use in a vector of the present invention, inserted between two coding regions. Once an mRNA is formed, two ribosomes (T) can begin translation at two distinct sites on the mRNA, resulting in the formation of two proteins (A and B). A and B can be, for example, a first polypeptide and an immunosuppressive compound. [Diagram 2] Figure 1 shows a 2A self-cleaving peptide co-expression element for use in a vector of the present invention, inserted between two genes. After transcription, one ribosome translates the mRNA and two proteins (A and B) are formed. The top part of the figure shows how a fusion protein is formed when the 2A sequence is not part of the coding sequence. A and B can be, for example, a first polypeptide and an immunosuppressive compound. [Diagram 3]1 shows a bidirectional promoter (P) co-expression element for use in a vector of the invention, located between two coding regions. One mRNA is formed, two ribosomes (T) can initiate translation on two different mRNAs, and two proteins (A and B) are formed. A and B can be, for example, a first polypeptide and an immunosuppressive compound. [Figure 4] Shown is a co-expression element for use in a vector of the invention, located in front of two promoters (P), i.e., two coding regions. Two mRNAs are formed, two ribosomes (T) can initiate translation on the two different mRNAs, and two proteins (A and B) are formed. A and B can be, for example, a first polypeptide and an immunosuppressive compound. [Figure 5A] 1 illustrates an embodiment of a construct encoded by a vector of the invention based on a first polypeptide. [Figure 5B] 1 illustrates an embodiment of a construct encoded by a vector of the invention based on a first polypeptide. [Figure 6A] Figure 2 shows ELISA results (protein expression and secretion) obtained after transient transfection of HEK293 cells with the DNA vectors VB5049* (vector according to the invention) or VB5052*. [Figure 6B] Figure 2 shows ELISA results (protein expression and secretion) obtained after transient transfection of HEK293 cells with the DNA vectors VB5049* (vector according to the invention) or VB5052*. [Figure 6C] Figure 2 shows ELISA results (protein expression and secretion) obtained after transient transfection of HEK293 cells with the DNA vectors VB5049* (vector according to the invention) or VB5052*. [Figure 6D] Figure 2 shows ELISA results (protein expression and secretion) obtained after transient transfection of HEK293 cells with the DNA vectors VB5049* (vector according to the invention) or VB5052*. [Figure 7A]1 shows the results of a Western blot analysis of supernatants from Expi293F cells transfected with the DNA vector VB5049* (a vector according to the invention) or VB5052*. [Figure 7B] 1 shows the results of a Western blot analysis of supernatants from Expi293F cells transfected with the DNA vector VB5049* (a vector according to the invention) or VB5052*. [Figure 8] Shown are the results of a FluoroSpot assay (IL-10 / IFN-γ ratio) on splenocytes obtained from mice following a single administration of either DNA vector VB5049* or VB5052* and restimulation with MOG(35-55) peptide. [Figure 9A] Figure 9 shows the percentage of splenic IFN-γ+ T cells (9A) and IL-17+ T cells (9B) in the total CD4+ T cell population as assessed by flow cytometry and measured after 16 h restimulation with MOG(35-55) peptide. C57BL / 6 mice were given a single intramuscular injection of either DNA vector VB5049*, VB5051* or VB5052* and spleens were harvested 7 days later. Data were generated from 5 mice / group and mouse spleens were pooled prior to analysis. [Figure 9B] Figure 9 shows the percentage of splenic IFN-γ+ T cells (9A) and IL-17+ T cells (9B) in the total CD4+ T cell population as assessed by flow cytometry and measured after 16 h restimulation with MOG(35-55) peptide. C57BL / 6 mice were given a single intramuscular injection of either DNA vector VB5049*, VB5051* or VB5052* and spleens were harvested 7 days later. Data were generated from 5 mice / group and mouse spleens were pooled prior to analysis. [Figure 10]Results of MOG tetramer staining and detection of MOG-specific T cells are shown. C57BL / 6 mice were intramuscularly injected with either DNA vector VB5049*, VB5051*, or VB5052* on day 0, followed by electroporation, and spleens were harvested on day 7 after injection. The percentage of splenic Foxp3+ cells in the CD4+MOG(35-55)tet+T cell population was detected by H-2 IAb / MOG(35-55) tetramer. Tetramer staining was performed ex vivo, and splenocytes were not restimulated with MOG(35-55) peptide. Data were generated from 5 mice / group, and mouse spleens were pooled before analysis. [Figure 11] The protein expression and secretion levels of the first polypeptide encoded by the indicated DNA vectors were detected by sandwich ELISA (capture antibody: anti-MOG antibody, detection antibody: anti-hIgG CH3 domain antibody) using the supernatant of Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): Supernatant from Expi293F cells treated with only the transfection reagent ExpiFectamine. [Figure 12A] Figure 1 shows the expression and secretion levels of immune inhibitory compounds encoded by the indicated DNA vectors, detected by sandwich ELISA using the supernatants of Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): Supernatants from Expi293F cells treated with the transfection reagent ExpiFectamine only. The following antibodies were used: (A) Capture antibody: 1 μg / mL rat anti-mouse IL-10 antibody, 100 μL / well, MAB417, R&D Systems. Detection antibody: 0.2 μg / mL goat anti-mouse IL-10 biotinylated antibody, 100 μL / well, BAF417, R&D Systems. [Figure 12B]Expression and secretion levels of immune inhibitory compounds encoded by the indicated DNA vectors were detected by sandwich ELISA using supernatants from Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): Supernatants from Expi293F cells treated with the transfection reagent ExpiFectamine only. The following antibodies were used: (B) Capture antibody: 2 μg / mL TGF-β1 antibody, 100 μL / well, MAB2402, RD Systems. Detection antibody: 0.8 μg / mL chicken anti-human TGF-β1 biotinylated antibody, 100 μL / well, BAF240, RD Systems. [Figure 12C] Expression and secretion levels of immune inhibitory compounds encoded by the indicated DNA vectors detected by sandwich ELISA using supernatants of Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): Supernatants from Expi293F cells treated with the transfection reagent ExpiFectamine only. The following antibodies were used: (C) Capture antibody: 0.8 μg / mL goat anti-mouse CTLA-4 antibody, 100 μL / well, AF476, RD Systems. Detection antibody: 0.8 μg / mL goat anti-mouse CTLA-4 biotinylated antibody, 100 μL / well, BAF476, RD Systems. [Figure 12D] Expression and secretion levels of immune inhibitory compounds encoded by the indicated DNA vectors detected by sandwich ELISA using supernatants from Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): Supernatants from Expi293F cells treated with the transfection reagent ExpiFectamine only. The following antibodies were used: (D) Capture antibody: 2 μg / mL rat anti-mouse IL-2 antibody, 100 μL / well, 503701, BioLegend. Detection antibody: 2 μg / mL rat anti-mouse IL-2 biotinylated antibody, 100 μL / well, 503803, BioLegend. [Figure 12E]Expression and secretion levels of immune inhibitory compounds encoded by the indicated DNA vectors detected by sandwich ELISA using supernatants of Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): Supernatants from Expi293F cells treated with transfection reagent ExpiFectamine only. The following antibodies were used: (E) Capture antibody: 2 μg / mL rat anti-mouse IFN-γ antibody, 100 μL / well, 505802, BioLegend. Detection antibody: 2 μg / mL rat anti-mouse IFN-γ biotinylated antibody, 100 μL / well, 505704, BioLegend. [Figure 13A] Figure 1 shows the distinct expression and secretion of the first polypeptide and immune inhibitory compound encoded by the indicated DNA vectors detected by sandwich ELISA using the supernatants of Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): Supernatant from Expi293F cells treated with the transfection reagent ExpiFectamine only. The following antibodies were used: 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 anti-IL-10 biotinylated antibody, 100 μL / well, BAF417, R&D Systems. [Figure 13B] Figure 1 shows the distinct expression and secretion of the first polypeptide and immune inhibitory compound encoded by the indicated DNA vectors detected by sandwich ELISA using the supernatant of Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): Supernatant from Expi293F cells treated with transfection reagent ExpiFectamine only. The following antibodies were used: Capture antibody: Mouse anti-MOG antibody, 0.25 μg / mL, 100 μL / well, sc-73330, Santa Cruz Biotechnology. Detection antibody: (B) 0.8 μg / mL chicken anti-human TGFβ1 biotinylated antibody, 100 μL / well, BAF240, RD Systems. [Figure 13C] Figure 1 shows the distinct expression and secretion of the first polypeptide and immune inhibitory compound encoded by the indicated DNA vectors detected by sandwich ELISA using the supernatants of Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): Supernatant from Expi293F cells treated with the transfection reagent ExpiFectamine only. The following antibodies were used: Capture antibody: Mouse anti-MOG antibody, 0.25 μg / mL, 100 μL / well, sc-73330, Santa Cruz Biotechnology. Detection antibody: (C) Goat anti-mouse CTLA-4 biotinylated antibody, 0.8 μg / mL, 100 μL / well, BAF476, RD Systems. [Figure 13D] Figure 1 shows the distinct expression and secretion of the first polypeptide and immune inhibitory compound encoded by the indicated DNA vectors detected by sandwich ELISA using the supernatants of Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): Supernatant from Expi293F cells treated with the transfection reagent ExpiFectamine only. The following antibodies were used: Capture antibody: Mouse anti-MOG antibody, 0.25 μg / mL, 100 μL / well, sc-73330, Santa Cruz Biotechnology. Detection antibody: (D) Rat anti-mouse IFN-γ biotinylated antibody, 2 μg / mL, 100 μL / well, 505704, BioLegend. [Figure 14A] Western blot analysis of supernatants from Expi293F cells transfected with the indicated DNA vectors. A: Reduced supernatant samples (35 μL loaded). Primary antibody: mouse anti-MOG (sc-73330). Secondary antibody: donkey anti-mouse, Dylight 800 (SA5-10172). Protein standards were detected in Chemidoc channel Dylight 650 (signals not shown). Chemidoc channel Dylight 800. Black arrows indicate the first polypeptides expressed from the respective DNA vectors as separate proteins and secreted from transfected cells. [Figure 14B]Western blot analysis of supernatants from Expi293F cells transfected with the indicated DNA vectors. B: Non-reduced supernatant sample (loaded 35 μL). Primary antibody: mouse anti-MOG (sc-73330). Secondary antibody: donkey anti-mouse, Dylight 800 (SA5-10172). Chemidoc channels Dylight 650 (for protein standards) and 800. Black arrows indicate dimeric proteins formed by two first polypeptide molecules expressed from the respective DNA vectors and secreted from transfected cells. [Figure 14C] Western blot analysis of supernatants from Expi293F cells transfected with the indicated DNA vectors. C: Reduced supernatant sample (35 μL loaded). Primary antibody: rat anti-IL10 (MAB417). Secondary antibody: donkey anti-rat, Dylight 488 (SA5-10026). Chemidoc channels Dylight 650 (for protein standard) and 488. Black arrows indicate IL-10 expressed as a separate protein from the respective DNA vector and secreted from transfected cells. [Figure 14D] Western blot analysis of supernatants from Expi293F cells transfected with the indicated DNA vectors. D: Reduced supernatant sample (35 μL loaded). Primary antibody: goat anti-CTLA-4 (AF476). Secondary antibody: donkey anti-goat, Dylight 800 (SA5-10092). Chemidoc channels Dylight 650 (for protein standards) and 800. Black arrows indicate CTLA-4 expressed as a separate protein from the respective DNA vector and secreted from transfected cells. [Figure 14E]Western blot analysis of supernatants from Expi293F cells transfected with the indicated DNA vectors. E: Reduced supernatant sample (35 μL loaded). Primary antibody: rat anti-IL2 (503702). Secondary antibody: donkey anti-rat, Dylight 650 (SA5-10029). Chemidoc channel Dylight 650. Black arrows indicate IL-2 expressed as a separate protein from the respective DNA vector and secreted from transfected cells. [Figure 15] Expression and secretion of MOG(27-63) encoded by DNA vector VB5051 detected by sandwich ELISA using supernatant from Expi293F cells transiently transfected with the DNA vector. Negative control (Neg ctrl): Supernatant from Expi293F cells treated with transfection reagent ExpiFectamine only. Detection antibody: Mouse anti-MOG antibody, 3.3 μg / mL, 100 μL / well, sc-73330, Santa Cruz Biotechnology. [Figure 16A] Results of a dual-color IL-10 / IFN-γ FluoroSpot assay are shown. C57BL / 6 mice were intramuscularly injected four times (days 0, 3, 7, and 10) with the indicated DNA vectors, followed by electroporation, and spleens were harvested 14 days after the first injection. Splenocytes were tested in a dual-color FluoroSpot assay for IL-10 and IFN-γ secretion (SFU / 106 splenocytes) either unstimulated (A) or restimulated with MOG(35-55) peptide for 44 h (B). Individual mice and mean ± SEM are shown (5 mice / group, *(p<0.05), two-tailed Mann-Whitney test). [Figure 16B]Results of a dual-color IL-10 / IFN-γ FluoroSpot assay are shown. C57BL / 6 mice were intramuscularly injected four times (days 0, 3, 7, and 10) with the indicated DNA vectors, followed by electroporation, and spleens were harvested 14 days after the first injection. Splenocytes were tested in a dual-color FluoroSpot assay for IL-10 and IFN-γ secretion (SFU / 106 splenocytes) either unstimulated (A) or restimulated with MOG(35-55) peptide for 44 h (B). Individual mice and mean ± SEM are shown (5 mice / group, *(p<0.05), two-tailed Mann-Whitney test). [Figure 17] IL-10 / IFN-γ ratios calculated from the data shown in Figure 16B are shown. Individual mice and mean ± SEM are shown (5 mice / group, *(p<0.05), two-tailed Mann-Whitney test). [Figure 18] Flow cytometry results of detection of Foxp3+-producing CD4+ T cells. C57BL / 6 mice were administered the indicated DNA vectors followed by electroporation four times on days 0, 3, 7, and 10, and spleens were harvested 14 days after the first administration. The percentage of splenic CD4+Foxp3+ T cells was determined upon restimulation with MOG(35-55) peptide for 16 hours. Data were generated from 5 mice / group, and mouse spleens were pooled before analysis. [Figure 19A] Flow cytometric detection of IFN-γ and IL-17. C57BL / 6 mice were administered four doses of the indicated DNA vectors followed by electroporation on days 0, 3, 7, and 10, and spleens were harvested 14 days after the first dose. The percentage of (A) IFN-γ+ T cells and (B) IL-17+ T cells in the total CD4+ T cell population was determined upon restimulation with MOG(35-55) peptide for 16 hours. Data were generated from 5 mice / group, and mouse spleens were pooled prior to analysis. [Figure 19B]Flow cytometric detection of IFN-γ and IL-17. C57BL / 6 mice were administered four doses of the indicated DNA vectors followed by electroporation on days 0, 3, 7, and 10, and spleens were harvested 14 days after the first dose. The percentage of (A) IFN-γ+ T cells and (B) IL-17+ T cells in the total CD4+ T cell population was determined upon restimulation with MOG(35-55) peptide for 16 hours. Data were generated from 5 mice / group, and mouse spleens were pooled prior to analysis. [Figure 20A] Results of a dual-color IL-10 / IFN-γ FluoroSpot assay are shown. C57BL / 6 mice were injected intramuscularly with the indicated DNA vectors on day 0, followed by electroporation, and spleens were harvested 7 days post-injection. Splenocytes were tested for IL-10 and IFN-γ secretion (SFU / 106 splenocytes) using dual-color FluoroSpot, either unstimulated (A) or restimulated with MOG(35-55) peptide for 44 h (B). Individual mice and mean ± SEM are shown (5 mice / group, **(p<0.01), two-tailed Mann-Whitney test). [Figure 20B] Results of a dual-color IL-10 / IFN-γ FluoroSpot assay are shown. C57BL / 6 mice were injected intramuscularly with the indicated DNA vectors on day 0, followed by electroporation, and spleens were harvested 7 days post-injection. Splenocytes were tested for IL-10 and IFN-γ secretion (SFU / 106 splenocytes) using dual-color FluoroSpot, either unstimulated (A) or restimulated with MOG(35-55) peptide for 44 h (B). Individual mice and mean ± SEM are shown (5 mice / group, **(p<0.01), two-tailed Mann-Whitney test). [Figure 21] IL-10 / IFN-γ ratios calculated from the data shown in Figure 20B are shown. Individual mice and mean±SEM are shown (5 mice / group, *(p<0.05), two-tailed Mann-Whitney test). [Figure 22]Results of MOG tetramer staining and detection of MOG-specific T cells are shown. C57BL / 6 mice were intramuscularly injected with the indicated DNA vectors on day 0, followed by electroporation, and spleens were harvested on day 7 after injection. The percentage of splenic Foxp3+ cells in the CD4+MOG(38-49)tet+T cell population was detected by H-2 IAb / MOG(38-49) tetramer. Tetramer staining was performed ex vivo, and splenocytes were not restimulated with MOG(35-55) peptide. Data were generated from 5 mice / group, and mouse spleens were pooled before analysis. [Figure 23A] Results of a dual-color IL-10 / IFN-γ FluoroSpot assay are shown. C57BL / 6 mice were injected intramuscularly with the indicated DNA vectors on day 0, followed by electroporation, and spleens were harvested 7 days post-injection. Splenocytes were tested for IL-10 and IFN-γ secretion (SFU / 106 splenocytes) using dual-color FluoroSpot without restimulation (control, A) or restimulated with MOG(35-55) peptide for 44 h (B). Individual mice and mean ± SEM are shown (5 mice / group, **(p<0.01), two-tailed Mann-Whitney test). [Figure 23B] Results of a dual-color IL-10 / IFN-γ FluoroSpot assay are shown. C57BL / 6 mice were injected intramuscularly with the indicated DNA vectors on day 0, followed by electroporation, and spleens were harvested 7 days post-injection. Splenocytes were tested for IL-10 and IFN-γ secretion (SFU / 106 splenocytes) using dual-color FluoroSpot without restimulation (control, A) or restimulated with MOG(35-55) peptide for 44 h (B). Individual mice and mean ± SEM are shown (5 mice / group, **(p<0.01), two-tailed Mann-Whitney test). [Figure 24] IL-10 / IFN-γ ratios calculated from the data shown in Figure 23B are shown. Individual mice and means ± SEM are shown (5 mice / group, *(p<0.01), two-tailed Mann-Whitney test). [Diagram 25]Results of MOG tetramer staining and detection of MOG-specific T cells are shown. C57BL / 6 mice were intramuscularly injected with the indicated DNA vectors on day 0, followed by electroporation, and spleens were harvested on day 7 after injection. The percentage of splenic Foxp3+ cells in the CD4+MOG(38-49)tet+T cell population was detected by H-2 IAb / MOG(38-49) tetramer. Tetramer staining was performed ex vivo, and splenocytes were not restimulated with MOG(35-55) peptide. Data were generated from 5 mice / group, and mouse spleens were pooled before analysis. [Figure 26] Results of detection of expanded Tregs are shown. C57BL / 6 mice were intramuscularly injected with the indicated DNA vectors on day 0, followed by electroporation, and spleens were harvested on day 7 after injection. The percentage of Ki67+ cells in the Treg (CD4+CD25+Foxp3+) cell population was detected ex vivo. Data were generated from 5 mice / group, and mouse spleens were pooled before analysis. [Figure 27A] Results of a dual-color IL-10 / IFN-γ FluoroSpot assay are shown. C57BL / 6 mice were injected intramuscularly with the indicated DNA vectors on day 0, followed by electroporation, and spleens were harvested 7 days post-injection. Splenocytes were tested for IL-10 and IFN-γ secretion (SFU / 106 splenocytes) using dual-color FluoroSpot without restimulation (control, A) or restimulated with MOG(35-55) peptide for 44 h (B). Individual mice and mean ± SEM are shown (5 mice / group, **(p<0.01), two-tailed Mann-Whitney test). [Figure 27B]Results of a dual-color IL-10 / IFN-γ FluoroSpot assay are shown. C57BL / 6 mice were injected intramuscularly with the indicated DNA vectors on day 0, followed by electroporation, and spleens were harvested 7 days post-injection. Splenocytes were tested for IL-10 and IFN-γ secretion (SFU / 106 splenocytes) using dual-color FluoroSpot without restimulation (control, A) or restimulated with MOG(35-55) peptide for 44 h (B). Individual mice and mean ± SEM are shown (5 mice / group, **(p<0.01), two-tailed Mann-Whitney test). [Figure 28] IL-10 / IFN-γ ratios calculated from the data shown in Figure 27B are shown. Individual mice and mean±SEM are shown (5 mice / group, *(p<0.01), two-tailed Mann-Whitney test). [Figure 29] Results of MOG tetramer staining and detection of MOG-specific T cells are shown. C57BL / 6 mice were intramuscularly injected with the indicated DNA vectors on day 0, followed by electroporation, and spleens were harvested on day 7 after injection. The percentage of splenic Foxp3+ cells in the CD4+MOG(38-49)tet+T cell population was detected by H-2 IAb / MOG(38-49) tetramer. Tetramer staining was performed ex vivo, and splenocytes were not restimulated with MOG(35-55) peptide. Data were generated from 5 mice / group, and mouse spleens were pooled before analysis. [Diagram 30] Results of detection of expanded Tregs are shown. C57BL / 6 mice were intramuscularly injected with the indicated DNA vectors on day 0, followed by electroporation, and spleens were harvested on day 7 after injection. The percentage of Ki67+ cells in the Treg (CD4+CD25+Foxp3+) cell population was detected ex vivo. Data were generated from 5 mice / group, and mouse spleens were pooled before analysis. [Diagram 31]Figure 1 shows the expression and secretion of the first polypeptide encoded by the indicated DNA vector, detected by sandwich ELISA using the supernatant of Expi293F cells transiently transfected with the DNA vector. Negative control (Neg ctrl): Supernatant from Expi293F cells treated with the transfection reagent ExpiFectamine only. Capture antibody: mouse anti-MOG antibody, detection antibody: anti-hIgG CH3 domain antibody. [Figure 32A] Figure 1 shows the expression and secretion levels of immune inhibitory compounds encoded by the indicated DNA vectors detected by sandwich ELISA using supernatants from Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): Supernatants from Expi293F cells treated with the transfection reagent ExpiFectamine only. The following conditions and antibodies were used: A) Supernatants diluted 1:100. Capture antibody: Mouse IL-10 antibody, 0.4 μg / mL, 100 μL / well, MAB417, R&D Systems. Detection antibody: Mouse IL-10 biotinylated antibody, 0.2 μg / mL, BAF417, R&D Systems. [Figure 32B] Expression and secretion levels of immune inhibitory compounds encoded by the indicated DNA vectors detected by sandwich ELISA using supernatants of Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): Supernatants from Expi293F cells treated with transfection reagent ExpiFectamine only. The following conditions and antibodies were used: B) Capture antibody: TGFβ1 antibody, 2 μg / mL, 100 μL / well, MAB2402, RD Systems. Detection antibody: Chicken anti-TGFβ1 biotinylated antibody, 0.8 μg / mL, 100 μL / well, BAF240, R&D Systems. [Figure 32C]Expression and secretion levels of immune inhibitory compounds encoded by the indicated DNA vectors detected by sandwich ELISA using supernatants from Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): Supernatant from Expi293F cells treated with transfection reagent ExpiFectamine only. The following conditions and antibodies were used: C) Capture antibody: Mouse GM-CSF antibody, 2 μg / mL, 100 μL / well, MAB415, R&D Systems. Detection antibody: Anti-GM-CSF biotinylated antibody, 0.8 μg / mL, BAF415, R&D Systems. [Figure 33A] Figure 1 shows the distinct expression and secretion of proteins encoded by the indicated DNA vectors detected by sandwich ELISA using supernatants from Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): Supernatant from Expi293F cells treated with transfection reagent ExpiFectamine only. Capture antibody: Mouse anti-MOG antibody, 0.25 μg / mL, 100 μL / well, sc-73330, Santa Cruz Biotechnology. The following detection antibodies were used: A) Mouse anti-IL-10 biotinylated antibody, 0.2 μg / mL, 100 μL / well, BAF417, R&D Systems. [Figure 33B] Figure 1 shows the distinct expression and secretion of proteins encoded by the indicated DNA vectors detected by sandwich ELISA using supernatants from Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): Supernatant from Expi293F cells treated with transfection reagent ExpiFectamine only. Capture antibody: Mouse anti-MOG antibody, 0.25 μg / mL, 100 μL / well, sc-73330, Santa Cruz Biotechnology. The following detection antibodies were used: B) Chicken anti-TGFβ1 biotinylated antibody, 0.8 μg / mL, 100 μL / well, BAF240, R&D Systems. [Figure 33C]Figure 1 shows the distinct expression and secretion of proteins encoded by the indicated DNA vectors detected by sandwich ELISA using supernatants from Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): Supernatant from Expi293F cells treated with transfection reagent ExpiFectamine only. Capture antibody: Mouse anti-MOG antibody, 0.25 μg / mL, 100 μL / well, sc-73330, Santa Cruz Biotechnology. The following detection antibodies were used: C) Anti-GM-CSF biotinylated antibody, 0.8 μg / mL, BAF415, R&D Systems. [Figure 34A] Western blot analysis of supernatants from Expi293F cells transfected with the indicated DNA vectors. A: Reduced supernatant samples (35 μL loaded). Primary antibody: mouse anti-MOG (sc-73330). Secondary antibody: donkey anti-mouse, Dylight 800 (SA5-10172). Protein standards were detected in Chemidoc channel Dylight 650 (signals not shown). Chemidoc channel Dylight 800. Black arrows indicate the first polypeptides expressed from the respective DNA vectors as separate proteins and secreted from transfected cells. [Figure 34B] Western blot analysis of supernatants from Expi293F cells transfected with the indicated DNA vectors. B: Non-reduced supernatant sample (loaded 35 μL). Primary antibody: mouse anti-MOG (sc-73330). Secondary antibody: donkey anti-mouse, Dylight 800 (SA5-10172). Chemidoc channels Dylight 650 (for protein standards) and 800. Black arrows indicate dimeric proteins formed by two first polypeptide molecules expressed from the indicated DNA vectors and secreted from transfected cells. [Figure 34C]Western blot analysis of supernatants from Expi293F cells transfected with the indicated DNA vectors. C: Reduced supernatant samples (35 μL loaded). Primary antibody: rat anti-IL10 (MAB417). Secondary antibody: donkey anti-rat, Dylight 488 (SA5-10026). Chemidoc channels Dylight 650 (for protein standard) and 488. Black arrows indicate IL-10 expressed as a separate protein from the respective DNA vector and secreted from transfected cells. In VB5044 and VB5054, the increased size of IL-10 is due to ribosome skipping resulting in the fusion of the P2A tail to IL-10. [Fig. 34D] Western blot analysis of supernatants from Expi293F cells transfected with the indicated DNA vectors. D: Reduced supernatant sample (35 μL loaded). Primary antibody: rabbit anti-TGF-β1 antibody (USB 1042777-biotin). Secondary antibody: donkey anti-rabbit, Dylight 650 (SA5-10041). Chemidoc channel Dylight 650. Black arrows indicate TGF-β1 expressed as separate proteins from the respective DNA vectors and secreted from transfected cells. [Figure 34E] Western blot analysis of supernatants from Expi293F cells transfected with the indicated DNA vectors. E: Reduced supernatant sample (35 μL loaded). Primary antibody: goat anti-mouse GM-CSF (BAF415). Secondary antibody: donkey anti-goat, Dylight 800 (SA5-10092). Chemidoc channels Dylight 650 (for protein standards) and 800. Black arrows indicate GM-CSF expressed as a separate protein from the respective DNA vector and secreted from transfected cells. [Diagram 35]1 shows protein expression and secretion levels of the first polypeptide detected by sandwich ELISA (capture antibody: anti-MOG antibody, detection antibody: anti-hIgG CH3 domain antibody) using supernatants from Expi293F cells transiently transfected with DNA vectors VB5068, VB5069, or VB5070. Negative control (Neg ctrl): Supernatant from Expi293F cells treated with only the transfection reagent ExpiFectamine. [Figure 36A] Figure 1 shows the expression and secretion of the first polypeptide encoded by the indicated DNA vector, detected by sandwich ELISA (capture antibody: anti-MOG antibody, detection antibody: anti-hIgG CH3 domain antibody) using supernatants of Expi293F cells transiently transfected with the DNA vector. Negative control (Neg ctrl): Supernatant from Expi293F cells treated with transfection reagent ExpiFectamine only. The following detection antibodies were used: A) Anti-mouse SCBG3A2 biotinylated antibody, 0.83 μg / mL, 100 μL / well, BAF3465, R&D Systems. [Figure 36B] Figure 1 shows the expression and secretion of the first polypeptide encoded by the indicated DNA vector, detected by sandwich ELISA (capture antibody: anti-MOG antibody, detection antibody: anti-hIgG CH3 domain antibody) using supernatants of Expi293F cells transiently transfected with the DNA vector. Negative control (Neg ctrl): Supernatant from Expi293F cells treated with transfection reagent ExpiFectamine only. The following detection antibodies were used: B) anti-mouse PD-1 biotinylated antibody, 0.72μg / mL, 100μL / mL, DY1021, R&D Systems). [Figure 37]Expression and secretion levels of IL-10 encoded by the indicated DNA vectors were detected by sandwich ELISA using supernatants from Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): Supernatants from Expi293F cells treated with transfection reagent ExpiFectamine only. Capture antibody: mouse IL-10 antibody, 0.4 μg / mL, 100 μL / well, MAB417, R&D Systems; Detection antibody: mouse anti-IL-10 biotinylated antibody, 0.2 μg / mL, 100 μL / well, BAF417, R&D Systems. [Figure 38] Figure 1 shows distinct expression and secretion of immune inhibitory compounds encoded by the indicated DNA vectors detected by sandwich ELISA using supernatants from Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): Supernatant from Expi293F cells treated with transfection reagent ExpiFectamine only. Supernatant dilution 1:100. Capture antibody: mouse anti-MOG antibody, 0.25 μg / mL, 100 μL / well, sc-73330, Santa Cruz Biotechnology; Detection antibody: mouse anti-IL-10 biotinylated antibody, 0.2 μg / mL, 100 μL / well, BAF417, R&D Systems. [Figure 39A] Western blot analysis of supernatants from Expi293F cells transfected with the indicated DNA vectors. A: Reduced supernatant samples (35 μL loaded). Primary antibody: mouse anti-MOG (sc-73330). Secondary antibody: donkey anti-mouse, Dylight 800 (SA5-10172). Chemidoc channel Dylight 800. Black arrows indicate intact first polypeptides expressed from the respective DNA vectors as separate proteins and secreted from transfected cells. [Figure 39B]Western blot analysis of supernatants from Expi293F cells transfected with the indicated DNA vectors. B: Reduced supernatant samples (35 μL loaded). Primary antibody: rat anti-IL10 (MAB417). Secondary antibody: donkey anti-rat, Dylight 488 (SA5-10026). Chemidoc channels Dylight 650 (for protein standard) and 488. Black arrows indicate IL-10 expressed as a separate protein from the respective DNA vector and secreted from transfected cells. [Figure 40A] Results of a dual-color IL-10 / IFN-γ FluoroSpot assay are shown. C57BL / 6 mice were injected intramuscularly with the indicated DNA vectors on day 0 followed by electroporation, and spleens were harvested 7 days post-injection. Splenocytes were tested for IL-10 and IFN-γ secretion (SFU / 106 splenocytes) using dual-color FluoroSpot without restimulation (control, A) or restimulated with MOG(35-55) peptide for 44 h (B). Individual mice and mean ± SEM are shown (5 mice / group). [Figure 40B] Results of a dual-color IL-10 / IFN-γ FluoroSpot assay are shown. C57BL / 6 mice were injected intramuscularly with the indicated DNA vectors on day 0 followed by electroporation, and spleens were harvested 7 days post-injection. Splenocytes were tested for IL-10 and IFN-γ secretion (SFU / 106 splenocytes) using dual-color FluoroSpot without restimulation (control, A) or restimulated with MOG(35-55) peptide for 44 h (B). Individual mice and mean ± SEM are shown (5 mice / group). [Diagram 41]Results of MOG tetramer staining and detection of MOG-specific T cells are shown. C57BL / 6 mice were intramuscularly injected with the indicated DNA vectors on day 0, followed by electroporation, and spleens were harvested on day 7 after injection. The percentage of Foxp3+ splenocytes among CD4+MOG(38-49)tet+ splenocytes was detected by H-2 IAb / MOG(38-49) tetramer. Tetramer staining was performed ex vivo, and splenocytes were not restimulated with MOG(35-55) peptide. Data were generated from 5 mice / group, and mouse spleens were pooled before analysis. [Diagram 42] Results of MOG tetramer staining and detection of MOG-specific T cells are shown. C57BL / 6 mice were intramuscularly injected with the indicated DNA vectors on day 0, followed by electroporation, and spleens were harvested on day 7 after injection. The percentage of MOG(38-49)tet+ splenocytes among CD4+CD25+Foxp3+ splenocytes was detected by H-2 IAb / MOG(38-49) tetramer. Tetramer staining was performed ex vivo, and splenocytes were not restimulated with MOG(35-55) peptide. Data were generated from 5 mice / group, and mouse spleens were pooled before analysis. [Diagram 43] Results of detection of expanded Tregs are shown. C57BL / 6 mice were intramuscularly injected with the indicated DNA vectors on day 0, followed by electroporation, and spleens were harvested on day 7 after injection. The percentage of Ki67+ splenocytes among the Treg (CD4+CD25+Foxp3+) cell population was detected ex vivo. Data were generated from 5 mice / group, and mouse spleens were pooled before analysis. [Diagram 44] Results are shown for detection of CD4+CD25+Foxp3+ cells by flow cytometry. C57BL / 6 mice were injected with the indicated DNA vectors on day 0 followed by electroporation, and spleens were harvested on day 7 post-injection. The percentage of CD4+CD25+Foxp3+ splenocytes was determined upon restimulation with MOG(35-55) peptide for 16 hours. Data were generated from 5 mice / group, and mouse spleens were pooled prior to analysis. [Diagram 45]Figure 1 shows the expression and secretion of the first polypeptide encoded by the indicated DNA vector as detected by sandwich ELISA (Capture antibody: Mouse anti-human IgG (CH3 domain), 1ug / mL, 100μL / well, MCA878G, BioRad; Detection antibody: CaptureSelect™ Biotin anti-IgG-Fc (human) Conjugate, 1μg / mL, 100μL / well, 7103262100, Thermo Fisher) using supernatants of Expi293F cells transiently transfected with the DNA vector. Negative control (Neg ctrl): Supernatant from Expi293F cells treated with only the transfection reagent ExpiFectamine. [Figure 46] Figure 1 shows the expression and secretion levels of IL-10 encoded by the indicated DNA vectors, detected by sandwich ELISA using supernatants from Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): Supernatants from Expi293F cells treated with transfection reagent ExpiFectamine only. Capture antibody: Mouse IL-10 antibody, 0.4 μg / mL, 100 μL / well, MAB417, R&D Systems. Detection antibody: Mouse IL-10 biotinylated antibody, 0.2 μg / mL, BAF417, R&D Systems. [Figure 47] Western blot analysis of supernatants from Expi293F cells transfected with the indicated DNA vectors. Reduced supernatant samples (35 μL loaded). Primary antibody: rat anti-IL-10 (MAB417). Secondary antibody: donkey anti-rat, Dylight 650 (SA5-10029). Chemidoc channel Dylight 650. Black arrow indicates IL-10 expressed as a separate protein from the indicated DNA vector and secreted from transfected cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The first polypeptide and / or the multimeric protein are also referred to herein as constructs.

[0019] In one embodiment, the construct is a tolerance-inducing construct.

[0020] 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 inflammatory immune response, but rather induces tolerance to T cell epitopes contained in the antigenic unit.

[0021] The term "tolerance" as used herein refers to a reduction in the level of an inflammatory immune response, a delay in the onset or progression of an inflammatory immune response, and / or a reduction in the risk of onset or progression of an inflammatory immune response to an antigen, such as an autoantigen, an allergen, or an allo- or xeno-antigen.

[0022] A "subject" is an animal, such as a mouse, or a human, preferably a human. The terms "mouse / murine" and "m" are used interchangeably herein to denote or refer to a mouse. The terms human and "h" are used interchangeably herein to denote or refer to 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, allergy, or transplant rejection, or may be a subject in need of prophylactic treatment or a subject suspected of having an immune disorder. The terms "subject" and "individual" are used interchangeably herein.

[0023] A "disease" is an abnormal medical condition, usually accompanied by certain signs and symptoms in a subject suffering from the disease. As used herein, an "immune disorder" refers to a condition involving an unwanted immune response, including autoimmune disease, allergy, or transplant rejection, i.e., rejection of an allograft or xenograft, such as rejection by a host of cells, tissues, or organs from the same (allo) or different (xeno) species that have been transplanted into the host.

[0024] "Treatment" refers to prophylactic or therapeutic treatment.

[0025] "Prophylactic treatment" refers to treatment administered to a subject who does not exhibit signs or symptoms of an immune disorder, or who exhibits only early signs or symptoms of an immune disorder, such that the treatment is administered with the intent of preventing or reducing the risk of developing an immune disorder. Prophylactic treatment functions as a preventative treatment against an immune disorder, or as a treatment that inhibits or reduces the further development or enhancement of an immune disorder and / or its associated symptoms. The terms prophylactic treatment, prevention and prevention are used interchangeably herein.

[0026] A "therapeutic treatment" is a treatment administered to a subject who exhibits symptoms or signs of an immune disease, which treatment is administered to the subject with the intent of reducing or eliminating those signs or symptoms, or slowing or halting the progression of the disease.

[0027] As used herein, "T cell epitope" refers to a discrete single T cell epitope, or a portion or region of an antigen that contains multiple T cell epitopes, e.g., multiple minimal T cell epitopes, such as a hotspot.

[0028] A "nucleotide sequence" is a sequence made up of nucleotides. The terms "nucleotide sequence" and "nucleic acid sequence" are used interchangeably herein.

[0029] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0030] vector A vector of the present invention can be any molecule suitable for carrying and expressing an exogenous nucleic acid sequence, such as DNA or RNA, inside a cell, ie, an expression vector.

[0031] In one embodiment, 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.

[0032] In another embodiment, the vector is an RNA vector, such as an RNA plasmid, or an RNA viral vector, such as a retroviral vector, for example a retroviral vector selected from the group consisting of an alphavirus, a lentivirus, a Moloney murine leukemia virus, and a rhabdovirus.

[0033] In a preferred embodiment, the vector is a DNA vector, more preferably a DNA plasmid.

[0034] DNA Plasmids A plasmid is a small extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. Plasmids are most often present in bacteria as small circular double-stranded DNA molecules. However, 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 the expression of the inserted gene of interest.

[0035] 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 of the DNA, near the transcription start site of the gene. A promoter can be about 100-1000 base pairs in length. The nature of the promoter usually depends on the gene and the transcript 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 the process, the mRNA is translated many times, so that when the ribosome translates the mRNA into a protein, many copies of the protein encoded by the gene of interest are produced. Generally, the ribosome facilitates the decoding by inducing the binding of tRNA anticodon sequences complementary to the codons of the mRNA. The tRNA carries the specific amino acids that are chained together into a polypeptide as the mRNA passes through the ribosome and is "read" by the ribosome. Translation proceeds in three stages: initiation, elongation, and termination. Following the translation process, the polypeptide is folded into an active protein and either carries out its function within the cell or is transported out of the cell and carries out its function elsewhere, sometimes after a significant number of post-translational modifications.

[0036] When a protein is transported outside a cell, the signal peptide guides the protein to the endoplasmic reticulum, where the signal peptide is cleaved and, after translation is completed, the protein is transported to the periplasm.

[0037] The DNA plasmid is 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.

[0038] Coexpression The vector of the present disclosure co-expresses several proteins. Such vectors (and plasmids) are also called multicistronic or polycistronic vectors (and multicistronic or polycistronic plasmids). Those skilled in the art know how to engineer vectors to contain sequences encoding several of these proteins and can choose different techniques so that these proteins are co-expressed from one vector as separate proteins.

[0039] Thus, one skilled in the art can construct a vector of the invention that co-expresses different proteins, ie, a first polypeptide and one or more immunoinhibitory compounds.

[0040] In a preferred embodiment, a vector of the invention comprises one or more co-expression elements, ie, nucleic acid sequences allowing the co-expression of a first polypeptide and one or more immunoinhibitory compounds from the same vector.

[0041] In one embodiment of the present disclosure, the vector comprises a co-expression element (or two or more co-expression elements) such that the first polypeptide and the one or more immunoinhibitory compounds are transcribed onto a single transcript but are independently translated into the first polypeptide and the one or more immunoinhibitory compounds. Thus, the presence of the co-expression element ultimately results in the production of separate translation products.

[0042] IRES In one embodiment of the present disclosure, the co-expression element is an IRES element, the concept of which is illustrated in FIG. 1. Internal ribosome entry site, abbreviated as IRES, is an RNA element that allows translation initiation in a cap-independent manner as part of the larger process of protein synthesis. In eukaryotic translation, initiation typically occurs at the 5' end of an mRNA molecule, since the assembly of the initiation complex requires recognition of the 5' cap. By placing an IRES element between two coding regions, an initiation complex is assembled at this site, allowing translation of the downstream coding region. Thus, in one embodiment of the present disclosure, the vector contains an IRES, and one transcript is produced from the vector and then translated into a separate protein.

[0043] The IRES element allows the co-expression of the first polypeptide and one or more immunoinhibitory compounds under the control of the same promoter. The promoter directs the transcription of a single mRNA containing the coding regions of the nucleic acid sequence encoding the first polypeptide and the nucleic acid sequence encoding one or more immunoinhibitory compounds. When two or more immunoinhibitory compounds are expressed from the vector of the present invention, the vector of the present invention must have an IRES element upstream of each nucleic acid sequence encoding an immunoinhibitory compound. Alternatively, when two or more immunoinhibitory compounds are expressed from the vector of the present invention, another type of co-expression element can be used.

[0044] The IRES element used in the vectors of the invention may be derived from a viral genome or from a cellular mRNA.Vectors containing IRES elements, such as DNA plasmids, are commercially available.

[0045] 2A self-cleaving peptide In another embodiment of the present disclosure, the co-expression element is a nucleic acid sequence encoding a 2A self-cleaving peptide (or abbreviated "2A peptide"), the concept of which is depicted in FIG.

[0046] In the context of this application, the terms "2A self-cleaving peptide" and "2A peptide" are used for peptides encoded by a nucleic acid sequence that, when located between two coding regions, causes the two coding regions to be transcribed as a single transcript but translated into two separate peptide chains. Generally, when the ribosome translates the mRNA, amino acids are covalently linked from the N-terminus to the C-terminus. The presence of a nucleic acid sequence encoding a 2A self-cleaving peptide causes the ribosome to skip synthesis of the peptide bond at the C-terminus of the 2A peptide, resulting in two separate peptide chains. 2A self-cleaving peptides are typically 18-22 amino acids long and often contain the consensus sequence DXEXNPGP (SEQ ID NO: 68), where X can be any amino acid.

[0047] In one embodiment of the invention, the ribosome skips the peptide bond between the glycine and proline residues found at the C-terminus of the 2A self-cleaving peptide, meaning that the upstream gene product has a few extra amino acid residues at its end, but the downstream gene product starts with a proline.

[0048] In one embodiment, the 2A self-cleaving peptide is a sequence of 18-22 amino acids in length that contains the consensus sequence DXEXNPGP (SEQ ID NO: 68), where X can be any amino acid.

[0049] Thus, the 2A self-cleaving peptide also allows for the co-expression of a first polypeptide and one or more immuno-inhibitory compounds under the control of the same promoter. Similar to the IRES element, when two or more immuno-inhibitory compounds are expressed from the vector of the present invention, a nucleic acid sequence encoding a 2A peptide must be present in the vector upstream of each nucleic acid sequence encoding an immuno-inhibitory compound. As an example, the vector comprises a first nucleic acid sequence encoding a first polypeptide, a second nucleic acid sequence encoding a first immuno-inhibitory compound, and a third nucleic acid sequence encoding a second immuno-inhibitory compound. The vector may comprise a nucleic acid sequence encoding a T2A peptide between the first and second nucleic acid sequences, and a nucleic acid sequence encoding a P2A peptide between the second and third nucleic acid sequences. Alternatively, when two or more immuno-inhibitory compounds are expressed from the vector of the present invention, another type of co-expression element may be used.

[0050] In a further embodiment, the 2A self-cleaving peptide is a 2A peptide selected from the group consisting of a T2A peptide, a P2A peptide, an E2A peptide, and an F2A peptide.

[0051] In one embodiment, the T2A peptide has an amino acid sequence identical to a T2A sequence listed in Table 1 or Table 2. In a further embodiment, the amino acid sequence DVEENPGP (SEQ ID NO:69) is present, but the remainder of the T2A amino acid sequence has 80% to 100% sequence identity, for example 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the T2A amino acid sequence in Table 1. In another embodiment, the T2A peptide has the amino acid sequence of SEQ ID NO:6.

[0052] In one embodiment, the P2A peptide has an amino acid sequence identical to a P2A sequence listed in Table 1 or Table 2. In a further embodiment, the amino acid sequence DVEENPGP (SEQ ID NO:69) is present, but the remainder of the P2A amino acid sequence has 80%-100% sequence identity, e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the P2A amino acid sequence of Table 1. In another embodiment, the P2A peptide has the amino acid sequence of SEQ ID NO:7.

[0053] In one embodiment, the E2A peptide has an amino acid sequence identical to an E2A sequence listed in Table 1 or Table 2. In a further embodiment, the amino acid sequence DVESNPGP (SEQ ID NO:70) is present, but the remainder of the E2A amino acid sequence has 80% to 100% sequence identity, e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the E2A sequence in Table 1. In another embodiment, the E2A peptide has the amino acid sequence of SEQ ID NO:8.

[0054] In one embodiment, the F2A peptide has an amino acid sequence identical to an F2A sequence listed in Table 1 or Table 2. In a further embodiment, the amino acid sequence DVESNPGP (SEQ ID NO:70) is present, but the remainder of the F2A amino acid sequence has 80%-100% sequence identity, e.g., 81%, 82%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the F2A sequence in Table 1. In another embodiment, the F2A peptide has the amino acid sequence of SEQ ID NO:9. [Table 1] For example, it is generally known that the cleavage and expression efficiency of 2A-peptides can be adjusted to be increased by inserting a GSG sequence before the N-terminus of the wild-type sequence, as shown in Table 2. [Table 2] In another embodiment, the vector of the present invention contains both an IRES element and a nucleic acid sequence encoding a 2A peptide. As an example, the vector comprises a first nucleic acid sequence encoding a first polypeptide, a second nucleic acid sequence encoding a first immunoinhibitory compound, and a third nucleic acid sequence encoding a second immunoinhibitory compound. The vector may comprise an IRES element between the first and second nucleic acid sequences, and a nucleic acid sequence encoding a 2A peptide between the second and third nucleic acid sequences. Alternatively, the vector may comprise a nucleic acid sequence encoding a 2A peptide between the first and second nucleic acid sequences, and an IRES element between the second and third nucleic acid sequences. Additional nucleic acid sequences encoding additional immunoinhibitory compounds may be included in the vector in the same manner.

[0055] In another embodiment, the vector of the present invention contains a nucleic acid sequence encoding two 2A peptides, i.e., as a contiguous sequence of two 2A peptides. As an example, the vector comprises a first nucleic acid sequence encoding a first polypeptide and a second nucleic acid encoding an immunosuppressant compound. The vector may comprise a nucleic acid sequence encoding two 2A peptides as a contiguous sequence between the first nucleic acid sequence and the second nucleic acid sequence.

[0056] Bidirectional promoter In one embodiment of the present disclosure, the vector contains a co-expression element (or two or more co-expression elements) such that the first polypeptide and the one or more immunoinhibitory compounds are transcribed as separate transcripts, resulting in separate transcription products and thus separate proteins.

[0057] In one embodiment of the present disclosure, the co-expression element is a bidirectional promoter, the concept of which is illustrated in Figure 3. A bidirectional promoter is typically a short (e.g., less than 1 kbp) intergenic region of DNA between the 5' ends of the genes in a bidirectional gene pair. A "bidirectional gene pair" refers to two adjacent genes encoded on opposite strands with their 5' ends oriented towards each other.

[0058] In one embodiment of the present disclosure, the bidirectional promoter is a back-to-back arrangement of a CAG promoter with four CMV enhancers (Sladitschek HL, Neveu PA et al. PLoS One 11(5), e0155177, 2016).

[0059] In one embodiment of the present disclosure, the bidirectional promoter is RPBSA (Kevin He et al, Int. J. Mo.l Sci. 21(23), 9256, 2020).

[0060] In one embodiment of the present disclosure, the bidirectional promoter is a back-to-back configuration of mouse Pgk1 and human eukaryotic translation elongation factor 1 alpha 1 promoters (Golding & Mann, Gene Therapy 18, 817-826, 2011).

[0061] In one embodiment, the vector of the invention is a plasmid comprising a first nucleic acid sequence encoding a first polypeptide and a second nucleic acid sequence encoding an immunoinhibitory compound as a bidirectional gene pair comprising a bidirectional promoter between their 5' ends.

[0062] Multiple promoters In another embodiment of the present disclosure, the co-expression elements are different promoters, i.e., the vector is, for example, a plasmid comprising a separate promoter for each of the nucleic acid sequences encoding the first polypeptide and the one or more immunoinhibitory compounds, i.e., a plasmid for separate transcription of each of the first polypeptide and the one or more immunoinhibitory compounds.

[0063] In one embodiment, each of the nucleic acid sequences has a different promoter, the concept of which is illustrated in Figure 4. In one embodiment, all of the nucleic acid sequences have the same promoter for equimolar expression. In an alternative embodiment, one nucleic acid sequence has a stronger promoter than the other nucleic acid sequence. That is, the nucleic acid sequence with the stronger promoter is more likely to be expressed at a higher level than the others.

[0064] A number of promoters are known in the art and are suitable for inclusion in the plasmids of the present disclosure. In one embodiment of the present disclosure, the promoter is derived from cytomegalovirus, such as the CMV promoter.

[0065] Combinations of different co-expression elements In one embodiment, the vector of the invention comprises one or more co-expression elements, preferably a co-expression element selected from the group consisting of an IRES element, a 2A peptide, a bidirectional promoter and a promoter.

[0066] The vectors of the invention may contain any kind of combination of co-expression elements.

[0067] As an example, the vector of the invention is a DNA plasmid comprising a first nucleic acid sequence encoding a first polypeptide, a second nucleic acid sequence encoding a first immunoinhibitory compound, and a third nucleic acid sequence encoding a second immunoinhibitory compound. In one embodiment, the DNA plasmid comprises an IRES and a 2A peptide, allowing for co-expression of the first polypeptide (under the control of a promoter) and the first and second immunoinhibitory compounds. In another embodiment, the DNA plasmid comprises a bidirectional promoter and another promoter.

[0068] Those skilled in the art will understand that the terms first, second, and third nucleic acid sequences in the above examples do not mean that the plasmid of the present invention comprises nucleic acid sequences in the order of the first, second, and third nucleic acid sequences. The second nucleic acid sequence can be downstream or upstream of the first or third nucleic acid sequence, the third nucleic acid sequence can be downstream or upstream of the first or second nucleic acid sequence, and the first nucleic acid sequence can be upstream or downstream of the second or third nucleic acid sequence. In another embodiment, the first and second nucleic acid sequences can be in opposite orientations on the same DNA strand, such as the first and third or second and third nucleic acid sequences. In a further embodiment, the nucleic acid sequences encoding the first polypeptide and the immunoinhibitory compound can be on opposite DNA strands.

[0069] Immunosuppressant Compounds The vectors of the present invention contain one or more nucleic acid sequences encoding one or more immunosuppressant compounds.

[0070] In one embodiment of the disclosure, the immunoinhibitory compound is a compound that induces, increases or maintains immune tolerance. In another embodiment of the disclosure, the immunoinhibitory compound is a compound known to induce, increase or maintain immune tolerance.

[0071] In yet another embodiment, the immunoinhibitory compound is a compound that favors the presentation of epitopes in antigenic units in a tolerance-inducing manner and / or a compound that favors the induction of tolerance-maintaining cells (not only regulatory T cells, anergy or apoptosis of effector T cells) and / or a compound that helps maintain such tolerance-maintaining cells.

[0072] In yet another embodiment, the immunoinhibitory compound is a compound that promotes and / or supports the presentation of epitopes in antigenic units in a tolerance-inducing manner and / or promotes and / or supports the induction of tolerance-maintaining cells (not only regulatory T cells, anergy or apoptosis of effector T cells) and / or helps maintain such tolerance-maintaining cells. In one embodiment of the present disclosure, the immune inhibitory compound is an extracellular portion, such as an extracellular domain, of an inhibitory checkpoint molecule. In one embodiment, the inhibitory checkpoint molecule is selected from the group consisting of CLTA-4, PD-1, BTLA, LAG3, NOX2, SIGLEC7, SIGLEC9, and TIM-3. In one embodiment, the inhibitory checkpoint molecule is CLTA-4. In one embodiment, the inhibitory checkpoint molecule is PD-1. In one embodiment, the inhibitory checkpoint molecule is BTLA. In one embodiment, the inhibitory checkpoint molecule is TIM-3.

[0073] In a preferred embodiment, the immune inhibitory compound is an extracellular portion, such as an extracellular domain, of a human (h) inhibitory checkpoint molecule, e.g., an extracellular portion, such as an extracellular domain, of a human inhibitory checkpoint molecule selected from the group consisting of hCLTA-4, hPD-1, hBTLA, hLAG3, hNOX2, hSIGLEC7, hSIGLEC9, and hTIM-3. In one embodiment, the inhibitory checkpoint molecule is hCLTA-4, such as hCTLA-4 having SEQ ID NO: 51. In one embodiment, the inhibitory checkpoint molecule is hPD-1, such as hPD-1 having SEQ ID NO: 52. In one embodiment, the inhibitory checkpoint molecule is hBTLA. In one embodiment, the inhibitory checkpoint molecule is hTIM-3. In one embodiment of the disclosure, the immune inhibitory compound is a cytokine selected from the group consisting of IL-10, TGF-β1, TGF-β2, TGF-β3, IL-27, IL-2, GM-CSF, FLT3L, IFN-γ, IL-37, and IL-35. In one embodiment, the cytokine is IL-10. In one embodiment, the cytokine is TGF-β1. In one embodiment, the cytokine is IL-27. In one embodiment, the cytokine is IL-2. In one embodiment, the cytokine is GM-CSF. In one embodiment, the cytokine is FLT3L. In one embodiment, the cytokine is IFN-γ. In one embodiment, the cytokine is IL-37. In one embodiment, the cytokine is IL-35.

[0074] In a preferred embodiment, the immune inhibitory compound is a human cytokine selected from the group consisting of hIL-10, hTGF-β1, hTGF-β2, hTGF-β3, hIL-27, hIL-2, hGM-CSF, hFLT3L, hIFN-γ, hIL-37, and hIL-35. In one embodiment, the cytokine is hIL-10, such as hIL-10 having SEQ ID NO:53. In one embodiment, the cytokine is hTGF-β1, such as hTGF-β1 having SEQ ID NO:54. In one embodiment, the cytokine is hTGF-β2, such as hTGF-β2 having SEQ ID NO:58. In one embodiment, the cytokine is hTGF-β3, such as hTGF-β3 having SEQ ID NO:59. In one embodiment, the cytokine is hIL-27. In one embodiment, the cytokine is hIL-2, such as hIL-2 having SEQ ID NO:55. In one embodiment, the cytokine is hGM-CSF, such as hGM-CSF having SEQ ID NO:56. In one embodiment, the cytokine is hFLT3L. In one embodiment, the cytokine is hIFN-γ, such as hIFN-γ having SEQ ID NO:57. In one embodiment, the cytokine is hIL-37. In one embodiment, the cytokine is hIL-35.

[0075] Vectors containing multiple nucleic acid sequences encoding multiple immunosuppressive compounds In one embodiment of the present disclosure, the vector comprises a nucleic acid sequence encoding 2, 3, 4, 5, 6, 7 or 8 immunoinhibitory compounds. In another embodiment, the vector comprises a nucleic acid sequence encoding 2-6 immunoinhibitory compounds, i.e., 2 or 3 or 4 or 5 or 6 immunoinhibitory compounds. The immunoinhibitory compounds may be the same or different, preferably different.

[0076] In a preferred embodiment, the different immunoinhibitory compounds generate or promote a tolerance-inducing environment at many different levels. By way of example, a vector of the invention comprises nucleic acid sequences encoding three different immunoinhibitory compounds, the first of which induces tolerance, the second of which increases tolerance, and the third of which maintains tolerance.

[0077] First Nucleic Acid Sequence The vector of the present disclosure comprises a first nucleic acid sequence that encodes a first polypeptide, i.e., DNA or RNA, including genomic DNA, cDNA and mRNA, either double-stranded or single-stranded. In one embodiment, the first nucleic acid sequence is DNA. In another embodiment, the first nucleic acid sequence is optimized for the species of the subject to which it is administered. When administered to humans, in one embodiment, the first nucleic acid sequence is optimized for human codons.

[0078] The first nucleic acid sequence encodes a first polypeptide comprising a targeting unit for targeting APCs, a multimerization unit, such as 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. Upon administration to a subject, the first polypeptide is expressed and forms a multimeric protein due to the presence of the multimerization unit, which induces a tolerogenic response against one or more T cell epitopes comprised in the antigenic unit.

[0079] Constructs The construct can be described as a polypeptide having an N-terminal start and a C-terminus (as shown in FIG. 5). The elements and units of the first polypeptide (targeting unit (TU), multimerization unit, e.g., in this FIG. 5, 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. 5a) or at the N-terminal start of the polypeptide (FIG. 5b). Preferably, the antigenic unit is located at the C-terminus of the polypeptide.

[0080] 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 to separate the T cell epitopes. A unit linker (UL) may connect a multimerization unit, such as a dimerization unit, with an antigenic unit. Figure 5 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 the multimeric protein and the first nucleic acid sequence encoding the first polypeptide.

[0081] In the following, the various units and elements of the first polypeptide / construct are described in detail, which are present in the first nucleic acid sequence as nucleic acid sequences encoding the units / elements, but in the first polypeptide or multimeric protein as amino acid sequences. For ease of reading, in the following, the units / elements are described mainly in relation to the first polypeptide / multimeric protein, i.e. based on their amino acid sequences.

[0082] Targeting Unit The first polypeptide encoded by the first nucleic acid contained in the vector of the present invention comprises a targeting unit that targets an APC.

[0083] The term "targeting unit" as used herein refers to a unit that delivers the construct of the present invention to an antigen-presenting cell and interacts with a surface molecule on the APC without activating the cell, e.g., binds to a surface receptor on the APC.

[0084] In another embodiment, the term "targeting unit" as used herein refers to a unit that delivers the construct of the present invention to an antigen-presenting cell and interacts with a surface molecule on the APC without inducing cell maturation, e.g., binds to a surface receptor on the APC.

[0085] The APCs internalize the construct and present the T cell epitopes contained in the antigenic units on the MHC on their surface in an anti-inflammatory, tolerogenic manner, for example by not upregulating costimulatory signals and / or by upregulating inhibitory surface molecules and / or by promoting secretion of inhibitory cytokines.

[0086] In one embodiment, the targeting unit is selected from the group consisting of TGFβ receptors (including TGFβR1, TGFβR2, and TGFβR3), IL-10R, such as IL-10RA and IL-10RB, IL-2R, IL-4R, IL-6R, IL-11R, IL-13R, IL-27R, IL-35R, IL-37R, GM-CSFR, FLT3, CCR7, CD11b, CD11c, CD103, CD14, CD36, CD205, CD109, VISTA, MARCO, MHCII, CD83, SIGLEC, Clec10A (MGL), ASGR, IL-10R ... (ASGR1 / ASGR2), CD80, CD86, Clec9A, Clec12A, Clec12B, DCIR2, Langerin, MR, DC-Sign, Treml4, Dectin-1, PDL1, PDL2, HVEM, CD163 and CD141.

[0087] In a preferred embodiment, the targeting unit is selected from the group consisting of hTGFβ receptors (including hTGFβR1, hTGFβR2, and hTGFβR3), hIL-10R, such as hIL-10RA and hIL-10RB, hIL-2R, hIL-4R, hIL-6R, hIL-11R, hIL-13R, hIL-27R, hIL-35R, hIL-37R, hGM-CSFR, hFLT3, hCCR7, hCD11b, hCD11c, hCD103, hCD14, hCD36, hCD205, hCD109, hVISTA, hIL-10R ... The invention comprises or consists of a portion that binds to a surface molecule on a human (h)APC selected from the group consisting of MARCO, hMHCII, hCD83, hSIGLEC, hClec10A (hMGL), hASGR (hASGR1 / hASGR2), hCD80, hCD86, hClec9A, hClec12A, hClec12B, hDCIR2, hLangerin, hMR, hDC-Sign, hTreml4, hDectin-1, hPDL1, hPDL2, hHVEM, hCD163 and hCD141.

[0088] The moiety may be a natural ligand, an antibody or part thereof, such as an scFv, or a synthetic ligand.

[0089] In one embodiment, the portion is an antibody or portion thereof, such as an scFv, having specificity for any of the aforementioned surface molecules, and upon binding to the surface molecule, presents T cell epitopes contained in the antigenic unit in an anti-inflammatory, tolerogenic manner.

[0090] In another embodiment, the moiety is a synthetic ligand with specificity for any of the aforementioned surface molecules, binding to which presents T cell epitopes contained in the antigenic unit in an anti-inflammatory, tolerogenic manner. Protein modeling can be used to design such synthetic ligands.

[0091] In yet another embodiment, the moiety is a natural ligand. In one embodiment, the natural ligand is selected from the group consisting of TGFβ, IL-10, IL-2, IL-4, IL-6, IL-11, IL-13, IL-27, IL-35, IL-37, GM-CSF, FLT3L, CCL19, CCL21, ICAM-1 (intercellular adhesion molecule 1, also known as CD54), keratin, VSIG-3, preferably the extracellular domain of VSIG-3, SCGB3A2, CTLA-4, preferably the extracellular domain of CTLA-4, PD-1, preferably the extracellular domain of PD-1, and BTLA, preferably the extracellular domain of BTLA.

[0092] In a preferred embodiment, the moiety is a human (h) natural ligand selected from the group consisting of hTGFβ, hIL-10, hIL-2, such as hIL-2 having SEQ ID NO:55, hIL-4, hIL-6, hIL-11, hIL-13, hIL-27, hIL-35, hIL-37, hGM-CSF, such as hGM-CSF having SEQ ID NO:56, hFLT3L, hCCL19, hCCL21, hICAM-1 (intercellular adhesion molecule 1, also known as CD54), hKeratin, hVSIG-3, preferably the extracellular domain of hVSIG-3, hSCGB3A2, hCTLA-4, preferably the extracellular domain of hCTLA-4, such as the extracellular domain of hCTLA4 having SEQ ID NO:51, hPD-1, preferably the extracellular domain of PD-1, such as the extracellular domain of hPD-1 having SEQ ID NO:52, and hBTLA, preferably the extracellular domain of hBTLA.

[0093] In another embodiment, the targeting unit is or comprises IL10 or TGFβ, preferably human IL-10 or human TGFβ, its isoforms TGFβ-1, TGFβ-2 and TGFβ-3.

[0094] In another embodiment, the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of human TGFβ. In one embodiment, the targeting unit comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 58 or SEQ ID NO: 59. In another embodiment, the targeting unit consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 58 or SEQ ID NO: 59.

[0095] In yet another embodiment, the targeting unit comprises an amino acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 58 or SEQ ID NO: 59. In an even further preferred embodiment, the targeting unit comprises the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 58 or SEQ ID NO: 59.

[0096] In yet another embodiment, the targeting unit consists of an amino acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 58 or SEQ ID NO: 59. In an even further preferred embodiment, the targeting unit consists of the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 58 or SEQ ID NO: 59.

[0097] In a preferred embodiment, the targeting unit comprises the amino acid sequence of SEQ ID NO:54 or SEQ ID NO:58 or SEQ ID NO:59, except that at most 80 amino acids, such as at most 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid have been substituted, deleted or inserted. In a preferred embodiment, the targeting unit consists of the amino acid sequence of SEQ ID NO:54 or SEQ ID NO:58 or SEQ ID NO:59, except that at most 80 amino acids, such as at most 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid have been substituted, deleted or inserted. In a preferred embodiment, the targeting unit comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence having SEQ ID NO:60 or SEQ ID NO:61 or SEQ ID NO:62. In a further preferred embodiment, the targeting unit comprises a nucleic acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to a nucleic acid sequence having SEQ ID NO: 60 or SEQ ID NO: 61 or SEQ ID NO: 62. In an even further preferred embodiment, the targeting unit comprises a nucleic acid sequence of SEQ ID NO: 60 or SEQ ID NO: 61 or SEQ ID NO: 62. In a more preferred embodiment, the targeting unit consists of a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence having SEQ ID NO:60 or SEQ ID NO:61 or SEQ ID NO:62. In a further preferred embodiment, the targeting unit consists of a nucleic acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 60 or SEQ ID NO: 61 or SEQ ID NO: 62. In yet another preferred embodiment, the targeting unit has the nucleic acid sequence of SEQ ID NO: 60 or SEQ ID NO: 61 or SEQ ID NO: 62.

[0098] In yet another embodiment, the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of human IL-10. In one embodiment, the targeting unit comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 53. In another embodiment, the targeting unit consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 53. In yet another embodiment, the targeting unit comprises an amino acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 53. In an even further preferred embodiment, the targeting unit comprises the amino acid sequence of SEQ ID NO: 53. In yet another embodiment, the targeting unit consists of an amino acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 53. In an even further preferred embodiment, the targeting unit consists of the amino acid sequence of SEQ ID NO: 53. In a preferred embodiment, the targeting unit comprises the amino acid sequence of SEQ ID NO: 53, except that at most 35 amino acids, such as at most 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids have been substituted, deleted or inserted. In a preferred embodiment, the targeting unit consists of the amino acid sequence of SEQ ID NO: 53, except that at most 35 amino acids, such as at most 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids have been substituted, deleted or inserted. In a preferred embodiment, the targeting unit comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence having SEQ ID NO:63. In a further preferred embodiment, the targeting unit comprises a nucleic acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to a nucleic acid sequence having SEQ ID NO: 63. In an even further preferred embodiment, the targeting unit comprises the nucleic acid sequence of SEQ ID NO: 63. In a more preferred embodiment, the targeting unit consists of a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence having SEQ ID NO:63. In a further preferred embodiment, the targeting unit consists of a nucleic acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 63. In yet another preferred embodiment, the targeting unit has the nucleic acid sequence of SEQ ID NO: 63.

[0099] In one embodiment, the targeting unit is or comprises an extracellular portion, such as the extracellular domain, of SCGB3A2 or VSIG-3, preferably human VSIG-3, preferably human VSIG-3 or human SCGB3A2.

[0100] In another embodiment, the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of human SCGB3A2. In one embodiment, the targeting unit comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 64. In another embodiment, the targeting unit consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 64.

[0101] In yet another embodiment, the targeting unit comprises an amino acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 64. In an even further preferred embodiment, the targeting unit comprises the amino acid sequence of SEQ ID NO: 64.

[0102] In yet another embodiment, the targeting unit consists of an amino acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 64. In an even further preferred embodiment, the targeting unit consists of the amino acid sequence of SEQ ID NO: 64.

[0103] In a preferred embodiment, the targeting unit comprises the amino acid sequence of SEQ ID NO: 64, except that at most 18 amino acids, such as at most 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids, have been substituted, deleted or inserted. In a preferred embodiment, the targeting unit consists of the amino acid sequence of SEQ ID NO: 64, except that at most 18 amino acids, such as at most 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids, have been substituted, deleted or inserted. In a preferred embodiment, the targeting unit comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence having SEQ ID NO:65. In a further preferred embodiment, the targeting unit comprises a nucleic acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to a nucleic acid sequence having SEQ ID NO: 65. In an even further preferred embodiment, the targeting unit comprises the nucleic acid sequence of SEQ ID NO: 65. In a more preferred embodiment, the targeting unit consists of a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence having SEQ ID NO:65. In a further preferred embodiment, the targeting unit consists of a nucleic acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 65. In yet another preferred embodiment, the targeting unit has the nucleic acid sequence of SEQ ID NO: 65.

[0104] In yet another embodiment, the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of the extracellular domain of human VSIG-3. In one embodiment, the targeting unit comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 66. In another embodiment, the targeting unit consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 66.

[0105] In yet another embodiment, the targeting unit comprises an amino acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 66. In an even further preferred embodiment, the targeting unit comprises the amino acid sequence of SEQ ID NO: 67. In yet another embodiment, the targeting unit consists of an amino acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 66. In an even more preferred embodiment, the targeting unit consists of the amino acid sequence of SEQ ID NO: 66. In a preferred embodiment, the targeting unit comprises the amino acid sequence of SEQ ID NO: 66, except that at most 86 amino acids, such as at most 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid have been substituted, deleted or inserted. In a preferred embodiment, the targeting unit consists of the amino acid sequence of SEQ ID NO: 66, except that at most 86 amino acids, for example at most 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid have been substituted, deleted or inserted. In a preferred embodiment, the targeting unit comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence having SEQ ID NO:67. In a further preferred embodiment, the targeting unit comprises a nucleic acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to a nucleic acid sequence having SEQ ID NO: 67. In an even further preferred embodiment, the targeting unit comprises the nucleic acid sequence of SEQ ID NO: 67. In a more preferred embodiment, the targeting unit consists of a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence having SEQ ID NO:67. In a further preferred embodiment, the targeting unit consists of a nucleic acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 67. In yet another preferred embodiment, the targeting unit has the nucleic acid sequence of SEQ ID NO: 67.

[0106] In yet another embodiment, the targeting unit is or comprises an antibody or portion thereof (eg, scFv) with specificity for CD205, such as anti-human CD205.

[0107] Multimerization Unit / Dimerization Unit The first polypeptide encoded by the first nucleic acid sequence contained in the vector of the present invention comprises a multimerization unit, such as a dimerization unit.

[0108] 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 connecting the antigenic unit and the targeting unit, the multimerization unit promotes / links the multimerization of multiple polypeptides, e.g., two, three, four or more polypeptides, into a multimeric protein, e.g., a dimeric protein, a trimeric protein or a tetrameric protein. Furthermore, the multimerization unit also provides flexibility to the multimeric protein, allowing the targeting unit to optimally bind to multiple surface molecules on the APC, even when located at variable distances. The multimerization unit can be any unit that meets one or more of these requirements.

[0109] Multimerization units that promote / link multimerization of two 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 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 comprising or consisting of a nucleic acid sequence having SEQ ID NO: 116, or an amino acid sequence encoded by said nucleic acid 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 comprising or consisting of the amino acid sequence of SEQ ID NO: 117. 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: 113, or an amino acid sequence encoded by said nucleic acid sequence, optionally further comprising a hinge region as described below.

[0110] 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 connecting the antigenic unit and the targeting unit, the dimerization unit also promotes / links the dimerization of two monomeric polypeptides into a dimeric protein. Furthermore, the dimerization unit also provides flexibility to the dimeric protein, allowing the targeting unit to optimally bind to multiple surface molecules on the APC, even when located at variable distances. The dimerization unit can be any unit that meets these requirements.

[0111] Thus, in one embodiment, the first polypeptide comprises a dimerization unit comprising a hinge region. In another embodiment, the dimerization unit comprises a hinge region and another domain that promotes dimerization. In yet another embodiment, the dimerization unit comprises a hinge region, a dimerization unit linker, and another domain that promotes dimerization, the dimerization unit linker connecting the hinge region and the other domain that promotes dimerization. In one embodiment, the dimerization unit linker is a glycine-serine rich linker, preferably GGGSSGGGSG (SEQ ID NO: 118), i.e., the dimerization unit comprises a glycine-serine rich dimerization unit linker, preferably the dimerization unit linker GGGSSGGGSG (SEQ ID NO: 118).

[0112] The term "hinge region" refers to an amino acid sequence contained in a dimerization unit that contributes to the binding of two polypeptides, i.e., promotes the formation of a dimeric protein. In the context of a multimerization unit that promotes / links the multimerization of two or more polypeptides, the term "hinge region" refers to an amino acid sequence contained in such a multimerization unit that contributes to the linking of two or more polypeptides, e.g., three or four polypeptides, and / or functions as a flexible spacer, allowing two targeting units of a multimeric protein to simultaneously bind to multiple surface molecules on an APC, even if they are located at variable distances. The hinge region can be from an Ig, e.g., from an IgG, e.g., from IgG1, IgG2 or IgG3. In one embodiment, the hinge region is from an IgM, e.g., comprises or consists of a nucleotide sequence having SEQ ID NO: 119 or an amino acid sequence encoded by said nucleic acid sequence.

[0113] The hinge region can contribute to dimerization through the formation of covalent bonds, such as disulfide bridges between cysteines. Thus, in one embodiment, the hinge region has the ability to form one or more covalent bonds. Preferably, the covalent bonds are disulfide bridges.

[0114] In one embodiment, the dimerization unit comprises or consists of hinge exon h1 and hinge exon h4 (human hinge region 1 and human hinge region 4) of IgG3, preferably having an amino acid sequence that has at least 80% sequence identity to amino acid sequence 1 to 27 of SEQ ID NO:1.

[0115] In a preferred embodiment, the dimerization unit comprises or consists of hinge exon h1 and hinge exon h4 having an amino acid sequence of at least 85% sequence identity to 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%, such as at least 98% or such as at least 99% sequence identity.

[0116] In a preferred embodiment, the dimerization unit comprises or consists of hinge exon h1 and hinge exon h4 having amino acid sequence 1-27 of SEQ ID NO:1. In a preferred embodiment, the dimerization unit comprises or consists of the amino acid sequence 1-27 of SEQ ID NO:1, except that at most 4 amino acids, such as at most 3 amino acids, such as at most 2 amino acids or such as at most 1 amino acid, have been substituted, deleted or inserted. In a preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence having SEQ ID NO:10. In a further preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 85% sequence identity to a nucleic acid sequence having SEQ ID NO: 10, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity. In an even more preferred embodiment, the dimerization unit comprises or consists of the nucleic acid sequence of SEQ ID NO:10.

[0117] In another embodiment, the dimerization unit comprises another domain that promotes dimerization, which is an immunoglobulin domain, for example 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 IgG. More preferably, the other domain that promotes dimerization is a carboxy-terminal C domain derived from IgG3.

[0118] In one embodiment, 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 acids 38 to 144 of SEQ ID NO:1.

[0119] 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 38 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%, such as at least 98% or such as at least 99% sequence identity.

[0120] In a preferred embodiment, the dimerization unit comprises a carboxy-terminal C domain derived from IgG3 having amino acid sequence 38 to 144 of SEQ ID NO:1.

[0121] In a preferred embodiment, the dimerization unit comprises or consists of the amino acid sequence 38-144 of SEQ ID NO:1, except that at most 16 amino acids, such as at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, are substituted, deleted or inserted.

[0122] In a preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence having SEQ ID NO:11.

[0123] In a further preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 85% sequence identity, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to a nucleic acid sequence having SEQ ID NO: 11. In an even further preferred embodiment, the dimerization unit comprises or consists of the nucleic acid sequence of SEQ ID NO: 11.

[0124] Immunoglobulin domains contribute to dimerization through non-covalent interactions, such as hydrophobic interactions. Thus, in one embodiment, an immunoglobulin domain has the ability to form a dimer through non-covalent interactions. Preferably, the non-covalent interactions are hydrophobic interactions.

[0125] If a dimerization unit comprises a CH3 domain, it preferably does not comprise a CH2 domain, and vice versa.

[0126] 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. In another 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.

[0127] In one embodiment, the dimerization unit comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:1.

[0128] In a preferred embodiment, the dimerisation 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%, such as at least 98% or such as at least 99% sequence identity.

[0129] In an even more preferred embodiment, the dimerization unit comprises the amino acid sequence of SEQ ID NO:1.

[0130] In a more preferred embodiment, the dimerization unit consists of an amino acid sequence having at least 80%, 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%, such as 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:1.

[0131] In an even more preferred embodiment, the dimerization unit consists of the amino acid sequence of SEQ ID NO:1.

[0132] In a preferred embodiment, the dimerization unit comprises or consists of the amino acid sequence of SEQ ID NO:1, except that up to 28 amino acids, such as up to 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids have been substituted, deleted or inserted.

[0133] In a preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence having SEQ ID NO:12. In a further preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 85% sequence identity to a nucleic acid sequence having SEQ ID NO: 12, such as at least 86% or at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity. In an even more preferred embodiment, the dimerization unit comprises or consists of the nucleic acid sequence of SEQ ID NO:12.

[0134] In the first polypeptide encoded by the first nucleic acid sequence, the multimerization unit, e.g., the dimerization unit, may have any orientation with respect to the antigenic unit and the targeting unit. In one embodiment, the antigenic unit is connected to the C-terminus of the multimerization / dimerization unit (e.g., via a unit linker), and the targeting unit is connected to the N-terminus of the multimerization / dimerization unit. In another embodiment, the antigenic unit is connected to the N-terminus of the multimerization / dimerization unit (e.g., via a unit linker), and the targeting unit is connected to the C-terminus of the multimerization / dimerization unit. It is preferred that the antigenic unit is connected to the C-terminus of the multimerization / dimerization unit, e.g., via a linker, preferably via a unit linker, and the targeting unit is connected to the N-terminus of the multimerization / dimerization unit.

[0135] Antigenic unit The first polypeptide encoded by the first nucleic acid sequence contained in the vector of the present invention comprises an antigenic unit comprising one or more T cell epitopes of an autoantigen, e.g., one or more epitopes of regulatory T cells (Treg) or one or more inhibitory neoantigens, an allergen, an alloantigen or a xenoantigen.

[0136] T cell epitopes suitable for inclusion in an antigenic unit may be known in the art, i.e., have been tested, proposed and / or verified to be involved and relevant to a particular immune disease and have been published, for example, in the scientific literature.

[0137] In one embodiment, 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 one embodiment, the multiple T cell epitopes are of the same autoantigen, i.e. comprised in the same autoantigen. In another embodiment, the multiple T cell epitopes are of different autoantigens, i.e. comprised in different autoantigens.

[0138] The terms "plurality," "multiple," and "several" are used interchangeably herein with "two or more."

[0139] 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 examined 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.

[0140] In another embodiment the antigenic unit comprises one or more T cell epitopes of an allergen, i.e. one T cell epitope of an allergen, or two or more T cell epitopes of an allergen, i.e. multiple T cell epitopes of an allergen. In one embodiment the multiple T cell epitopes are of the same allergen, i.e. comprised in the same allergen. In another embodiment the multiple T cell epitopes are of different allergens, i.e. comprised in different allergens.

[0141] By way of example, Fel d 1, Fel d 4 and Fel d 7 are three of the most prominent cat allergens and account for the majority of cat allergies in humans, and an antigenic unit may, for example, comprise one or more T cell epitopes of Fel d 1, i.e. one T cell epitope of Fel d 1 or multiple T cell epitopes of Fel d 1. Furthermore, an antigenic unit may, for example, comprise multiple T cell epitopes of Fel d 4 and Fel d 7, e.g. one or more T cell epitopes of Fel d 4 and one or more T cell epitopes of Fel d 7.

[0142] In another embodiment, 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 two or more T cell epitopes of an allo / xenoantigen, i.e. multiple T cell epitopes of an allo / xenoantigen. In one embodiment, the multiple T cell epitopes are of the same allo / xenoantigen, i.e. comprised in the same allo / xenoantigen. In another embodiment, the multiple T cell epitopes are of different allo / xenoantigens, i.e. comprised in different allo / xenoantigens.

[0143] In one embodiment, the antigenic unit comprises one T cell epitope, hi another embodiment, the antigenic unit comprises two or more T cell epitopes, i.e. a plurality of T cell epitopes.

[0144] In one embodiment, the vectors of the invention / constructs encoded by such vectors are for use in personalized therapy, i.e., therapy specifically designed for a particular subject / single patient, hi another embodiment, the vectors of the invention / constructs encoded by such vectors are for general use in a patient population or patient, i.e., off-the-shelf therapy.

[0145] Individualized constructs For individualized constructs, T cell epitopes optimized for a patient receiving treatment with a vector encoding such a construct are selected to be included in the antigenic unit, which will increase the therapeutic effect compared to off-the-shelf treatments that include the construct.

[0146] The antigenic units of the individualized construct can be designed as follows, as exemplified for a patient suffering from MS: 1) Determine the patient's HLA class I and / or HLA class II alleles. 2) Identifying that the T cell epitope is contained in one or more autoantigens (e.g., autoantigens that have been tested, 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) Design and produce one or more test constructs, optionally placing T cell epitopes into the antigenic units of the constructs as described in this application.

[0147] 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 a predictive HLA binding algorithm. 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.

[0148] Any suitable HLA binding algorithm can be used, for example one of the following: Available software analyses 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 Off-the-shelf tolerance-inducing constructs The antigenic units of the prefabricated constructs encoded by the vectors of the invention may contain discrete T cell epitopes, minimal T cell epitope hotspots, or both. Such antigenic units preferably contain minimal T cell epitope hotspots, i.e., one or more regions of an antigen that contain multiple minimal T cell epitopes (e.g., having a length of 7-15 amino acids) predicted to be presented by different HLA alleles, covering a wide range of subjects, e.g., ethnic populations or even the world population. The inclusion of such hotspots maximizes the likelihood that the construct will induce tolerance in a wide range of subjects.

[0149] Further embodiments of the antigenic unit The T cell epitope contained in the antigenic unit of the first polypeptide encoded by the vector of the present invention has a length of 7 to about 200 amino acids, and longer T cell epitopes may contain minimal T cell epitope hotspots.

[0150] In one embodiment, the antigenic unit comprises one or more T cell epitopes 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.

[0151] A T cell epitope having a length of about 60 to 200 amino acids may be split into shorter sequences and included in an antigenic unit separated by a linker, such as a linker described herein. As an example, a T cell epitope having a length of 150 amino acids may be split into three sequences of 50 amino acids each and included in an antigenic unit with a linker separating the three sequences from each other.

[0152] In one embodiment, the length of one T cell epitope is adjusted so that the protein containing the T cell epitope is not folded correctly. For example, Fel d 1, the most prominent cat allergen, is a protein formed by two heterodimers, each dimer consisting 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 mast cells and basophils of a subject bind to the antigenic unit of the construct containing the T cell epitope.

[0153] Thus, if a longer T cell epitope is included in the antigenic unit, folding of the protein can be tested in vitro, such as by ELISA using an antibody against the protein (e.g., cat allergen) to determine whether the antibody binds to the T cell epitope. If the antibody binds to the T cell epitope, its length can be adjusted or split into shorter sequences as described herein.

[0154] In one embodiment, 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 a preferred embodiment, the antigenic unit comprises a T cell epitope having a length suitable for specific presentation on MHC class I or MHC class II. In one embodiment, the T cell epitope has a length of 7-11 amino acids for MHC class I presentation. In another embodiment, the T cell epitope has a length of about 15 amino acids for MHC class II presentation.

[0155] Location and details of T cell epitopes The number of T cell epitopes in an antigenic unit may vary and depends on other elements contained in the antigenic unit, such as the length and number of T cell epitope linkers described in this application.

[0156] In one embodiment, 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.

[0157] In one embodiment the antigenic unit comprises 1 to 10 T cell epitopes, such as 1, 2, 3, 4, 5, 6, 7, 8 or 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, 22, 23, 24, 25, 26, 27, 28, 29 or 30 T cell epitopes, or 31 to 40 T cell epitopes, such as 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 T cell epitopes, or 41 to 50 T cell epitopes, such as 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 T cell epitopes.

[0158] In one embodiment, the T cell epitopes are randomly arranged in the antigenic unit, in another embodiment, one or more of the following methods for arranging them in the antigenic unit can be used:

[0159] In one embodiment, the T cell epitopes are arranged from more antigenic to less antigenic in the direction from the multimerization unit (such as the dimerization unit) to the termini of the antigenic unit (see FIG. 5). Alternatively, 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 termini of the multimerization unit or antigenic unit, particularly when the hydrophilicity / hydrophobicity of the T cell epitopes varies significantly.

[0160] Since exact central location of an 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, in which the most hydrophobic T cell epitope is located as close to the middle as possible.

[0161] As an example, an antigenic unit may contain five T cell epitopes, which are: 1-2-3 * - Arranged like 4-5, 1, 2, 3 * , 4 and 5 are different T cell epitopes, and - is a T cell epitope linker; * represents the most hydrophobic T cell epitope, which is located in the center of the antigenic unit.

[0162] In another example, the antigenic unit comprises six T cell epitopes, which are as follows: 1-2-3 * -4-5-6, or: 1-2-4-3 * - Arranged like 5-6, 1, 2, 3 * , 4, 5 and 6 are T cell epitopes, and - is a T cell epitope linker; * represents the most hydrophobic T cell epitope, which is located essentially in the center of the antigenic unit.

[0163] Alternatively, T cell epitopes can be arranged alternately between hydrophilic and hydrophobic T cell epitopes. Optionally, the GC-rich sequences encoding T cell epitopes are arranged to avoid GC clusters. In one embodiment, the GC-rich sequences encoding T cell epitopes are arranged such that there is at least one non-GC-rich sequence between them.

[0164] T cell epitope linker When an antigenic unit contains multiple T cell epitopes, the epitopes are preferably separated by T cell epitope linkers, which allows each T cell epitope to be presented to the immune system in an optimal manner. 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.

[0165] In one embodiment, the T cell epitope linker is designed to be non-immunogenic. The T cell epitope linker can be a rigid linker, which means that the two amino acid sequences that the linker connects do not allow substantially free movement relative to each other. Alternatively, the T cell epitope linker can be a flexible linker, i.e., a linker that allows the two amino acid sequences that the linker connects to move substantially freely relative to each other. Both types of linkers are useful. In one embodiment, the linker is a flexible linker, which can present the T cell epitopes to the immune system in an optimal manner, even if the antigenic unit contains multiple T cell epitopes.

[0166] In one embodiment, the T cell epitope linker is a peptide consisting of 4 to 40 amino acids, for example, 35, 30, 25 or 20 amino acids, for example, 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, or 10 to 15 amino acids, or 8 to 12 amino acids. In one embodiment, the T cell epitope linker consists of 10 amino acids.

[0167] In one embodiment, all T cell epitope linkers contained in an antigenic unit are identical. However, if one or more T cell epitopes contain a sequence similar to that of a linker, it may be advantageous to replace adjacent T cell epitope linkers with linkers of different sequences. In addition, if it is predicted that a T cell epitope / linker junction constitutes an epitope by itself, it is preferable to use T cell epitope linkers of different sequences.

[0168] In one embodiment, the T cell epitope linker is a flexible linker, preferably a flexible linker comprising small non-polar (e.g., glycine, alanine or leucine) or polar (e.g., serine or threonine) amino acids. These amino acids are flexible due to their small size, allowing movement of the connected amino acid sequence. The incorporation of serine or threonine reduces unfavorable interactions between the linker and the antigen, since it can form hydrogen bonds with water molecules and maintain the stability of the linker in aqueous solution. In one embodiment, the flexible linker is a serine (S) and / or glycine (G) rich linker, i.e., a linker comprising several serine and / or several glycine residues. Preferred examples are GGGGSGGGSS (SEQ ID NO: 75), GGGSG (SEQ ID NO: 76), GGSGG (SEQ ID NO: 77), SGSSGS (SEQ ID NO: 78), or multiple variants thereof, such as GGGGSGGGGS (SEQ ID NO: 79), (GGGGS)m (SEQ ID NO: 80), (GGGS)m (SEQ ID NO: 81), (GGSGG)m (SEQ ID NO: 82), (GGGSG)m (SEQ ID NO: 83)) or (SGSSGS)m (SEQ ID NO: 84), where m is an integer from 1 to 5, such as 1, 2, 3, 4 or 5, and in a preferred embodiment, m is 2. In another preferred embodiment, 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, such as 1, 2, 3 or 4 leucine residues.

[0169] In one embodiment, the T cell epitope linker comprises or consists of LGGGS (SEQ ID NO: 85), GLGGS (SEQ ID NO: 86), GGLGS (SEQ ID NO: 87), GGGLS (SEQ ID NO: 88) or GGGGL (SEQ ID NO: 89). In another embodiment, the T cell epitope linker comprises or consists of LGGSG (SEQ ID NO: 90), GLGSG (SEQ ID NO: 91), GGLSG (SEQ ID NO: 92), GGGLG (SEQ ID NO: 93) or GGGSL (SEQ ID NO: 94). In yet another embodiment, the T cell epitope linker comprises or consists of LGGSS (SEQ ID NO: 95), GLGSS (SEQ ID NO: 96) or GGLSS (SEQ ID NO: 97).

[0170] In yet another embodiment, the T cell epitope linker comprises or consists of LGLGS (SEQ ID NO: 98), GLGLS (SEQ ID NO: 99), GLLGS (SEQ ID NO: 100), LGGLS (SEQ ID NO: 101) or GLGGL (SEQ ID NO: 102). In yet another embodiment, the T cell epitope linker comprises or consists of LGLSG (SEQ ID NO: 103), GLLSG (SEQ ID NO: 104), GGLSL (SEQ ID NO: 105), GGLLG (SEQ ID NO: 106) or GLGSL (SEQ ID NO: 107). In yet another embodiment, the T cell epitope linker comprises or consists of LGLSS (SEQ ID NO: 108) or GGLLS (SEQ ID NO: 109).

[0171] 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.

[0172] In one embodiment, the T cell epitope linker comprises or consists of LGGGSGGGGS (SEQ ID NO: 110), GLGGSGGGGS (SEQ ID NO: 111), GGLGSGGGGS (SEQ ID NO: 112), GGGLSGGGGS (SEQ ID NO: 155), or GGGGLGGGGS (SEQ ID NO: 114). In another embodiment, the T cell epitope linker comprises or consists of LGGSGGGGSG (SEQ ID NO: 115), GLGSGGGGSG (SEQ ID NO: 156), GGLSGGGGSG (SEQ ID NO: 157), GGGLGGGGSG (SEQ ID NO: 158), or GGGSLGGGSG (SEQ ID NO: 159). In yet another embodiment, the T cell epitope linker comprises or consists of LGGSSGGGSS (SEQ ID NO: 121), GLGSSGGGSS (SEQ ID NO: 122), GGLSSGGGSS (SEQ ID NO: 123), GGGLSGGGSS (SEQ ID NO: 124), or GGGSLGGGSS (SEQ ID NO: 125).

[0173] In a further embodiment, the T cell epitope linker comprises or consists of LGGGSLGGGS (SEQ ID NO: 126), GLGGSGLGGS (SEQ ID NO: 127), GGLGSGGLGS (SEQ ID NO: 128), GGGLSGGGLS (SEQ ID NO: 129) or GGGGLGGGGL (SEQ ID NO: 130). In another embodiment, the T cell epitope linker comprises or consists of LGGSGLGGSG (SEQ ID NO: 131), GLGSGGLGSG (SEQ ID NO: 132), GGLSGGGLSG (SEQ ID NO: 133), GGGLGGGGLG (SEQ ID NO: 134) or GGGSLGGGSL (SEQ ID NO: 135). In yet another embodiment, the T cell epitope linker comprises or consists of LGGSSLGGSS (SEQ ID NO: 136), GLGSSGLGSS (SEQ ID NO: 137) or GGLSSGGLSS (SEQ ID NO: 138).

[0174] In yet another embodiment, the subunit linker comprises or consists of GSGGGA (SEQ ID NO: 139), GSGGGAGSGGGA (SEQ ID NO: 140), GSGGGAGSGGGAGSGGGA (SEQ ID NO: 141), GSGGGAGSGGGAGSGGGAGSGGGA (SEQ ID NO: 142), or GENLYFQSGG (SEQ ID NO: 143). In yet another embodiment, the subunit linker comprises or consists of SGGGSSGGGS (SEQ ID NO: 144), SSGGGSSGGG (SEQ ID NO: 145), GGSGGGGSGG (SEQ ID NO: 146), GSGSGSGSGS (SEQ ID NO: 147), GGGSSGGGSG (SEQ ID NO: 118), GGGSSS (SEQ ID NO: 149), GGGSSGGGSSGGGSS (SEQ ID NO: 150), or GLGGLAAA (SEQ ID NO: 151).

[0175] In another embodiment, the T cell epitope linker is a rigid linker. Such a rigid linker may help to efficiently separate (larger) T cell epitopes and prevent them from interfering with each other. In one embodiment, the T cell epitope linker comprises or consists of KPEPKPAPAPKP (SEQ ID NO: 152), AEAAAKEAAAKA (SEQ ID NO: 153), (EAAAK)m (SEQ ID NO: 154), PSRLEEELRRRLTEP (SEQ ID NO: 160) or SACYCELS (SEQ ID NO: 161).

[0176] In yet another embodiment, the T cell epitope linker comprises or consists of the sequence TQKSLSLSPGKGLGGL (SEQ ID NO: 162). In yet another embodiment, the T cell epitope linker comprises or consists of the sequence SLSLSPGKGLGGL (SEQ ID NO: 163).

[0177] In yet another embodiment, the T cell epitope linker comprises or consists of GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 164) or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 165) or EPKSCDTPPPCPRCP (SEQ ID NO: 166).

[0178] In yet another embodiment, the T cell epitope linker is a cleavable linker, e.g., a linker that contains one or more recognition sites for an endopeptidase, e.g., a furin, a caspase, a cathepsin, or other endopeptidase.

[0179] Examples of T cell epitope linkers are disclosed in paragraphs

[0098] to

[0099] and cited sequences of WO 2020 / 176797(A1), paragraphs

[0135] to

[0139] of U.S. Patent Application Publication No. 2019 / 0022202(A1), WO 2017 / 118695(A1) and WO 2021 / 219897(A1), all of which are incorporated by reference herein.

[0180] Allergens Vectors encoding the constructs described herein are useful for inducing tolerance to T cell epitopes of a variety of different protein allergens, including, for example, protein allergens that undergo post-translational modifications, which may be encoded by nucleic acid sequences contained in the first nucleic acid sequence of the vector of the invention.

[0181] One or more T cell epitopes contained in an antigenic unit may be derived from the following allergens: In some embodiments, the allergen is a food allergen. In some embodiments, the allergen is a crustacean allergen. In some embodiments, the allergen is tropomyosin, and in other embodiments, the allergen is arginine kinase, myosin light chain, sarcoplasmic reticulum calcium binding protein, troponin C, or triosephosphate isomerase, or actin. In some embodiments, the allergen is Pan b 1. In some embodiments, the antigenic unit comprises a Pan b 1 T cell epitope (251-270). In some embodiments, the antigenic unit comprises Met e 1. In some embodiments, the antigenic unit comprises one or more of Met e 1 T cell epitopes (241-260), (210-230), (136-155), (76-95), (46-65), and (16-35). In some embodiments, the antigenic unit includes all of the Met e 1 T cell epitopes (241-260), (210-230), (136-155), (76-95), (46-65), and (16-35).

[0182] In some embodiments, the allergen is a cow's milk allergen, hi some embodiments, the cow's milk allergen is 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.

[0183] 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 d 3, egg lysozyme, or ovomucin.

[0184] One T cell epitope known in the art and that has been tested in the context of egg allergy is OVA(257-264), which has the amino acid sequence SIINFEKL (SEQ ID NO: 120).

[0185] In one embodiment, the antigenic unit of the construct encoded by the vector of the present invention comprises the T cell epitope OVA(257-264). Such a vector or a pharmaceutical composition comprising such a vector can be used in the treatment of egg allergy.

[0186] 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.

[0187] In some embodiments, the allergen is a fruit allergen, hi 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, β1,3 glucanase, germin-like protein, alkaline serine protease, pathogenesis-related protein 1, actinidin, phytocystatin, quiwellin, major latex protein, cupin, or 2S albumin. In some embodiments, the allergen is a plant allergen. In some embodiments, the plant allergen is pathogenesis-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.

[0188] In some embodiments, the allergen is a wheat allergen. In some embodiments, the wheat allergen is 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. In some embodiments, the allergen is a soybean allergen. In some embodiments, the soybean allergen is Gly m 1, Gly m 2, Gly m 3, Gly m 4, Gly m 5, Gly m 6, Gly m 7, or Gly m 8. In other embodiments, the soy allergen is Gly m agglutinin, Gly m Bd28K, Gly m 30 kD, Gly m CPI or Gly m TI.

[0189] In some embodiments, the allergen is a peanut allergen. In some embodiments, the peanut allergen is Ara h 1, Ara h 2, Ara h 3, Ara h 4, Ara h 5, Ara h 6, Ara h 7, Ara h 8, Ara h 9, 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. 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.

[0190] In other embodiments, the food allergen is an allergen selected from the group consisting of buckwheat, celery, food coloring, egg, fish, fruit, garlic, gluten, oats, beans, corn, milk, mustard, tree nuts, peanuts, poultry, meat, rice, sesame, shellfish, soy, tree nuts, and wheat.

[0191] 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 / icarapin, 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.

[0192] In some embodiments, the allergen is a latex allergen, hi some embodiments, the latex allergen is 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.

[0193] In some embodiments, the allergen is a dust mite allergen. In some embodiments, the allergen is a house dust mite allergen. In some embodiments, the allergen is a storage dust allergen. In some embodiments, the dust mite allergen is Der p 1, Der p 2, Der p 3, Der p 4, Der p 5, Der p 7, Der p 8, Der p 10, Der p 11, Der p 21, or Der p 23. In some embodiments, the antigenic unit comprises a Der p 1 T cell epitope (111-139). In some embodiments, the dust mite allergen is Der f 1, Der f 2, Der f 3, Der f 7, Der f 8, or Der f 10. In some embodiments, the Dermatophagoides allergen is Blot t 1, Blot t 2, Blot t 3, Blot t 4, Blot t 5, Blot t 8, Blot t 10, Blot t 12, or Blot t 21.

[0194] In some embodiments, the allergen is a cockroach allergen. In some embodiments, the cockroach allergen is Bla g 1, Bla g 2, Bla g 3, Bla g 4, Bla g 5, Bla g 6, Bla g 7, Bla g 8, or Bla g 11. In some embodiments, the cockroach allergen is Per a 1, Per a 2, Per a 3, Per a 6, Per a 7, Per a 9, or Per a 10.

[0195] 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 f 1, Asp f 2, Asp f 3, Asp f 4, Asp f 5, Asp f 6, Asp f 7, Asp f 8, Asp f 9, Asp f 10, Asp f 11, Asp f 12, Asp f 13, Asp f 14, Asp f 15, Asp f 16, Asp f 17, Asp f 18, Asp f 22, Asp f 23, Asp f 27, Asp f 28, Asp f 29, or Asp f 34.

[0196] 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 f 1, Mala f 2, Mala f 3, Mala f 4, Mala f 5, Mala f 6, Mala f 7, Mala f 8, Mala f 9, Mala f 10, Mala f 11, Mala f 12, or Mala f 13, or MGL_1204.

[0197] In some embodiments, the allergen is a furry animal allergen. In some embodiments, the allergen is a dog allergen. In some embodiments, the dog allergen is Can f 1, 2, 3, 4, 5, or 6. In some embodiments, the allergen is a horse allergen. In some embodiments, the horse allergen is Ecu c 1, 2, 3, or 4. In some embodiments, the allergen is a cat allergen. In some embodiments, the cat allergen is Fel d 1, Fel d 2, Fel d 3, Fel d 4, Fel d 5, Fel d 6, Fel d 7, or Fel d 8. In some embodiments, the allergen is a laboratory animal allergen. In some embodiments, the allergen is lipocalin, urinary prealbumin, secretoglobulin, or serum albumin.

[0198] 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 Timothy grass, Orchard grass, Chinese grass, Perennial rye, Japanese yarrow, Bahiagrass, Corn sorghum, or Corngrass allergen. In some embodiments, the grass pollen allergen is Phl p 1, Phl p 2, Phl p 3, Phl p 4, Phl p 5, Phl p 6, Phl p 7, Phl p 11, Phl p 12, or Phl p 13.

[0199] In some embodiments, the allergen is a tree pollen allergen, hi some embodiments, the tree pollen allergen is an alder, birch, hornbeam, hazel, ash, chestnut, beech, white oak, ash, privet, olive, lilac, cypress, or cedar pollen allergen. In some embodiments, the tree pollen allergen is Aln g 1 or Aln g 4, Bet v 1, Bet v 2, Bet v 3, Bet v 4, Bet v 6 or Bet v 7, Car b 1, Cor a 1, Cor a 2, Cor a 6, Cor a 8, Cor a 9, Cor a 10, Cor a 11, Cor a 12, 1 Cor a 3, Cor a 14, Ost c 1, Cas 1, Cas 5, Cas 8 or Cas 9, Fag s 1, Que a 1, Fra e 1, Lig v 1, Ole e 1, Ole e 2, Ole e 3, Ole e 4, Ole e 5, Ole e 6, Ole e 7, Ole e 8, Ole e 9, Ole e 10, Ole e 11 or Ole e 12, Syr v 1, Cha o 1, Cha o 2, Cry j 1, Cry j 2, Cup s 1, Cup s 3, Jun a 1, Jun a 2, Jun a 3, Jun o 4, Jun v 1, Jun v 3, Pla a 1, Pla a 2 or Pla a 3. In some embodiments, the antigenic unit comprises the Bet v 1 T cell epitope (139-152).

[0200] In some embodiments, the allergen is a weed pollen allergen. In some embodiments, the weed allergen is a ragweed, artemisia, sunflower, feverfew, pyrethrium, plantain, annual mercury, pigweed, Russian thistle, or amaranth pollen allergen. In some embodiments, the ragweed pollen allergen is Amb a 1, Amb a 4, Amb a 6, Amb a 8, Amb a 9, Amb a 10, or Amb a 11. In some embodiments, the artemisia pollen allergen is Art v 1, Art v 3, Art v 4, Art v 5, or Art v 6. In some embodiments, the sunflower pollen allergen is Hel a 1 or Hel a 2. In some embodiments, the pyrethrium pollen allergen is Par j 1, Par j 2, Par j 3, or Par j 4. In some embodiments, the plantain lanceolate pollen allergen is Pla l 1. In some embodiments, the annual mountain ash pollen allergen is Mer a 1. In some embodiments, the pigweed pollen allergen is Che a 1, Che a 2, or Che a 3. In some embodiments, the Russian thistle pollen allergen is Sal k 1, Sal k 4, or Sal k 5. In some embodiments, the amaranth pollen allergen is Ama r 2.

[0201] In still other embodiments, the allergen is selected from environmental allergens such as insects, cockroaches, dust mites, or molds.

[0202] In some embodiments, the vectors of the present invention may be used in the treatment of allergic diseases selected from the group consisting of allergic rhinitis, asthma, atopic dermatitis, allergic gastrointestinal disease, contact dermatitis and drug allergy or a combination thereof.

[0203] Allergies to drugs affect more than 7% of the general population. The constructs encoded by the vectors of the present invention can be used to induce tolerance to immunogenic T cell epitopes present in such drugs, thus allowing affected patients to continue and benefit from treatment with the drug.

[0204] Thus, in some embodiments, the allergen is included in a drug that has undesirable immunogenicity. In some embodiments, the allergen is Factor VIII. In some embodiments, the allergen is insulin. In some embodiments, the allergen is a monoclonal antibody used in therapy.

[0205] autoantigen In another embodiment, the vector of the invention encodes a construct containing one or more T cell epitopes 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.

[0206] In some embodiments, the autoimmune disease is 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). In some embodiments, the construct encoded by the vector of the invention comprises one or more T cell epitopes derived from one or more of the aforementioned autoantigens.

[0207] The MS-associated T cell epitope is a T cell epitope 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. Another MOG T cell epitope is MOG(27-63).

[0208] Other MS-associated T-cell epitopes known in the art and that have been tested include those listed in Table 3 below: [Table 3] * T cell epitope-induced EAE was observed. In a preferred embodiment, the antigenic unit of the construct encoded by the vector of the invention 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). Such vectors or pharmaceutical compositions comprising such vectors may be used in the treatment of MS.

[0209] In some embodiments, the autoimmune disease is type 1 diabetes. In some embodiments, the autoantigen is glutamic acid decarboxylase 65-kilodalton isoform (GAD65), which is an autoantigen involved in type 1 diabetes. In some other embodiments, the autoantigen is insulin, IA-2, or ZnT8. In yet some other embodiments, the autoantigen is IGRP, ChgA, IAPP, peripherin, tetraspanin-7, GRP78, urocortin-3, or insulin gene enhancer protein isl-1. In some embodiments, the construct encoded by the vector of the invention comprises one or more T cell epitopes derived from one or more of the aforementioned autoantigens. 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). In some embodiments, the construct encoded by the vector of the invention comprises one or more T cell epitopes derived from one or more of the aforementioned autoantigens.

[0210] 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). In some embodiments, the construct encoded by the vector of the invention comprises one or more T cell epitopes derived from one or more of the aforementioned autoantigens. In one embodiment, the autoimmune disease is chronic inflammatory demyelinating polyneuropathy (CIDP) and the autoantigen is neurofascin 155. In another embodiment, the autoimmune disease is Hashimoto's thyroiditis (HT) and the autoantigen is thyroid peroxidase and / or thyroglobulin. In another embodiment, the autoimmune disease is pemphigus foliaceus and the autoantigen is desmosome-associated glycoprotein. In another embodiment, the autoimmune disease is pemphigus vulgaris and the autoantigen is desmoglein 3. In another embodiment, the autoimmune disease is thyroid eye disease (TED) and the autoantigen is calcium-binding protein (calsequestrin). In another embodiment, the autoimmune disease is Graves' disease and the autoantigen is thyroid-stimulating hormone receptor. In another embodiment, the autoimmune disease is primary biliary cirrhosis (PBC) and the autoantigen is antimitochondrial antibody (AMA), antinuclear antibody (ANA), rim-like / membrane (RL / M) and / or multinuclear 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 psoriasis and the autoantigen is selected from the group consisting of 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. In another embodiment, the autoimmune disease is rheumatoid arthritis and the autoantigen is selected from the group consisting of citrullinated proteins, homocitrullinated proteins and the Fc portion of IgG. In some embodiments, the construct encoded by the vector of the invention comprises one or more T cell epitopes derived from one or more of the aforementioned autoantigens.

[0211] Unit Linker The antigenic unit is preferably connected to the multimerization unit by a unit linker. Thus, in one embodiment, the first nucleic acid sequence contained in the vector of the present invention encodes a unit linker that connects the antigenic unit to the multimerization unit.

[0212] The unit linker may include a restriction site to facilitate the construction of the first nucleic acid sequence. In one embodiment, the unit linker is GLGGL (SEQ ID NO: 102) or GLSGL (SEQ ID NO: 174). In another embodiment, the unit linker comprises or consists of GGGGS (SEQ ID NO: 175), GGGGSGGGGS (SEQ ID NO: 79), (GGGGS)m (SEQ ID NO: 80), EAAAK (SEQ ID NO: 176), (EAAAK)m (SEQ ID NO: 154), (EAAK)MgS (SEQ ID NO: 178), (EAAAK)MgS (SEQ ID NO: 177), GPSRLEEELRRRLTEPG (SEQ ID NO: 179), AAY, or HEYGAEALERAG (SEQ ID NO: 173).

[0213] Signal peptide In one embodiment of the present disclosure, at least one of the first nucleic acid sequence and the one or more further nucleic acid sequences encoding one or more immunoinhibitory compounds also encodes 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 first polypeptide. Furthermore, the signal peptide is located at the N-terminus of the immunoinhibitory compound. The signal peptide is designed to allow secretion of the first polypeptide and / or the immunoinhibitory compound from cells containing the vector of the present invention. Preferably, each of the first nucleic acid sequence and the one or more further nucleic acid sequences encoding one or more immunoinhibitory compounds also encodes a signal peptide.

[0214] Any suitable signal peptide can be used. For the first polypeptide, an example of a suitable signal peptide is the human Ig VH signal peptide, preferably when the targeting unit is an antibody or a part thereof, such as an scFv. In one embodiment, the signal peptide is the natural leader sequence of the protein that is the targeting unit, i.e. the signal peptide that is naturally present at the N-terminus of the protein encoded by the vector of the invention as the targeting unit. Examples of such signal peptides are the signal peptide of human IL-10 (wherein the targeting unit is human IL-10) or the signal peptide of human TGF-β1 (wherein the targeting unit is human TGF-β1).

[0215] For one or more of the immunoinhibitory compounds, the signal peptide is preferably the natural leader sequence of the immunoinhibitory compound, i.e. the signal peptide that is naturally present at the N-terminus of the immunoinhibitory compound. Examples of such signal peptides are the signal peptide of CTLA-4 (wherein the immunoinhibitory compound is CTLA-4, preferably the extracellular domain of CTLA-4) or the signal peptide of GM-CSF (wherein the immunoinhibitory compound is GM-CSF).

[0216] Thus, in one embodiment, a vector of the invention comprises a first nucleic acid sequence encoding a human IL-10 signal peptide, such as a human IL-10 signal having SEQ ID NO: 69. In a preferred embodiment, such a vector comprises a first nucleic acid sequence encoding a human IL-10 targeting unit and also encoding a human IL-10 signal peptide. In another embodiment, a vector of the invention comprises a first nucleic acid sequence encoding a human Ig VH signal peptide, such as a human Ig VH signal peptide having SEQ ID NO: 2. In a preferred embodiment, such a vector comprises a first nucleic acid sequence encoding a scFv targeting unit, such as an anti-human CD205 targeting unit, and also encoding a human Ig VH signal peptide.

[0217] In one embodiment, the vector of the invention is a human Ig The signal peptide may include a first nucleic acid sequence encoding a signal peptide selected from the group consisting of a VH signal peptide, a signal peptide of hTGF-β1, a signal peptide of hTGF-β2, a signal peptide of hTGF-β3, a signal peptide of hIL-10, a signal peptide of hIL-2, a signal peptide of hIL-4, a signal peptide of hIL-6, a signal peptide of hIL-11, a signal peptide of hIL-13, a signal peptide of hIL-27, a signal peptide of hIL-35, a signal peptide of hIL-37, a signal peptide of hGM-CSF, a signal peptide of hFLT3L, a signal peptide of hCCL19, a signal peptide of hCCL21, a signal peptide of hICAM-1, a signal peptide of hKeratin, a signal peptide of hVSIG-3, a signal peptide of hSCGB3A2, a signal peptide of hCTLA-4, a signal peptide of hPD-1, and a signal peptide of hBTLA, for example, any of the foregoing signal peptides listed in the "Sequence Summary" section herein.

[0218] In another embodiment, the vector of the present invention comprises one or more additional nucleic acid sequences encoding one or more immune inhibitory compounds and further encoding a signal peptide selected from the group consisting of the signal peptide of hCLTA-4, the signal peptide of hPD-1, the signal peptide of hBTLA, the signal peptide of hLAG3, the signal peptide of hNOX2, the signal peptide of hSIGLEC7, the signal peptide of hSIGLEC9, the signal peptide of hTIM-3, the signal peptide of hIL-10, the signal peptide of hTGF-β1, the signal peptide of hTGF-β2, the signal peptide of hTGF-β3, the signal peptide of hIL-27, the signal peptide of hIL-2, the signal peptide of hGM-CSF, the signal peptide of hFLT3L, the signal peptide of hIFN-γ, and the signal peptide of hIL-37 and the signal peptide of hIL-35, e.g., any of the foregoing signal peptides listed in the "Sequence Summary" section herein.

[0219] Sequence identity Sequence identity can be determined as follows: a high sequence identity indicates that the second sequence is more likely to be derived from the first sequence. Amino acid sequence identity requires identical amino acid sequences between the 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 using computer analysis, such as, but not limited to, the ClustalW computer alignment program (Higgins D., Thompson J., Gibson T., Thompson JD, Higgins DG, Gibson TJ, 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673-4680) and the default parameters proposed therein. When used with default settings, this program aligns the mature (biologically active) portions of the query and reference polypeptides. The number of perfectly conserved residues is counted and divided by the length of the reference polypeptide. In doing so, any tag or fusion protein sequences that form part of the query sequence are disregarded in the alignment and subsequent sequence identity determination.

[0220] The ClustalW algorithm can similarly be used to align nucleotide sequences. Sequence identity can be calculated in a similar manner as shown for amino acid sequences.

[0221] Another preferred mathematical algorithm used for comparing sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated in 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 ends of the sequences. When utilizing the ALIGN and Align0 programs for comparing amino acid sequences, it is preferred to use the BLOSUM50 substitution matrix with gap opening / extension penalties of -12 / -2. Amino acid sequence variants can be prepared by introducing appropriate changes into the nucleotide sequence encoding the first polypeptide and / or one or more immunostimulatory compounds or by peptide synthesis. Such modifications include, for example, deletion from and / or insertion into and / or substitution of residues within the amino acid sequence. The terms substituted / substitution, deleted / deletion and inserted / insertion as 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 used to arrive at the final first polypeptide and / or one or more immunostimulatory compounds, provided that the final protein has the desired properties. For example, deletion, insertion or substitution of an amino acid residue may produce a silent change and result in a functionally equivalent polypeptide / immunostimulatory compound.

[0222] Deliberate amino acid substitutions can be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues, so long as the desired properties of the protein in question are retained. 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. Included herein are conservative substitutions, i.e., like substitutions such as basic with basic, acidic with acidic, polar with polar, and non-conservative substitutions, i.e., substitutions of one class of residue for another class of residue, or involving the inclusion of unnatural amino acids such as ornithine, diaminobutyric acid ornithine, norleucine, ornithine, pyriylalanine, thienylalanine, naphthylalanine, and phenylglycine. Conservative substitutions that may be made are, 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). Substitution with unnatural amino acids is also possible. * and alpha-disubstituted * Amino acids, N-alkyl amino acids * , lactic acid * , halide derivatives of natural amino acids, e.g., trifluorotyrosine * , p-Cl-phenylalanine * , p-Br-phenylalanine * , pI-phenylalanine * , L-allyl-glycine * , 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 * , methyl derivatives of phenylalanine (Phe), e.g., 4-methylPhe * Pentamethyl-Phe * , L-Phe(4-amino)#, L-Tyr(methyl) * , L-Phe(4-isopropyl) * , L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) * , L-diaminopropionic acid * , and L-Phe(4-benzyl) * Examples include: In the above paragraph, * indicates the hydrophobicity of the substituted residue, # indicates the hydrophilicity of the substituted residue, * indicates the amphipathic nature of the substituted residue. Variant amino acid sequences may include 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 spacers such as glycine or β-alanine residues. A further form of variation includes the presence of one or more amino acid residues in peptoid form.

[0223] Polypeptides and multimeric / dimeric proteins The vector of the invention encodes the first polypeptide described above. The polypeptide (and one or more immunosuppressive compounds) are expressed in vivo as a result of administration of the vector to a subject.

[0224] The presence of a multimerization unit (eg, a dimerization unit) results in the formation of a multimeric protein when the polypeptide is expressed.

[0225] The multimeric protein may be a homomultimer or a heteromultimer, for example, if the protein is a dimeric protein, the dimeric protein may be a homodimer, i.e., a dimeric protein in which the two polypeptide chains are identical and therefore contain identical units and therefore T cell epitopes, or the dimeric protein may be a heterodimer comprising two polypeptide chains, where 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 the antigenic unit exceeds the upper limit size of the antigenic unit. Preferably, the multimeric protein is a homomultimeric protein.

[0226] Preparation of host cells and vectors The vectors of the present invention are generally vectors suitable for transfecting a host cell to a) express a first polypeptide encoded by a first nucleic acid sequence and form a multimeric protein composed of a plurality of such first polypeptides, and b) express one or more immunoinhibitory compounds encoded by further nucleic acid sequences. In one embodiment, the host cell comprising the vector of the invention is a cell of a cell culture, e.g., a bacterial cell, and the protein encoded by the vector is expressed in vitro. In another embodiment, the host cell comprising the vector of the invention is a cell of a subject, and the protein encoded by the vector is expressed in the subject, i.e., in vivo, as a result of administration of the vector to the subject.

[0227] Suitable host cells for in vitro transfection include prokaryotic cells, yeast cells, insect cells or higher eukaryotic cells. Suitable host cells for in vivo transfection are, for example, muscle cells.

[0228] In one embodiment, the vector is a CpG-free vector. In another embodiment, the vector is a pALD-CV77 vector.

[0229] Methods for engineering and producing vectors of the invention, e.g., expression vectors such as DNA and RNA plasmids or viral vectors, are well known and a person skilled in the art would be able to engineer / produce the vectors of the invention using such known methods. In addition, various commercial manufacturers offer services for vector design and production.

[0230] In one aspect, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as 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; (b) one or more additional nucleic acid sequences encoding one or more immunoinhibitory compounds, 1. A method for producing a vector allowing for the co-expression of a first polypeptide and one or more immunosuppressive compounds as separate molecules, comprising: a) transfecting a cell in vitro with a vector; b) culturing the cells; and c) optionally lysing the cells to release the vectors from the cells; d) recovering and optionally purifying the vector or DNA plasmid.

[0231] Pharmaceutical Compositions In one embodiment of the disclosure, the vector, such as a DNA plasmid, is for use as a pharmaceutical.

[0232] Thus, in one embodiment of the present disclosure, the vector is provided in a pharmaceutical composition comprising the vector and a pharma- ceutically acceptable carrier or diluent.

[0233] Thus, in one aspect, the present disclosure provides a pharmaceutical composition comprising: (i) a pharma- ceutically acceptable carrier or diluent; and (ii) (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as 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; (b) one or more additional nucleic acid sequences encoding one or more immunoinhibitory compounds, and a vector allowing for the co-expression of the first polypeptide and one or more immunoinhibitory compounds as separate molecules.

[0234] Suitable pharma- ceutically acceptable carriers or diluents include, but are not limited to, saline, buffered saline such as PBS, dextrose, water, glycerol, ethanol, isotonic aqueous buffer, and combinations thereof.

[0235] In one embodiment, the pharma- ceutically acceptable carrier or diluent is an aqueous buffer solution. In another embodiment, the aqueous buffer solution is Tyrode's buffer, e.g., Tyrode's buffer containing 140 mM NaCl, 6 mM KCl, 3 mM CaCl2, 2 mM MgCl2, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) pH 7.4, and 10 mM glucose.

[0236] The pharmaceutical composition may include a molecule that facilitates transfection of a host cell, ie, a transfection agent.

[0237] The pharmaceutical composition may include an adjuvant. In one embodiment, the adjuvant is selected from the group consisting of dexamethasone, the B subunit of the enterotoxin cholera toxin (CTB), a TLR2 ligand, an excretory / secretory (ES) product from a helminth, a rapamycin vitamin D3 analog, and an aryl hydrocarbon receptor ligand.

[0238] In some specific embodiments, the pharmaceutical compositions comprise a pharma- ceutically acceptable amphiphilic block copolymer comprising blocks of poly(ethylene oxide) and poly(propylene oxide).

[0239] 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.

[0240] "Poloxamer" refers to a linear amphiphilic block copolymer composed of one poly(ethylene oxide) block bonded to one poly(propylene oxide) block bonded to one PEO block, i.e., a structure of 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 usually named by using a three-digit identifier, where the first two digits multiplied by 100 indicate the approximate molecular weight of the PPO content, and the last digit multiplied by 10 indicates the approximate percentage of the PEO content. For example, "Poloxamer 188" refers to a polymer containing a PPO block of molecular weight about 1800 (corresponding to a PPO with b of about 31) and about 80% (w / w) PEO (corresponding to a of 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 according to the manufacturer's datasheet, show a large variation in molecular weight (7,680-9,510), 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 copolymer, and means that the values ​​of a and b are found on average in the final formulation.

[0241] "Poloxamines" or "sequential poloxamines" (sold under the name Tetronic®) are X-shaped block copolymers in which four PEO-PPO arms are connected 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 poloxamines are similarly X-shaped block copolymers in which four PPO-PEO arms are connected 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.

[0242] Preferred amphiphilic block copolymers are poloxamers or poloxamines. Poloxamers 407 and 188 are preferred, 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 proportion of PEO of about 40%; Tetronic® 704 has a total average molecular weight of 5500, a total average weight of PPO units of 3300, and a proportion of PEO 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 proportion of PEO of about 40%.

[0243] In one embodiment, the pharmaceutical composition comprises the amphiphilic block copolymer in an amount of from 0.2% w / v to 20% w / v, such as 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, from 0.2% w / v to 4% w / v, from 0.4% w / v to 18% w / v, from 0.6% w / v to 18% w / v, from 0.8% w / v to 18% w / v, from 1% w / v to 18% w / v, from 2% w / v to 18% w / v, from 1% w / v to 5% w / v, or from 2% w / v to 4% w / v. Particularly preferred is an amount in the range of 0.5% w / v to 5% w / v. In another embodiment, the pharmaceutical composition comprises the amphiphilic block copolymer in an amount of 2% w / v to 5% w / v, for example about 3% w / v.

[0244] The pharmaceutical compositions can be formulated in any manner suitable for administration to a subject, such as, for example, a liquid formulation for injection, for example, intradermal or intramuscular injection.

[0245] The pharmaceutical compositions may be administered in any manner suitable for administration to a subject, such as by intradermal, intramuscular, or subcutaneous injection, or by mucosal or epidermal application, such as intranasal or oral.

[0246] In a preferred embodiment, the pharmaceutical composition is administered by intramuscular or intradermal injection.

[0247] The amount of vector, e.g., a DNA plasmid, in a pharmaceutical composition may vary depending on whether the pharmaceutical composition is being administered for prophylactic or therapeutic treatment.

[0248] A pharmaceutical composition of the invention typically comprises a vector, such as a DNA plasmid, in the range of 0.1 to 10 mg, for example about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 mg, or for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg.

[0249] In a preferred embodiment, the pharmaceutical composition is a sterile pharmaceutical composition.

[0250] treatment In some embodiments of the disclosure, the vectors, e.g., DNA plasmids, are for use in the prophylactic or therapeutic treatment of conditions involving an unwanted immune response, i.e., immune disorders, such as autoimmune disorders, allergic disorders, and transplant rejection.

[0251] Thus, in a further aspect, the present invention provides a method for treating a subject having or in need of prevention of an immune disorder selected from the group consisting of an autoimmune disease, an allergic disease and transplant rejection, comprising the steps of: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as 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; (b) one or more additional nucleic acid sequences encoding one or more immunoinhibitory compounds, The method includes administering to a subject a vector that allows for the co-expression of a first polypeptide and one or more immunoinhibitory compounds as separate molecules.

[0252] In one embodiment, the present invention provides a method for treating a subject having or in need of prevention of an autoimmune disease, comprising: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit, the antigenic unit comprising one or more T cell epitopes of an autoantigen; (b) one or more additional nucleic acid sequences encoding one or more immunoinhibitory compounds, The method includes administering to a subject a vector that allows for the co-expression of a first polypeptide and one or more immunoinhibitory compounds as separate molecules.

[0253] In another embodiment, the present invention provides a method for treating a subject having or in need of prevention of an allergic disease, comprising: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit, the antigenic unit comprising one or more T cell epitopes of an allergen; (b) one or more additional nucleic acid sequences encoding one or more immunoinhibitory compounds, The method includes administering to a subject a vector that allows for the co-expression of a first polypeptide and one or more immunoinhibitory compounds as separate molecules.

[0254] In one embodiment, the present invention provides a method for treating a subject having or in need of prevention of transplant rejection, comprising: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit that targets an antigen-presenting cell, a multimerization unit, such as a dimerization unit, and an antigenic unit, the antigenic unit comprising one or more T cell epitopes of an alloantigen or a xenoantigen; (b) one or more additional nucleic acid sequences encoding one or more immunoinhibitory compounds, The method includes administering to a subject a vector that allows for the co-expression of a first polypeptide and one or more immunoinhibitory compounds as separate molecules.

[0255] In the method of treatment, the vector is preferably administered in a therapeutically or prophylactically effective amount, which may be administered in a single dose, i.e., in a single administration, or in several doses, i.e., in repeated administrations, or in a series of doses, for example over a period of days, weeks or months or years.

[0256] The actual amount administered will depend on and may vary depending on parameters such as whether the treatment is prophylactic or therapeutic, the severity of the immune disorder being treated, the age, weight, sex, medical history, pre-existing conditions, and general condition of the subject, as well as the judgment of a medical professional.

[0257] In the methods of treatment, the vectors can be administered in the form of pharmaceutical compositions and by the modes of administration described herein.

[0258] Treatment methods according to the present invention may be continued for as long as the clinician overseeing the patient's care determines that the method is effective and that treatment is necessary.

[0259] 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 ELISPOT when stimulated with the antigenic unit / T cell epitope within the antigenic unit, and a decrease in the level of basophil activation in the basophil activation test (BAT).

[0260] Radioallergosorbent testing (RAST) can also be used to compare allergen-specific IgE antibody levels in a subject's blood samples before and after administration of the immunotherapeutic construct, with lower allergen-specific IgE antibody levels indicating successful tolerance induction.

[0261] Also disclosed herein is a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as 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; (b) one or more additional nucleic acid sequences encoding one or more immunoinhibitory compounds, A vector that allows for the co-expression of a first polypeptide and one or more immune inhibitory compounds as separate molecules and is administered to a subject for use in treating a subject having an immune disease selected from the group consisting of an autoimmune disease, an allergic disease, and a transplant rejection reaction, or a subject in need of prevention thereof.

[0262] Also disclosed herein is a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as 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; (b) one or more additional nucleic acid sequences encoding one or more immunoinhibitory compounds, The use of a vector that allows for the co-expression of a first polypeptide and one or more immune inhibitory compounds as separate molecules for the manufacture of a medicament for use in treating a subject having an immune disorder selected from the group consisting of an autoimmune disease, an allergic disease, and a transplant rejection reaction, or a subject in need of prevention thereof, which medicament is administered to the subject.

[0263] Also disclosed herein is a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as 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; (b) one or more additional nucleic acid sequences encoding one or more immunoinhibitory compounds, The use of a vector that allows for the co-expression of a first polypeptide and one or more immune inhibitory compounds as separate molecules to treat a subject having an immune disease selected from the group consisting of an autoimmune disease, an allergic disease, and a transplant rejection reaction, or a subject in need of prevention thereof.

[0264] Also disclosed herein is a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as 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; (b) one or more additional nucleic acid sequences encoding one or more immunoinhibitory compounds, A vector that allows for the co-expression of a first polypeptide and one or more immune inhibitory compounds as separate molecules when used in the therapeutic or prophylactic treatment of an immune disease selected from the group consisting of an autoimmune disease, an allergic disease and a transplant rejection reaction.

[0265] Also disclosed herein is a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as 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; (b) one or more additional nucleic acid sequences encoding one or more immunoinhibitory compounds, The use of a vector that allows for the co-expression of a first polypeptide and one or more immunoinhibitory compounds as separate molecules for the therapeutic or prophylactic treatment of an immune disorder selected from the group consisting of an autoimmune disease, an allergic disease and a transplant rejection reaction.

[0266] Also disclosed herein is a pharmaceutical agent for treating or preventing an immune disease selected from the group consisting of an autoimmune disease, an allergic disease, and a transplant rejection reaction, comprising administering to a subject having the disease or in need of prevention of the disease: (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as 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; (b) one or more additional nucleic acid sequences encoding one or more immunoinhibitory compounds, This is a pharmaceutical agent, which is administered to a subject a vector that allows the first polypeptide and one or more immune inhibitory compounds to be co-expressed as separate molecules. EXAMPLES

[0267] The foregoing written description is believed to be sufficient to enable one skilled in the art to practice the present invention. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any manner. Indeed, various modifications of the present invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

[0268] Example 1: A DNA vector was designed and generated that contains a nucleotide sequence encoding mouse myelin oligodendrocyte glycoprotein (MOG) 27-63 and further encoding the following elements / units: [Table 4] * The MOG(27-63) sequence was taken from Krienke et al., Science 371, 145-153, 2021.

[0269] Myelin oligodendrocyte glycoprotein (MOG) is a protein expressed in the central nervous system. The MOG(27-63) sequence contained in the antigenic unit of the DNA vector contains MOG(35-55), the immunodominant 35-55 T cell epitope of MOG, which is the primary 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.

[0270] In the following, "m", "murine" and "mouse" are used interchangeably, and "h" and human are used interchangeably.

[0271] DNA vector VB5049, a vector according to the present invention *(SEQ ID NO:3) encodes a first polypeptide comprising a targeting unit, a dimerization unit, a unit linker, and an antigenic unit as described above in Table 4, and IL-10 as an immunoinhibitory compound. Due to the presence of the co-expression element T2A, the first polypeptide and the immunoinhibitory compound are expressed as separate molecules.

[0272] DNA vector VB5052 * (SEQ ID NO: 4) encodes a first polypeptide comprising a human macrophage inflammatory protein alpha variant targeting unit (also called hCCL3L1, LD78β or hMIP1α) which is known to target APCs in an immunogenic manner, i.e., first polypeptides / dimeric proteins comprising such targeting units are known to induce a proinflammatory immune response in subjects to which they are administered (see, for example, WO 2011 / 161244(A1)). VB5052 * is VB5049 * and is used as a comparison with the constructs of the present invention.

[0273] DNA vector VB5051 * (SEQ ID NO:5) encodes only a single protein / peptide, i.e., the antigenic unit, MOG(27-63); * is used as a comparison for the constructs of the present invention.

[0274] Production of DNA vectors All DNA vectors in this Examples section were produced by ordering sequences as described in the tables in this Examples section from Genscript Biotech BV, Netherlands, and cloning them into the expression vector pALD-CV77, a DNA plasmid.

[0275] Example 2: The aim of this study was to characterize the expression of the DNA vector VB5049 after transient transfection into mammalian cells. * and VB5052* The objective of this study was to evaluate the expression and secretion characteristics of the protein encoded by .

[0276] HEK293 cells were obtained from ATCC and VB5049 * or VB5052 * The cells were transiently transfected with DNA vectors. Briefly, 2 × 10 5 Cells / well were plated in 24-well tissue culture plates containing 10% FBS growth medium and transfected with 1 μg of each DNA vector using Lipofectamine® 2000 reagent (Invitrogen, Thermo Fischer Scientific). The transfected cells were then maintained at 5% CO2 and 37°C for 5 days, after which the cell supernatant was harvested and VB5049 was added. * or VB5052 * Characterization of the secreted protein encoded by was performed by sandwich ELISA of the supernatants using an antibody against hIgG CH3 domain (detection antibody, 100 μL / well, 0.1 μg / mL mouse anti-human IgG Fc secondary antibody, biotin (05-4240, Invitrogen)) and an antibody against MOG (capture antibody, 100 μL / well, 0.25 μg / mL mouse anti-MOG antibody (NYRMOG, sc-73330, Santa Cruz Biotechnology)). The results are shown in Figure 6A.

[0277] VB5049 * Secretion of IL-10 encoded by IL-10 was measured by sandwich ELISA using antibodies against mouse IL-10 (capture antibody, 100 μL / well, 0.4 μg / mL rat anti-mouse IL-10 antibody (MAB417, R&D Systems), detection antibody, 100 μL / well, 0.2 μg / mL goat anti-mouse IL-10 biotinylated antibody (BAF417, R&D Systems)). The results are shown in Figure 6B.

[0278] VB5049 *Secretion of the first polypeptide and IL-10 as two separate proteins from the mouse was demonstrated by sandwich ELISA using an antibody against mouse MOG (capture antibody, 100 μL / well, 0.25 μg / mL mouse anti-MOG antibody (NYRMOG, sc-73330, Santa Cruz Biotechnology) and an antibody against mouse IL-10 (detection antibody, 100 μL / well, 0.2 μg / mL goat anti-mouse IL-10 biotinylated antibody (BAF417, R&D Systems)). The results are shown in FIG. 6C.

[0279] VB5052 * Secretion of the first polypeptide encoded by was further demonstrated by sandwich ELISA using an antibody against hIgG CH3 domain (capture antibody, 100 μL / well, 1 μg / mL, mouse anti-human IgG (CH3 domain), 153272, Biorad) and an antibody against hCCL3L1 (detection antibody, 100 μL / well, 0.2 μg / mL, goat anti-human CCL3L1 biotin antibody). The results are shown in FIG. 6D.

[0280] As is clear from FIG. 6A (capture antibody: anti-MOG antibody, detection antibody: anti-hIgG CH3 domain antibody) and FIG. 6D (capture antibody: anti-hIgG CH3 domain, detection antibody: anti-human CCL3L1), VB5049 * and VB5052 * Both of the first polypeptides encoded by were secreted at high levels.

[0281] Figure 6B shows the level of IL-10 secretion in the undiluted supernatant. By comparing the dilution curve of the supernatant with that of recombinant IL-10, the secretion level in the supernatant was estimated to be 2.25 μg / mL.

[0282] Figure 6C shows the ELISA results obtained using anti-MOG antibody as the capture antibody and anti-mouse IL-10 antibody as the detection antibody. As expected, VB5049 *showed a very weak signal and the presence of the T2A peptide confirmed that the first polypeptide and IL-10 were expressed as two separate molecules.

[0283] The results shown in Figures 6A to 6D show that the DNA vector VB5049 * and VB5052 * The first polypeptide / dimer protein (comprising a targeting unit, a dimerization unit and an antigenic unit) encoded by VB5049 is secreted from transfected cells; and * For example, we show that IL-10 is also expressed and secreted as a separate protein.

[0284] To further characterize the expressed proteins, Western blot (WB) analysis was performed. Briefly, Expi293F cells (3 × 10 6 Cells / mL, 1.6 mL) were seeded into 6-well culture plates. Cells were transfected with 1 μg / mL of plasmid DNA using ExpiFectamine 293 reagent (Thermo Fisher Sci.) and plates were incubated on an orbital shaker (19 mm diameter, 125 rpm) in a humidified CO2 cell incubator (8% CO2, 37 °C). After 18 h of incubation, ExpiFectamine 293 Transfection Enhancer (Thermo Fisher Sci.) was added to each well. Plates were incubated for an additional 28 h, after which the supernatants were harvested.

[0285] Samples were prepared by mixing 105 μL of supernatant from transfected Expi293F cells with 37.5 μL of 4× Laemmli sample buffer (Bio-Rad) with 7.5 μL of DTT (Thermo Fisher Sci.) or 7.5 μL of ultrapure water for reducing and non-reducing conditions, respectively. Samples (reduced or non-reduced) were heated at 70 °C for 10 min, and then 10 μL was loaded onto a 4%-20% Criterion TGX Stain-Free precast gel (Bio-Rad). SDS-PAGE was performed in 1× Tris / glycine / SDS running buffer (Bio-Rad) using Precision Plus Protein All Blue Prestained protein standards (Bio-Rad). Proteins were transferred from the gel to an EtOH-activated low-fluorescence (LF) 0.45 μm PVDF membrane (Bio-Rad) using a Tran-Blot Turbo semi-dry transfer system (Bio-Rad). The PVDF membrane was blocked in EveryBlot buffer (Bio-Rad) for 5 min and probed with mouse anti-MOG (sc-73330, Santa Cruz Biotechnology) and rat anti-mouse IL-10 antibody (MAB417, R&D systems) to detect the first polypeptide / dimer protein and IL-10, respectively. The membrane was incubated with fluorescent dye-conjugated secondary antibody at room temperature for 1 h, then washed and dried. Images were acquired by using a ChemiDoc™ MP Imaging System (settings Dylight 488 and 800, Auto Optimal).

[0286] 7A and 7B show VB5049 * and VB5052 * A first polypeptide / dimeric protein encoded by VB5049 * 1 shows successful expression and secretion of IL-10 encoded by

[0287] Figure 7A shows the VB5052 * and VB5049 *The results show that proteins expressed by VB5052 and secreted from transfected cells could be detected by SDS-PAGE under reducing and non-reducing conditions. Membranes were probed with mouse anti-mouse MOG. Under reducing conditions, VB5052 * The first polypeptide encoded by VB5049 was detected as a monomer (31 kDa), whereas under non-reducing conditions it was detected as both a monomer (31 kDa, first polypeptide) and a dimer (61 kDa, dimeric protein). * The first polypeptide encoded by was detected as a monomer (51 kDa), but under non-reducing conditions, it was detected as both a monomer (51 kDa, first polypeptide) and a dimer (101 kDa, dimeric protein).

[0288] FIG. 7B shows the detection of IL-10 (18 kDa). The 71 kDa protein was not detected, indicating that IL-10 was expressed as a separate protein in VB5049. * It was confirmed that it was expressed from

[0289] Taken together the ELISA and Western blot data demonstrated that intact first polypeptide / dimer protein could be co-expressed with mIL10 from a DNA vector using the T2A peptide as a co-expression element.

[0290] Example 3: VB5049 was evaluated by measuring IL-10 (an anti-inflammatory cytokine associated with immune tolerance) and IFN-γ (a marker for inducing an inflammatory immune response) and calculating the IL-10 / IFN-γ ratio. * The tolerance-inducing ability of VB5049 was evaluated. * These results show the extent to which the immune response induced by is biased towards a tolerogenic profile.

[0291] 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 facilities of the Radium Hospital (Oslo, Norway). All animal protocols were approved by the Food Safety Authority (Oslo, Norway). * , VB5052 * and VB5051 * Five mice / group were used for the study and two mice / group for the negative control (PBS only). * is VB5049 * It is a pro-inflammatory version of VB5049 * It is included as a comparison to VB5049. * VB5052, a pro-inflammatory version of * is predicted to induce IFN-γ production. * VB5049 * was included as a comparison. 50 μg of each DNA vector dissolved in sterile PBS was administered once into each tibial muscle by intramuscular injection (2×25 μL, 1000 μg / mL), followed by electroporation using an AgilePulse in vivo electroporation system (BTX, USA).

[0292] Seven days after administration, spleens were harvested and crushed with 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 in a 96-well IFN-γ / IL-10 dual-color FluoroSpot plate. 5Splenocytes 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 with dual-color FluoroSpot 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 triplicates of splenocytes.

[0293] As can be seen in Figure 8, VB5049 * The IL-10 / IFN-γ ratio obtained with VB5049 was high. * This indicates that VB5052 induces significantly higher levels of the immune-inhibitory cytokine IL-10 than the inflammatory cytokine IFN-γ, which showed an IL-10 / IFN-γ ratio of approximately 1. * This is not the case, however, showing that both cytokines are produced at comparable levels after restimulation with the MOG(35-55) peptide.

[0294] Total splenocytes obtained from mice administered the DNA vector were also analyzed by multicolor flow cytometry. Briefly, cells were restimulated with MOG(35-55) peptide for 16 h, then harvested and counted. 2 × 10 per sample 6Cells were used for flow cytometry analysis. Cells were first stained with fixable viability dye (eFluor780) for 10 min at room temperature in the dark. After this, the cells were washed with 1x PBS, centrifuged (400g / 10min / 4°C) and incubated with the surface staining antibody (Ab) mix (anti-CD3 BUV395, anti-CD4 BV785, and anti-CD8) for 30min at 4°C in the dark. After incubation, the cells were washed and the cell pellet was resuspended in flow buffer (PBS, 10% FBS and 2mM EDTA). The cells were then fixed and permeabilized (eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set) for 45-60min at 4°C in the dark. Subsequently, the cells were washed with 1x permeabilization buffer, centrifuged as before, and the cell pellet was resuspended in the intracellular staining Ab mix (anti-IFN-γ APC, anti-IL-17 Alexa fluor 488, BV421, anti-Foxp3 PE). The cells were incubated with the intracellular staining Ab mix for 30min at 4°C in the dark. Afterwards, the cells were stained with BD Symphony Cells were washed and resuspended in flow buffer until flow cytometry was performed on an A5 flow cytometer. Compensation was set using single-stained Ultra comp eBeads for fluorescent dye-conjugated Abs and ArC-reactive beads for fixable viability dyes. Flow cytometry files were analyzed using FlowJo software.

[0295] Both IFN-γ and IL-17 are proinflammatory cytokines that contribute to the pathogenesis of chronic inflammatory and autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis. Thus, tolerance-inducing constructs must reliably induce tolerance without inadvertently sensitizing autoantigen immune responses, for example, by inducing proinflammatory cytokines that could exacerbate autoimmunity.

[0296] 9A and 9B show that VB5049, as observed by FluoroSpot assay, *Flow cytometry demonstrated a lack of IFN-γ production upon administration of VB5049. * We also demonstrated that VB5052 did not induce IL-17 production. * Administration of VB5051 induced the production of IFN-γ and IL-17, as would be expected. * Treatment induced some IFN-γ production in splenocytes, but levels of IL-17 were very low.

[0297] The generation of MOG-specific regulatory T cells (Tregs), T cells that act to suppress and control MOG-specific inflammatory immune responses, thereby maintaining self-tolerance, was identified by MOG-specific tetramer staining and flow cytometry (CD4+MOG(35-55)-tet+Foxp3+ cells).

[0298] Briefly, 1–2 × 10 pooled from each group 6Splenocytes were transferred to 96-well V-bottom plates. Tetramers and antibodies were diluted in PBS with 5% FBS before use and protected from light. All steps that required cell washing were performed with PBS with 5% FBS unless otherwise stated. Cells were first stained with T-Select MHC Class II Tetramers specific for MOG(35-55) (10 μL / well, H-2 IAb MOG(35-55) Tetramer-PE, TS-M704-1, MBL International Corporation) or ProT2® MHC Class II Tetramers specific for MOG(38-49) (1 μg / mL, H-2 IAb-GWYRSPFSRVVH-ProT2® Tetramer PE, 2958, Proimmune) according to the manufacturer's instructions. Without washing the cells, Fc receptors were blocked for 5 min 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, cells were stained for 30 min on ice 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, Thermo Fischer / eBioscience) and Anti-Mouse CD25 PerCP-Cy5.5 (0.25 μg / mL, Clone:PC61, 102030, BioLegend). Cells were washed twice with PBS. Cells were then stained with fixable viability dye (150 μL per well, diluted 1:8000 in PBS, Fixable Viability Stain 780, 565388, BD biosciences) for 10 min on ice.Cells were washed twice with PBS containing 5% FBS and fixed and permeabilized using Foxp3 / Transcription Factor Staining Buffer Set according to the manufacturer's instructions (200 μL per well, 00-5523-00, Thermo Fischer / eBioscience). Cells were washed and stained for 30 min on ice with an intracellular Ab cocktail containing Anti-Mouse FOXP3 eFluor 660 (0.25 μg / mL, Clone:FJK-16s, 50-5773-82, Thermo Fischer / eBioscience) and Anti-Mouse Ki-67 Alexa Fluor 488 (0.25 μg / mL, Clone:Clone:11F6, 151204, BioLegend). Cells were washed twice, resuspended in 200 μL PBS containing 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): unstained controls (cells were not treated with Abs) and Fluorescence Minus One (FMO) controls (samples stained with all fluorescently labeled Abs used herein minus one fluorescently labeled Ab being gated).

[0299] As shown in Figure 10, VB5052 * and VB5051 * Compared to VB5049 * A higher proportion of MOG(35-55)-specific Foxp3+ cells was detected after a single dose of VB5052. * In the case of * This is the result of a natural feedback loop on the inflammatory immune response induced by B. difficile (discussed in more detail in Example 5).

[0300] Therefore, Example 3 is VB5049 * but the proinflammatory control construct VB5052 *Example 3 further shows that VB5049 induced a higher anti-inflammatory to pro-inflammatory cytokine ratio (IL-10 / IFN-γ) compared to VB5049, indicating a lack of inflammatory IFN-γ production. * But VB5052 * or VB5051 * These results indicate that VB5049 induces more MOG(35-55)-specific Foxp3+ cells compared to VB5049. * Administration of VB5051 * and VB5052 * It is shown that administration of 100 mg / kg / day elicited a greater antigen-specific tolerogenic response compared to administration of 100 mg / kg / day.

[0301] Example 4 A DNA vector was designed and generated that contains a nucleotide sequence encoding MOG(27-63) and further encoding the following elements / units: [Table 5] Additionally, DNA vectors VB5049 (SEQ ID NO: 32), VB5052 (SEQ ID NO: 33) and VB501 (SEQ ID NO: 34) were designed and produced. * , VB5052 * and VB501 * (Table 4) but with SEQ ID NO: 16 MOG(27-63) * Instead of containing a nucleotide sequence encoding MOG(27-63) of SEQ ID NO:18, it contains a nucleotide sequence encoding MOG(27-63).

[0302] The DNA vectors VB5049, VB5052 and VB5062 to VB5065 encode the same first polypeptide comprising an antigenic unit having MOG(27-63): VB5049 also encodes mIL-10, which is expressed as a separate molecule VB5062 also encodes mTGF-β1, which is expressed as a separate molecule VB5063 also encodes the extracellular domain of mCTLA-4, which is expressed as a separate molecule VB5064 additionally encodes mIL-2, which is expressed as a separate molecule VB5065 additionally encodes mIFN-γ, which is expressed as a separate molecule VB5048 only encodes the first polypeptide, but not the immunosuppressant compound, and serves as a comparison with the vector of the present invention. · VB5052 is a "proinflammatory" version of the aforementioned vector and serves as a comparison to the vectors of the present invention. · VB5051 encodes only MOG(27-63) and serves as a comparison to the vectors of the present invention. Characterization of expression and secretion of proteins encoded by DNA vectors Characterization of expression and secretion of the proteins encoded by VB5049, VB5052, VB5062, VB5063, VB5064 and VB5065 was performed as follows. 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 of plasmid DNA using ExpiFectamine293 reagent (Thermo Fisher Sci.), and plates were incubated on an orbital shaker (3 mm diameter, 900 rpm) in a humidified CO2 cell incubator (8% CO2, 37 °C). Supernatants were harvested 72 h after transfection.

[0303] Characterization of the secreted first polypeptides / dimer proteins was performed in a sandwich ELISA of the supernatants using an antibody against MOG (capture antibody, mouse anti-MOG antibody, 0.25 μg / mL, 100 μL / well, sc-73330, Santa Cruz Biotechnology) and an antibody against hIgG CH3 domain (detection antibody, mouse anti-human IgG Fc secondary antibody, biotin, 0.1 μg / mL, 100 μL / well, 05-4240, Invitrogen). As shown in FIG. 11, all the first polypeptides / dimer proteins were highly expressed and secreted.

[0304] Characterization of expression and secretion of the encoded immune inhibitory compounds mIL-10, mTGF-β1, mCTLA-4, mIL-2, and mIFN-γ was performed by sandwich ELISA using antibodies against mIL-10, hTGF-β1 (which also binds mTGF-β1), mCTLA-4, mIL-2, and mIFN-γ, respectively, and the results are shown in Figures 12A-E.

[0305] The following antibodies were used: (A) Capture antibody: 1 μg / mL rat anti-mouse IL-10 antibody, 100 μL / well, MAB417, R&D Systems. Detection antibody: 0.2 μg / mL goat anti-mouse IL-10 biotinylated antibody, 100 μL / well, BAF417, R&D Systems. (B) Capture antibody: 2 μg / mL TGF-β1 antibody, 100 μL / well, MAB2402, RD Systems. Detection antibody: 0.8 μg / mL chicken anti-human TGF-β1 biotinylated antibody, 100 μL / well, BAF240, RD Systems. (C) Capture antibody: 0.8 μg / mL goat anti-mouse CTLA-4 antibody, 100 μL / well, AF476, RD Systems. Detection antibody: 0.8 μg / mL goat anti-mouse CTLA-4 biotinylated antibody, 100 μL / well, BAF476, RD Systems. (D) Capture antibody: 2 μg / mL rat anti-mouse IL-2 antibody, 100 μL / well, 503701, BioLegend. Detection antibody: 2 μg / mL rat anti-mouse IL-2 biotinylated antibody, 100 μL / well, 503803, BioLegend. (E) Capture antibody: 2 μg / mL rat anti-mouse IFN-γ antibody, 100 μL / well, 505802, BioLegend. Detection antibody: 2 μg / mL rat anti-mouse IFN-γ biotinylated antibody, 100 μL / well, 505704, BioLegend.

[0306] As shown in Figures 12A-12E, all five immunoinhibitory compounds were expressed and secreted from their respective vectors.

[0307] Sandwich ELISA using antibodies against MOG and the immune inhibitory compounds mIL-10, mTGF-β1, mCTLA-4, and mIL-2, respectively, confirmed that the first polypeptide / dimer protein and the immune inhibitory compound were expressed and secreted as separate proteins. The results are shown in Figures 13A to 13D. The following antibodies were used: 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 anti-IL-10 biotinylated antibody, 100 μL / well, BAF417, R&D Systems. (B) 0.8 μg / mL chicken anti-human TGF-β1 biotinylated antibody, 100 μL / well, BAF240, RD Systems. (C) 0.8 μg / mL goat anti-mouse CTLA-4 biotinylated antibody, 100 μL / well, BAF476, RD Systems. (D) 2 μg / mL Rat anti-mouse IFN-γ biotinylated antibody, 100 μL / well, 505704, BioLegend As shown in Figures 13A-13D, the first polypeptide and the immunoinhibitory compound are expressed and secreted as separate molecules, rather than being expressed as a fusion protein.

[0308] Characterization of intact proteins expressed from DNA vectors Western blot analysis was performed on supernatant samples from transfected Expi293F cells to further characterize the proteins encoded by VB5049, VB5062, VB5063 and VB5064, which encode the same first polypeptide but different immunoinhibitory compounds. VB5048, which encodes the same first polypeptide as the above DNA vector but does not encode an immunoinhibitory compound, was included as a comparison.

[0309] Samples were prepared by mixing 14 μL of supernatant from transfected Expi293F cells with 5 μL of 4× Laemmli sample buffer (Bio-Rad) with 1 μL of DTT (Cayman Chemical) or 1 μL of ultrapure water for reducing and non-reducing conditions, respectively (total sample volumes scaled up at the indicated ratios). 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 in 1× Tris / glycine / SDS running buffer (Bio-Rad) using Precision Plus Protein All Blue Prestained protein standards (Bio-Rad). Proteins were transferred from the gel to EtOH-activated low-fluorescence (LF) 0.45 μm PVDF membranes (Bio-Rad) 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), goat anti-mouse CTLA-4 (AF467, R&D Systems) or rat anti-mouse IL-2 (503702) to detect MOG, mIL-10, mCTLA-4 or mIL-2, respectively. Membranes were incubated with fluorochrome-conjugated species-specific secondary antibodies for 1 h at room temperature, then washed and dried. For mIL10 detection in Dylight channel 488, 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. The results are shown in Figure 14.

[0310] Western blot analysis confirmed the ELISA results and showed that VB5049, VB5062, VB5063 and VB5064 express two proteins, a first polypeptide / dimeric protein (Figures 14A and B, reducing and non-reducing conditions) and an immunoinhibitory compound as a second protein, namely mIL-10 (Figure 14C), mCTLA-4 (Figure 14D) or mIL-2 (Figure 14E). Importantly, no additional bands were observed on membranes probed with anti-IL-10, anti-CTLA-4 and anti-IL-2, indicating successful ribosome skipping at the T2A sequence and expression of the first polypeptide and the immunoinhibitory compound as separate proteins from a single DNA plasmid.

[0311] Taken together, the ELISA and Western blot data demonstrated that an intact dimeric protein containing a targeting unit, a dimerization unit and an antigenic unit can be co-expressed from a DNA plasmid with an immunoinhibitory compound by using the T2A peptide as a co-expression element.

[0312] Characterization of expression and secretion of MOG(27-63) peptide from vector VB5051 Protein expression and secretion of MOG(27-63) encoded by vector VB5051 was determined as described above in this Example 4.

[0313] Secretion of MOG(27-63) peptide was characterized by direct ELISA, coating with supernatant, and detection using an antibody against MOG (capture antibody, 100 μL / well, 3.3 μg / mL mouse anti-MOG antibody, sc-73330, Santa Cruz Biotechnology). Figure 15 shows that MOG(27-63) peptide is expressed from VB5051 and secreted from mammalian cells transfected with the vector.

[0314] Example 5: The tolerance-inducing capacity of VB5049 was evaluated in the spleens of mice administered VB5049 (see Example 4) and determined by the IL-10 / IFN-γ ratio calculated from IL-10 (an anti-inflammatory cytokine associated with immune tolerance) and IFN-γ (a marker for inducing an inflammatory immune response) signals produced in a two-color FluoroSpot assay after restimulation with MOG(35-55) peptide, as well as by detection of CD4+Foxp3+ T cells. Furthermore, the absence of IFN-γ and IL-17 production, two pro-inflammatory cytokines known to be involved in the pathology of MS, was demonstrated. The results obtained were compared with the responses induced by administration of VB5052 or VB5051 (for units / elements and description of these vectors, see Example 4).

[0315] The study design and methods applied were similar to those in Example 3, except that different dosing schedules for VB5049 were tested to validate the results obtained in Example 3 and to further extend the data beyond the single dosing regimen. * The results were similar to those described for

[0316] Briefly, 50 μg of DNA vector VB5049, VB5051 or VB5052 was administered intramuscularly four times (D0, D3, D7 and D10) followed by electroporation and spleens were harvested 14 days after the first administration. Spleens were crushed with a cell strainer to obtain single cell suspensions and splenocytes were either restimulated with MOG(35-55) peptide for 44 h or not, and then tested for IFN-γ and IL-10 production in a two-color FluoroSpot assay performed as described in Example 3.

[0317] As shown in Figure 16A, some production of IL-10 was detected in unrestimulated splenocytes taken from mice administered VB5049, VB5051 or VB5052, but only low background levels of IFN-γ were observed. Upon MOG(35-55) restimulation of splenocytes, no significant IFN-γ production was detected in the groups of mice administered either VB5049 or VB5051, whereas, in contrast, significantly elevated levels of IFN-γ were detected in splenocytes from mice administered VB5052 (Figure 16B). To avoid excessive inflammation and ultimately resolve inflammation, it is important that the production of proinflammatory cytokines such as IFN-γ is regulated by a negative feedback mechanism that includes the production of anti-inflammatory cytokines such as IL-10 (e.g., Sugimoto et al., Front Immunol 2016, Vol. 7, Article 160). Therefore, the increased levels of IL-10 observed in response to VB5052 may be explained by such a feedback mechanism to control the induced inflammatory response. As shown in Figure 17, a significantly higher IL-10 / IFN-γ ratio was detected in VB5049 compared to VB5052, indicating a higher immunosuppressive potential of VB5049 compared to VB5052.

[0318] Detection of Foxp3, IFN-γ and IL-17 by flow cytometry: The presence of Foxp3-expressing CD4+ T cells was identified in the splenocyte population by flow cytometry. CD4+Foxp3+ T cells can suppress effector T cell and inflammatory immune responses, thereby maintaining self-tolerance. Flow cytometry was performed as described in Example 3.

[0319] As shown in Figure 18, a higher percentage of CD4+Foxp3+ T cells was detected in response to VB5049 administration compared to VB5051 administration, indicating higher levels of suppressor T cells induced by VB5049 compared to VB5051. The proinflammatory cytokines IFN-γ and IL-17 were not detected by flow cytometry in response to VB5049 administration, as shown in Figures 19A and 19B, respectively. In contrast, both proinflammatory cytokines were detected in response to administration of the proinflammatory comparison vector VB5052.

[0320] Thus, Example 5 shows that administration of VB5049, encoding a first polypeptide comprising an anti-DEC205 targeting unit, a dimerization unit, and an antigen comprising MOG(27-63), and further encoding IL-10, results in a higher anti-inflammatory to pro-inflammatory cytokine ratio (IL-10 / IFN-γ) compared to administration of VB5051, encoding only MOG(27-63). Furthermore, administration of VB5049 results in a lack of pro-inflammatory IFN-γ and IL-17 cytokine production and a higher percentage of CD4+Foxp3+T cells compared to VB5051. Taken together, these results show that VB5049, in contrast to its pro-inflammatory version VB5052, can induce a greater antigen-specific tolerogenic response in an anti-inflammatory manner compared to the antigen alone (VB5051). These results also verify the results of Example 3 and show that the tolerogenic properties of VB5049 are preserved after repeated administration to mice.

[0321] Example 6: The tolerance-inducing ability of VB5062 was evaluated in the spleens of mice administered VB5062 (see Table 5 / Example 4) and was determined by the IL-10 / IFN-γ ratio calculated from the IL-10 (anti-inflammatory cytokine associated with immune tolerance) and IFN-γ (marker for inducing an inflammatory immune response) signals produced in a two-color FluoroSpot assay after restimulation with MOG(35-55) peptide, and by the detection of Foxp3+MOG(38-49)-specific regulatory T cells (Tregs) detected ex vivo using MOG(38-49)-specific tetramers. The results obtained were compared with the immunogenicity induced by VB5052 and the tolerance-inducing ability of VB5051 (see Example 4 for the units / elements and description of these vectors).

[0322] Briefly, mice (5 per group) were intramuscularly administered 50 μg of DNA vector VB5062, VB5051 or VB5052, followed by electroporation. Seven days after administration, spleens were harvested and crushed with a cell strainer to obtain single cell suspensions. Splenocytes were either restimulated with MOG(35-55) peptide for 44 hours or not, and then tested for IFN-γ and IL-10 production in a two-color FluoroSpot assay performed as described in Example 3.

[0323] As shown in FIG. 20A, IL-10 production was detected in non-restimulated splenocytes harvested from mice administered VB5062, VB5051 or VB5052, but only low background levels of IFN-γ were observed. Upon MOG(35-55) restimulation of splenocytes, no significant IFN-γ production was detected in the groups of mice administered either VB5062 or VB5051, whereas, in contrast, significantly elevated levels of IFN-γ were detected in splenocytes from mice administered VB5052 (FIG. 20B). The increase in IL-10 levels observed in response to VB5052 may be explained by a potential feedback mechanism to control the induced inflammatory response, as described in Example 5. As shown in FIG. 21, a significantly higher IL-10 / IFN-γ ratio was detected in VB5062 compared to VB5052, indicating a higher immunosuppressive potential of VB5062 compared to VB5052.

[0324] MOG(38-49) tetramer staining and flow cytometry were performed as described in Example 3. Dual-color FluoroSpot assays detected similar IL-10 levels for VB5062 and VB5051 (Figures 20A-B), but further single-cell analysis showed an increased percentage of CD4+MOG(38-49)-specific Foxp3+ cells in mice treated with VB5062 compared to mice treated with VB5051, as shown in Figure 22.

[0325] Thus, Example 6 shows that administration of VB5062, encoding a first polypeptide comprising an anti-DEC205 targeting unit, a dimerization unit, and an antigenic unit comprising MOG(27-63), and further encoding the immunoinhibitory compound TGFβ1, results in a higher anti-inflammatory to pro-inflammatory cytokine ratio (IL-10 / IFN-γ). Furthermore, splenocytes from mice administered VB5062 showed a lack of pro-inflammatory IFN-γ cytokine production compared to splenocytes obtained from mice administered the pro-inflammatory version of VB5052. Administration of VB5062 induced a higher percentage of MOG(38-49)-specific Foxp3+ cells compared to VB5051. Taken together, these results show that VB5062, in contrast to its pro-inflammatory version VB5052, can induce a greater antigen-specific tolerogenic response in an anti-inflammatory manner compared to the antigen alone (VB5051).

[0326] Example 7: The tolerance induction capacity of VB5063 was measured as described in Example 6 and compared with the immunogenicity of VB5052 and the tolerance induction capacity of VB5051 (see Table 5 / Example 4 for units / elements and description of these vectors).

[0327] As shown in FIG. 23A, in unstimulated splenocytes taken from mice administered VB5062, VB5051 or VB5052, production of IL-10 was detected, whereas only low background levels of IFN-γ were observed. Upon MOG(35-55) restimulation of splenocytes, no significant IFN-γ production was detected in groups of mice administered either VB5063 or VB5051, whereas, in contrast, significantly elevated levels of IFN-γ were detected in splenocytes from mice administered VB5052 (FIG. 23B). Significantly higher IL-10 levels were detected in both unstimulated and MOG(35-55) restimulated splenocytes taken from mice administered VB5063, compared to the levels detected in mice administered VB5051. The increase in IL-10 levels observed in response to VB5052 may be explained by a potential feedback mechanism for controlling inflammatory responses, as described in Example 5. As shown in FIG. 24, a significantly higher IL-10 / IFN-γ ratio was detected in VB5063 compared to VB5052, indicating that VB5063 has a higher immunosuppressive ability than VB5052.

[0328] As shown in Figure 25, a higher percentage of MOG(38-49)-specific Foxp3+ cells was detected in response to VB5063 compared to VB5051, indicating increased generation of immunosuppressive Tregs in response to VB5063 administration.

[0329] Furthermore, to determine the percentage of actively proliferating Treg cells induced in response to VB5063 treatment, the expression of Ki67 (a nuclear marker strictly associated with dividing cells) was analyzed in splenocytes harvested from mice treated with VB5063 or VB5051. As shown in Figure 26, a higher percentage of Ki67+ cells within the Treg (CD4+CD25+Foxp3+) population was detected ex vivo in mice treated with VB5063 compared to VB5051.

[0330] Thus, Example 7 shows that administration of VB5063, encoding a first polypeptide comprising an anti-DEC205 targeting unit, a dimerization unit, and an antigenic unit comprising MOG(27-63), and further encoding the immunoinhibitory compound CTLA-4, results in a higher anti-inflammatory to pro-inflammatory cytokine ratio (IL-10 / IFN-γ). Furthermore, splenocytes from mice administered VB5063 showed a lack of pro-inflammatory IFN-γ cytokine production compared to the pro-inflammatory version VB5052. Furthermore, VB5063 induced a higher percentage of both MOG(38-49)-specific Foxp3+ and CD4+CD5+Foxp3+Ki67+ expanded Treg cells compared to VB5051. Taken together, these results indicate that VB5063, in contrast to its proinflammatory version, VB5052, is able to induce greater antigen-specific tolerogenic responses compared to antigen alone (VB5051) in an anti-inflammatory manner.

[0331] Example 8: The tolerance induction capacity of VB5064 was measured as described in Example 6 and compared with the immunogenicity of VB5052 and the tolerance induction capacity of VB5051 (see Table 5 / Example 4 for units / elements and description of these vectors).

[0332] As shown in FIG. 27A, IL-10 production was detected in unrestimulated splenocytes harvested from mice administered VB5064, VB5051, or VB5052, but only low background levels of IFN-γ were observed. Upon MOG(35-55) restimulation of splenocytes, no significant IFN-γ production was detected in groups of mice administered either VB5064 or VB5051, whereas, in contrast, significantly elevated levels of IFN-γ were detected in splenocytes from mice administered VB5052 (FIG. 27B). The increase in IL-10 levels observed in response to VB5052 may be explained by a potential feedback mechanism to control the inflammatory response, as described in Example 5. As shown in FIG. 28, a significantly higher IL-10 / IFN-γ ratio was detected in VB5064 compared to VB5052, indicating a higher immunosuppressive potential of VB5064 compared to VB5052.

[0333] Dual-color FluoroSpot assays detected similar IL-10 levels with VB5064 and VB5051 (Figure 27A,B), but a higher percentage of MOG(38-49)-specific Foxp3+ cells in response to VB5064 compared to VB5051 (Figure 29).

[0334] Furthermore, to determine the percentage of actively proliferating Treg cells induced in response to VB5064 treatment, the expression of Ki67 (a nuclear marker strictly associated with dividing cells) was analyzed in splenocytes harvested from mice treated with VB5064 or VB5051. As shown in Figure 30, a higher percentage of Ki67+ cells within the Treg (CD4+CD25+Foxp3+) population was detected ex vivo in splenocytes from mice treated with VB5064 compared to VB5051.

[0335] Thus, Example 8 shows that administration of VB5064, encoding a first polypeptide comprising an anti-DEC205 targeting unit, a dimerization unit and an antigenic unit comprising MOG(27-63), and further encoding the immunoinhibitory compound IL-2, results in a higher anti-inflammatory to pro-inflammatory cytokine ratio (IL-10 / IFN-γ). Furthermore, splenocytes from mice administered VB5064 showed a lack of pro-inflammatory IFN-γ cytokine production compared to splenocytes obtained from mice administered the pro-inflammatory version of VB5052. Furthermore, VB5064 induced a higher percentage of both MOG(38-49)-specific Foxp3+ and CD4+CD5+Foxp3+Ki67+ expanded Treg cells compared to VB5051. Taken together, these results indicate that VB5064, in contrast to its proinflammatory version, VB5052, is able to induce greater antigen-specific tolerogenic responses compared to antigen alone (VB5051) in an anti-inflammatory manner.

[0336] Example 9 A DNA vector was designed and generated that contains a nucleotide sequence encoding MOG(27-63) and further encoding the following elements / units: [Table 6] The DNA vectors VB5049, VB5044 and VB5054 encode the same first polypeptide comprising an antigenic unit having MOG(27-63) and further encode the following immunoinhibitory compounds that are expressed as separate molecules due to the presence of the co-expression elements listed in Table 6: VB5049: mIL-10 (serves as a control in Western blot analysis) VB 5044: mIL-10 and mTGF-β1 VB 5054: mIL-10 and mTGF-β1 and mGM-CSF Characterization of expression and secretion of proteins encoded by DNA vectors Characterization of expression and secretion of proteins encoded by VB5044 and VB5054 was performed as described in Example 4. As shown in Figure 31, the first polypeptide / dimer protein from both vectors was highly expressed and secreted.

[0337] Characterization of expression and secretion of the encoded immunoinhibitory compounds mIL-10, mTGF-β1, and mGM-CSF was performed by sandwich ELISA using antibodies against mIL-10, hTGF-β1, and mGM-CSF, respectively. The results are shown in Figures 32A-C and show that all immunoinhibitory compounds encoded by VB5044 or VB5054 were expressed and secreted. Even the third immunoinhibitory compound encoded by vector VB5054, mGM-CSF, was highly expressed and secreted (Figure 32C).

[0338] Sandwich ELISA using antibodies against MOG and immune inhibitory compounds mIL-10, mTGF-β1 and mGM-CSF, respectively, confirmed that the first polypeptide / dimer protein and immune inhibitory compounds were expressed and secreted as separate proteins. The results show that the ribosome skipping peptides T2A and P2A are highly effective, and that all the first polypeptide / dimer proteins and immune inhibitory compounds were expressed and secreted as separate proteins (Figure 33A-C).

[0339] Characterization of intact proteins expressed from VB5044 and VB5054 by Western blot Western blot (WB) analysis was performed on supernatants from transfected Expi293F cells to further characterize the proteins encoded by VB5044 and VB5054. VB5049, encoding the same first polypeptide and the immune inhibitory compound IL-10, was included as a control.

[0340] WB was performed as described in Example 4. PVDF membranes were probed with mouse anti-MOG (sc-73330, Santa Cruz Biotechnology), rat anti-mouse IL-10 (MAB417, R&D Systems), rabbit anti-TGFβ1 (USB1042777-Biotin, United States Biological) or goat anti-mouse GM-CSF (BAF415, R&D Systems) to detect the first polypeptide / dimer protein, mIL-10, mTGF-β1 and mGM-CSF, respectively. The results are shown in Figures 34A-E.

[0341] WB analysis confirmed the ELISA results, showing that VB5044 and VB5054 express three and four proteins, respectively, the first polypeptide / dimeric protein (Figure 34A and Figure 34B, reducing and non-reducing conditions) and two or three immunoinhibitory compounds, namely mIL-10 (Figure 34C), mTGF-β1 (Figure 34D) and mGM-CSF (Figure 34E). Importantly, no additional bands were observed on membranes probed with anti-IL-10, anti-TGF-β1 and anti-GM-CSF, indicating successful ribosome skipping at the 2A sequence and expression of the first polypeptide and the immunoinhibitory compounds as separate proteins from a single DNA plasmid.

[0342] Taken together, the ELISA and WB data demonstrated that intact dimeric proteins containing the targeting unit, dimerization unit and antigenic unit could be co-expressed from DNA vectors with several immunoinhibitory compounds by using different 2A peptides as co-expression elements.

[0343] Example 10 A DNA vector was designed and generated that contains a nucleotide sequence encoding MOG(27-63) and further encoding the following elements / units: [Table 7] DNA vectors VB5068, VB5069 and VB5070 encode a first polypeptide comprising an antigenic unit with MOG(27-63) and further encode the immune inhibitory compound IL-10, which is expressed as a separate molecule due to the presence of the T2A peptide co-expression element. The first polypeptides encoded by these vectors contain different targeting units: ·VB5068:mSCGB3A2 VB5069: mVSIG3 extracellular domain VB5070: mPD-1 extracellular domain Characterization of expression and secretion of proteins encoded by DNA vectors Characterization of expression and secretion of proteins encoded by VB5068, VB5069 and VB5070 was performed as described in Example 4. As shown in Figure 35, the first polypeptide / dimer proteins from all three vectors were highly expressed and secreted.

[0344] Expression and secretion of the full-length first polypeptide / dimer proteins encoded by VB5068 and VB5070 was verified by sandwich ELISA using antibodies against MOG and the respective targeting units, mSCGB3A2 and mPD-1. Figures 36A and 36B show that the full-length proteins were highly expressed and secreted from both vectors.

[0345] Taken together, these results demonstrated that cells transfected with DNA vectors VB5068 and VB5070 expressed and secreted proteins containing the targeting unit (mSCGB3A2 or mPD-1), the CH3-containing dimerization unit, and the mouse MOG(27-63) antigen.

[0346] Secretion characterization of the immune inhibitory compound mIL-10 encoded by VB5068 and VB5069 was performed by sandwich ELISA using an antibody against mouse IL-10. Figure 37 shows that mIL-10 was highly expressed and secreted from both vectors.

[0347] Sandwich ELISA using antibodies against MOG and mouse IL-10 confirmed that the first polypeptide / dimer protein and the immune inhibitory compound IL-10 were expressed and secreted as separate proteins. The results show that the ribosome skipping peptide T2A is highly effective and that the first polypeptide / dimer protein and mIL-10 were expressed and secreted as separate proteins (Figure 38).

[0348] Characterization of intact proteins expressed from VB5069 and VB5070 Western blot analysis was performed on supernatants from transfected Expi293F cells to further characterize the proteins encoded by DNA vectors VB5069 and VB5070.

[0349] Western blots were performed as described in Example 4. PVDF membranes were probed with mouse anti-MOG (sc-73330, Santa Cruz Biotechnology) to detect the first polypeptide / dimer protein and with rat anti-mouse IL-10 (MAB417, R&D Systems) to detect the immune inhibitory compound mIL-10. The results are shown in Figures 39A and 39B.

[0350] Western blot analysis confirmed the ELISA results and demonstrated that VB5069 and VB5070 express two proteins, the first polypeptide (Figure 39A, reduced supernatant sample) and mIL-10 (Figure 39B). Importantly, no additional bands were observed in membranes probed with anti-IL-10, indicating successful ribosome skipping at the T2A sequence and expression of two distinct proteins from a single DNA vector.

[0351] Considering the foregoing examples disclosed herein, taken together with the ELISA and WB data, it has been demonstrated that an intact first polypeptide comprising a targeting unit distinct from an scFv having specificity for mouse CD205 can be co-expressed from a DNA vector together with an immune inhibitory compound (mIL-10) by using the T2A peptide as a co-expression element.

[0352] Example 11. The tolerance-inducing ability of VB5068 (see Table 7) was measured as described in Example 6 and compared with the immunogenicity of VB5052 (a pro-inflammatory version of VB5068) and the tolerance-inducing ability of VB5051 (see Example 4 for units / elements and description of these vectors).

[0353] As shown in Figure 40A, IL-10 production was detected in unrestimulated splenocytes harvested from mice administered VB5068, VB5051 or VB5052, whereas only low background levels of IFN-γ were observed. Upon MOG(35-55) restimulation of splenocytes, no IFN-γ production was detected in groups of mice administered VB5068 or VB5051, whereas, in contrast, significantly elevated levels of IFN-γ were detected in splenocytes from mice administered VB5052 (Figure 40B). The increase in IL-10 levels observed in response to VB5052 may be explained by a potential feedback mechanism for controlling inflammatory responses, as described in Example 5.

[0354] Dual-color FluoroSpot assays detected similar IL-10 levels in splenocytes from mice administered either VB5068 or VB5051 (Figure 40A / B), whereas flow cytometry detected a higher percentage of MOG(38-49)-specific Foxp3+ cells in response to VB5068 compared to VB5051 (Figure 41).

[0355] The observed higher percentage of MOG(38-49)-specific Foxp3+ cells indicative of Tregs detected in response to VB5068 (Figure 41) was verified and confirmed by the inclusion of an additional phenotypic marker, CD25, to identify Tregs. Splenocytes harvested from mice treated with VB5068 and analyzed by flow cytometry showed higher levels of CD4+CD25+Foxp3+MOG(38-49)-tet+Tregs compared to the levels detected in the spleens of mice treated with either VB5051 or VB5052 (Figure 42).

[0356] Furthermore, to determine the percentage of actively proliferating Treg cells induced in response to VB5064 treatment, the expression of Ki67 (a nuclear marker strictly associated with dividing cells) was analyzed in splenocytes harvested from mice treated with VB5068 or VB5051. As shown in Figure 43, a higher percentage of Ki67+ cells within the Treg (CD4+CD25+Foxp3+) population was detected ex vivo in mice treated with VB5068 compared to VB5051 and VB5052.

[0357] To evaluate and confirm the percentage of Tregs induced and detected ex vivo after VB5068 administration (Figures 41 and 42), the induction of Tregs after MOG(35-55) restimulation of splenocytes was also evaluated. Splenocytes from mice administered VB5068, VB5051, or VB5052 were restimulated with MOG(35-55) peptide for 16 hours and analyzed by flow cytometry in further experiments. As shown in Figure 44, a higher percentage of CD4+CD25+Foxp3+Tregs was detected in splenocytes harvested from mice administered VB5068 compared to those detected in the spleens of mice administered either VB5051 or VB5052.

[0358] Example 11 shows that administering VB5068 to mice, which encodes a first polypeptide comprising an SCGB3A2 targeting unit, a dimerization unit, and an antigenic unit comprising MOG(27-63), and further encodes the immunoinhibitory compound IL-10, results in a higher anti-inflammatory cytokine to pro-inflammatory cytokine ratio (IL-10 / IFN-γ). Furthermore, splenocytes from mice administered VB5068 showed a lack of pro-inflammatory IFN-γ cytokine production compared to splenocytes from mice administered the pro-inflammatory version of VB5052. Furthermore, VB5068 induced a higher percentage of both MOG(38-49)-specific Foxp3+ Treg and CD4+CD5+Foxp3+Ki67+ expanded Treg cells compared to both VB5051 and VB5052. Taken together, these results show that VB5064, in contrast to its proinflammatory version VB5052, is able to induce a greater antigen-specific tolerogenic response compared to the antigen alone (VB5051) in an anti-inflammatory manner. Furthermore, the data presented in Example 11 show the versatility of the vectors of the invention, demonstrating that different targeting units (anti-CD205 and SCGB3A2 targeting units) allow targeting of the antigen contained in the antigenic unit to APCs in a tolerogenic manner.

[0359] Example 12 A vector was designed and produced that contains a nucleotide sequence encoding a T cell epitope of the shrimp allergen tropomyosin from the species Metapenaeus ensis and further encoding the following elements / units: [Table 8] Tropomyosin is a major allergen in crustaceans. Six major T cell epitopes were identified for tropomyosin (Met e 1 allergen) from shrimp species Metapenaeus ensis 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 et al.,Int J Mol Sci 20(18),4656,2015).

[0360] The DNA vector VB5076 encodes a first polypeptide comprising an antigenic unit containing T cell epitopes derived from Met e 1. The six T cell epitopes ((241-260), (210-230), (136-155), (76-95), (46-65), and (16-35)) are separated from each other by the T cell epitope linker GGGGSGGGGS. VB5076 further encodes an immunoinhibitory compound mIL10.

[0361] Characterization of expression and secretion of protein encoded by DNA vector VB5076 Characterization of expression and secretion of the protein encoded by VB5076 was performed as described in Example 4. As shown in Figure 45, the first polypeptide / dimer protein from VB5076 was expressed and secreted at high levels.

[0362] Secretion characterization of the immune inhibitory compound mIL-10 encoded by VB5076 was performed by sandwich ELISA using an antibody against mouse IL-10. Figure 46 shows that mIL-10 was highly expressed and secreted from VB5076.

[0363] Characterization of intact protein expressed from VB5076 Western blot analysis was performed on the supernatants from transfected Expi293F cells to further characterize the protein encoded by VB5076. No commercially available antibody against the first polypeptide / dimer protein expressed by VB5076 was identified that was compatible with Western blotting. Thus, only the immune inhibitory compound mIL-10 could be detected.

[0364] Western blots were performed as described in Example 4. PVDF membranes were probed with rat anti-mouse IL-10 (MAB417, R&D Systems) to detect the immune inhibitory compound mIL-10. The results are shown in Figure 47.

[0365] In membranes probed with anti-IL-10, one major band of the expected molecular weight for IL-10 was observed, indicating successful ribosomal skipping at the T2A sequence and expression of mIL10 as a molecule separate from VB5076.

[0366] Taken together, the ELISA and Western blot data demonstrated that a first polypeptide / dimeric protein containing an antigenic unit with several T cell epitopes can be co-expressed from a DNA plasmid with another protein (an immunoinhibitory compound) by using the T2A peptide as a co-expression element.

[0367] Overview of Arrays SEQ ID NO:1 Amino acid sequence of an embodiment of a dimerization unit consisting of hinge region 1 (amino acids 1-12) derived from human IgG3, hinge region 4 (amino acids 13-27) derived from human IgG3, a glycine-serine linker (amino acids 28-37), and a CH3 domain (amino acids 38-144) derived from human IgG3. ELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK SEQ ID NO:2 Signal peptide of mIg VH MNFGLRLIFLVLTLKGVQC SEQ ID NO:3 VB5049 * The amino acid sequence of Amino acids 1-19: mouse immunoglobulin heavy chain signal peptide; amino acids 20-265: mouse single chain variable fragment (scFv) anti-DEC205; amino acids 266-409: dimerization unit (SEQ ID NO:1): hinge region 1 from human IgG3, hinge region 4 from human IgG3, glycine-serine linker, CH3 domain from human IgG3; amino acids 410-414: unit linker; amino acids 415-451 mouse MOG 27-63 (MOG 35-55 underlined), amino acids 452-454: linker; amino acids 455-472 co-expression element T2A peptide; amino acids 473-490: signal peptide mouse IL-10, natural leader sequence; amino acids 491-650: mouse IL-10 [Table 9] SEQ ID NO:4 VB5052 * The amino acid sequence of Amino acids 1-23: signal peptide human CCL3L1; amino acids 24-93: human CCL3L1; amino acids 94-237: dimerization unit (SEQ ID NO: 1): hinge region 1 from human IgG3, hinge region 4 from human IgG3, glycine-serine linker, CH3 domain from human IgG3; amino acids 238-242: unit linker; amino acids 243-279: mouse MOG 27-63 (MOG 35-55 underlined) [Table 10] SEQ ID NO:5 VB5051 * The amino acid sequence of Amino acids 1-19: mouse immunoglobulin heavy chain signal peptide; amino acids 20-56: mouse MOG 27-63 (MOG 35-55 underlined). [Table 11] SEQ ID NO:6 T2A EGRGSLLTCGDVEENPGP SEQ ID NO:7 P2A ATNFSLLKQAGDVEENPGP SEQ ID NO:8 E2A QCTNYALLKLAGDVESNPGP SEQ ID NO:9 F2A VKQTLNFDLLKLAGDVESNPGP SEQ ID NO:10 Nucleotide sequence encoding amino acids 1 to 27 of SEQ ID NO:1 GAGCTCAAAACCCCACTTGGTGACACAACTCACACAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCA SEQ ID NO:11: Nucleotide sequence encoding amino acids 38 to 144 of SEQ ID NO:1 GGACAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAGCGGGCAGCCGGAGAACAACTACAACACCA CGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA SEQ ID NO:12: A nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1. GAGCTCAAAACCCCACTTGGTGACACAACTCACACAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCAGGCGGTGGAAGCAGCGGAGGTGGAAGTGGAGGACAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGC GACATCGCCGTGGAGTGGGAGAGCAGCGGGCAGCCGGAGAACAACTACAACACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTTCACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA SEQ ID NO:13 Signal peptide hCCL3L1 MQVSTAALAVLLCTMALCNQVLS SEQ ID NO:14 hCCL3L1 APLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSA SEQ ID NO:15 scFv with specificity for mouse CD205 DIQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLELKGGGGSGGGGSGGGGSE VKLLESGGGLVQPGGSLRLSCAASGTFNDFYMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISRDNTQNILYLQMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSS SEQ ID NO:16 MOG(27-63) * SPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEAQP SEQ ID NO:17 Signal peptide mIL-10 MPGSALLCCLLLLTGMRI SEQ ID NO:18 mIL-10 SRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS SEQ ID NO:19 MOG(27-63) SPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAP SEQ ID NO:20 VB5062 MNFGLRLIFLVLTLKGVQCDIQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYC QHYQSFPWTFGGGTKLELKGGGGSGGGGSGGGGSEVKLLESGGGLVQPGGSLRLSCAASGTFNDFYMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISR DNTQNILYLQMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSSELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLSPGKNATGMEVGWYRSP FSRVVHLYRNGKDQDAEQAPGSGEGRGSLLTCGDVEENPGPMPPSGLRLLPLLLPLPWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQG EVPPGPLPEAVLALYNSTRDRVAGESADPEPEPEADYYAKEVTRVLMVDRNNAIYEKTKDISHSIYMFFNTSDIREAVPEPPLLSRAELRLQRLKSSVEQHVELYQKY SNNSWRYLGNRLLTPTDTPEWLSFDVTGVVRQWLNQGDGIQGFRFSAHCSCDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMATPLERAQHLHSSRHRRALDT NYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASASPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS SEQ ID NO:21 VB5063 MNFGLRLIFLVLTLKGVQCDIQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLELKGGGGSGGGGSGGGGSEVKLLESGGGLVQPGGS LRLSCAASGTFNDFYMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISRDNTQNILYLQMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSSELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPS REEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLSPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAPGSGEGRGSLLTCGDVEENPGPMA CLGLRRYKAQLQLPSRTWPFVALLTLLFIPVFSEAIQVTQPSVVLASSHGVASFPCEYSPSHNTDEVRVTVLRQTNDQMTEVCATTFTEKNTVGFLDYPFCSGTFNESRVNLTIQGLRAVDTGLYLCKVELMYPPPYFVGMGNGTQIYVIDPEPCPDSD SEQ ID NO:22 VB5064 MNFGLRLIFLVLTLKGVQCDIQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLELKGGGGSGGGGSGGGSEVKL LESGGGLVQPGGSLRLSCAASGFTFNDFYMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISRDNTQNILYLQMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSSELKTPLGDTTHTEPKSCDTPPPCPRC PGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLMYSMQLASCVTLTLVLLVNSAPT SSSTSSSTAEAQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ SEQ ID NO:23 VB5065 MNFGLRLIFLVLTLKGVQCDIQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLELKGGGGSGGGGSGGGGSE VKLLESGGGLVQPGGSLRLSCAASGTFNDFYMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISRDNTQNILYLQMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSSELKTPLGDTTHTEPKSCDT PPPCPRCPGGGSSGGGSGGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLMNATHCILALQL FLMAVSGCYCHGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQIISFYLRLFEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSRC SEQ ID NO:24 Signal peptide mTGF-β1 MPPSGLRLLPLLLPLPWLLVLTPGRPAAG SEQ ID NO:25 mTGF-β1 LSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESADPEPEPEADYYAKEVTRVLMVDRNNAIYEKTKDISHSIYMFFNTSDIREAVPEPPLLSRAELRLQRLKSSVEQHVELYQKYSNNSWRYLGNRLLTPTDTPEWLSFDVTGVVRQWLN QGDGIQGFRFSAHCSCDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMATPLERAQHLHSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASASPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS SEQ ID NO:26 Signal peptide mCLTA-4 MACLGLRRYKAQLQLPSRTWPFVALLTLLFIPVFS SEQ ID NO:27 mCLTA-4 EAIQVTQPSVVLASSHGVASFPCEYSPSHNTDEVRVTVLRQTNDQMTEVCATTFTEKNTVGFLDYPFCSGTFNESRVNLTIQGLRAVDTGLYLCKVELMYPPPYFVGMGNGTQIYVIDPEPCPDSD SEQ ID NO:28 Signal peptide mIL-2 MYSMQLASCVTLTLVLLVNS SEQ ID NO:29 mIL-2 APTSSSTSSSTAEAQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ SEQ ID NO:30 Signal peptide mIFN-γ MNATHCILALQLFLMAVSGCYC SEQ ID NO:31 mIFN-γ HGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQIISFYLRLFEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSRC SEQ ID NO:32 VB5049 MNFGLRLIFLVLTLKGVQCDIQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLELKGGGGSGGGGSGGGSEVKLLESGGGLVQPGGSLRLS CAASGFTFNDFYMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISRDNTQNILYLQMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSSELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLSPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAPGSGEGRGSLLTCGDVEENPGPMPGSALLCCLLLLTG MRISRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS SEQ ID NO:33 VB5052 MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWAVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVY TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLSPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAP SEQ ID NO:34 VB5051 MNFGLRLIFLVLTLKGVQCSPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAP SEQ ID NO:35 VB5048 MNFGLRLIFLVLTLKGVQCDIQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQS FPWTFGGGTKLELKGGGGSGGGGSGGGGSEVKLLESGGGLVQPGGSLRLSCAASGFTFNDFYMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISRDNTQNILYLQ MNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSSELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLSPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAP SEQ ID NO:36 VB5044 SEQ ID NO:37 VB5054 SEQ ID NO:38 Signal peptide mGM-CSF MWLQNLLFLGIVVYSLS SEQ ID NO:39 mGM-CSF APTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNEFSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADFIDSLKTFLTDIPFECKKPVQK SEQ ID NO:40 VB5068 MKLVSIFLLVTIGICGYSATALLINRLPVVDKLPVPLDDIIPSFDPLKMLLKTLGISVEHLVTGLKKCVDELGPEASEAVKKLLEALSHLVELKTPLGDTTHTEPKSCDTPPPCPRCPG GGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLG GLSPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAPGSGEGRGSLLTCGDVEENPGPMPGSALLCCLLLLTGMRISRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKD QLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS SEQ ID NO:41 VB5069 MTRRRSAPASWLLVSLLGVATSLEVSESPGSVQVARGQTAVLPCAFSTSAALLNLNVIWMVIPLSNANQPEQVILYQGGQMFDGALRFHGRVGFTGTMPATNVSIFINNTQLSDTGTYQCLVNNLPDRGGRNIGVTGLTVLVPPSAPQCQIQGSQD LGSDVILLCSSEEGIPRPTYLWEKLDNTLKLPPTATQDQVQGTVTIRNISALSSGLYQCVASNAIGSTCLLDLQVISPQPRSVELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLSPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAPGSGEGRGSLLTCGDVEENPGPMPGSALLCCLLLLTGMRISRG QYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS SEQ ID NO:42 VB5070 MWVRQVPWSFTWAVLQLSWQSGWLLEVPNGPWRSLTFYPAWLTVSEGANATFTCSLSNWSEDLMLNWNRLSPSNQTEKQAAFCNGLSQPVQDARFQIIQLPNRHDFHMNILDTRRNDSGIYLCGAISLHPKAKIEESP GAELVVTERILETSTRYPSPSPKPEGRFQGMELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLT VDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLSPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAPGSGEGRGSLLTCGDVEENPGPMPGSALLCCLLLLTGMRISRGQYSREDNNCTHFPVGQSH MLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS SEQ ID NO:43 Signal peptide mSCGB3A2 [Table 12] SEQ ID NO:44 mSCGB3A2 LLINRLPVVDKLPVPLDDIIPSFDPLKMLLKTLGISVEHLVTGLKKCVDELGPEASEAVKKLLEALSHLV SEQ ID NO:45 Signal peptide mVSIG3 MTRRRSAPASWLLVSLLGVATS SEQ ID NO:46 mVSIG3 extracellular domain LEVSESPGSVQVARGQTAVLPCAFSTSAALLNLNVIWMVIPLSNANQPEQVILYQGGQMFDGALRFHGRVGFTGTMPATNVSIFINNTQLSSDTGTYQCLVNNLPDRGGR NIGVTGLTVLVPPSAPQCQIQGSQDLGSDVILLCSSEEGIPRPTYLWEKLDNTLKLPPTATQDQVQGTVTIRNISALSSGLYQCVASNAIGSTCLLDLQVISPQPRSV SEQ ID NO:47 Signal peptide mPD-1 MWVRQVPWSFTWAVLQLSWQSGWL SEQ ID NO:48 mPD-1 extracellular domain LEVPNGPWRSLTFYPAWLTVSEGANATFTCSLSNWSEDLMLNWNRLSPSNQTEKQAAFCNGLSQPVQDARFQIIQLPNRHDFHMNILDTRRNDSGIYLCGAISLHPKAKIESPGAELVVTERILETSTRYPSPSPKPEGRFQGM SEQ ID NO:49 VB5076 MNFGLRLIFLVLTLKGVQCDIQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLELKGGGGSGGGGSGGGGSEVKLLESGGGLVQPGGSLRLSCAASGFTFNDFYMNWIRQPPGQAPEWLGVIRNKG NGYTTEVNTSVKGRFTISRDNTQNILYLQMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSSELKTPLGDTTHTEPKSCDTPPPPRCPGGGSSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGPYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFLYSKLTVDKSRWQQGNIFSCSVMHE ALHNRFTQKSLSLSPGKGLGLKEVDRLEDELVNEKEKYKSIGGGGSGGGGSAYKEQIKTLTNKLKAAEARAEGGGGSGGGGSNQLKEARFLAEEADRKYDEVGGGGSGGGGSAALNRRIQLLEEDLERSEERGGGGSGGGGSDLDQVQESLLKANNQLVEKDGGGGSGGGGSEQQNKEANNRAEKSEEEVHNGSG EGRGSLLTCGDVEENPGPMPGSALLCCLLLLTGMRISRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIKFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS sequence number 50 Met 1 KEVDRLEDELVNEKEKYKSIGGGGSGGGGSAYKEQIKTLTNKLKAAEARAEGGGGSGGGGSNQLKEARFLAEADRKYDEVGGGGGSGGGGSAALNRRIQLLEEDLERSEERGGGGSGGGGSDLDQVQESLLKANNQLVEKDGGGGSGGGGSEQQNKEANNRAEKSEEEVHN SEQ ID NO:51 hCTLA4 extracellular domain, signal peptide underlined [Table 13] SEQ ID NO:52 hPD-1 extracellular domain, signal peptide underlined [Table 14] SEQ ID NO:53 hIL-10, signal peptide underlined [Table 15] SEQ ID NO:54 hTGFβ-1, signal peptide underlined [Table 16] SEQ ID NO:55 hIL-2, signal peptide underlined [Table 17] SEQ ID NO:56 hGM-CSF, signal peptide underlined [Table 18] SEQ ID NO:57 hIFN-γ, signal peptide underlined [Table 19] SEQ ID NO:58 hTGFβ-2 MHYCVLSAFLILHLVTVALSLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPEDYPEPEEVPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPP FFPSETVCPVVTTPSGSVGSLCSRQSQVLCGYLDAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTR AEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRA LDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS SEQ ID NO:59 hTGFβ-3 MKMHLQRALVVLALLNFATVSLSLSTCTTLDFGHIKKKRVEAIGQILSKLRLTSPPEPTVMTHVPYQVLALYNSTRELLEEMHGEREEGCTQENTESEYYAKEIHKFDMIQGLAEHNELAVCPKGITSKVFRFNVSSVEKNRTNLFRAEFRVLRVPNPSSKRNEQRIELFQILRPDEHIAKQRYIGGKNLPTRGTAEWLSFDVTD TVREWLLRRESNLGLEISIHCPCHTFQPNGDILENIHEVMEIKFKGVDNEDDHGRGDLGRLKKQKDHHNPHLILMMIPPHRLDNPGQGGQRKKRALDTNYCFR NLEENCCVRPLYIDFRQDLGWKWVHEPKGYYANFCSGPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLSNMVVKSCKCS SEQ ID NO:60 hTGFβ-1 SEQ ID NO:61 hTGFβ-2 sequence no.62 hTGFβ-3 ATGAAGATGCACTTGCAAAGGGCTCTGGTGGTCCTGGCCCTGCTGAACTTTGCCACGGTCAGCCTCTCTCTGTCCACTTGCACCACCTTGACTTCGGCCACATCAAGAAGAAGAGGGTGGAAGCCATTAGGGGACAGATCTTGAGCAAGCTCAGGCTCACCAGCCCCCCTGAGCCAACGGTGATCCACGTCCCCTATCAGGTCCTGGCCCTTTACAACAGCACCCGGG AGCTGCTGGAGGAGATGCATGGGGAGAGGGAGGAAGGCTGCACCAGGAAAACACCGAGTCGGAATACTATGCCAAAGAAATCCATAAATTCGACATGATCCAGGGGCTGGCGGAGACACAACGAACTGGCTGTCTGCCCTAAAGGAATTACCTCCAAGGTTTTCCGCTTCAATGTGTCCTCAGTGGAGAAAAAATAGAACCAACCTATTCCGAGCAGAATTCCGGGGTCTTGCGG GTGCCCAACCCCAGCTCTAAGCGGAATGAGCAGAGGATCGAGCTCTTCCAGATCCTTCGGCCAGATGAGCACATTGCCAAACAGCGCTATATCGGTGGGCAAGAATCTGCCCACACGGGGCACTGCCGAGTGGCTGTCCTTTGATGTCACTGACACTGTGCGTGAGTGGCTGTTGAGAAGAGAGTCCAACTTAGGTCTAGAAATCAGCATTCACTGTCCATGTCACACCTTTC AGCCCAATGGAGATATCCTGGAAAACATTCACGAGGTGATGGAAATCAAATTCAAAGGCGTGGCAATGAGGATGACCATGGCCGTGGAGATCTGGGGCGCCTCAAGAAGCAGAAGGATCACCACAACCCTCATCTAATCCTCATGATGATTCCCCCACACCGGCTCGACAACCCGGGCCAGGGGGGTCAGAGAGAAGCGGGCTTTGGACACCAATTACTGCTTCCGGTGGA sequence no. 63 hIL-10 ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCAGCCCAGGCCAGGGCACCCAGTCTGAGAACAGCTGCACCCACTTCCCAGGCAACCTGCCTAACATGCTTCGAGATCTCCGAGATGCCTTCAGCAGAGTGAAGACTTTCTTTCAAATGAAGGATCAGCTGGACAACTTGTTGTTAAAGGAGTCCTTGCTGGAGGACTTTAAGGGTTACCTGGGTTGCCAAGCCTTGTCTGAGATGATCCAGTTTTACCTGGAGGAGGTGATGCCCCAAGCTGAGAACCAAGACCCAGACATCAAGGCGCATGTGAACTCCCTGGGGGAGAACCTGAAGACCCTCAGGCTGAGGCTACGGCGCTGTCATCGATTTCTTCCCTGTGAAAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAATGCCTTTAATAAGCTCCAAGAGAAAGGCATCTACAAAGCCATGAGTGAGTTTGACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAACTGA SEQ ID NO: 64 hSCGB3A2, signal peptide underlined

Table 20

[0368] Embodiment 1. (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as 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; (b) one or more additional nucleic acid sequences encoding one or more immunoinhibitory compounds, A vector that allows for the co-expression of the first polypeptide and one or more immunoinhibitory compounds as separate molecules.

[0369] 2. The vector of embodiment 1, wherein the one or more immunoinhibitory compounds induce immune tolerance.

[0370] 3. The vector of embodiment 1, wherein the one or more immunoinhibitory compounds increase immune tolerance.

[0371] 4. The vector of embodiment 1, wherein the one or more immunoinhibitory compounds maintain immune tolerance.

[0372] 5. The vector of embodiment 1, wherein the one or more immunoinhibitory compounds induce immune tolerance and / or increase immune tolerance and / or maintain immune tolerance.

[0373] 6. A vector according to any one of embodiments 1 to 5, wherein the one or more immunoinhibitory compounds favour the presentation of one or more T cell epitopes in the antigenic unit in a tolerance-inducing manner, e.g. promote and / or support the presentation of one or more T cell epitopes in the antigenic unit in a tolerance-inducing manner.

[0374] 7. A vector according to any one of embodiments 1 to 6, wherein the one or more immunoinhibitory compounds favour the induction of tolerance-maintaining cells, e.g. promote and / or support the induction of tolerance-maintaining cells.

[0375] 8. The vector according to any one of embodiments 1 to 7, wherein the one or more immunosuppressive compounds serve to maintain tolerance-maintaining cells.

[0376] 9. The vector according to any one of embodiments 1 to 8, wherein the immune inhibitory compound is an extracellular portion, such as an extracellular domain, of an inhibitory checkpoint molecule.

[0377] 10. The vector of embodiment 9, wherein the inhibitory checkpoint molecule is selected from the group consisting of CLTA-4, PD-1, BTLA, LAG3, NOX2, SIGLEC7, SIGLEC9 and TIM-3.

[0378] 11. The vector of embodiment 9 or 10, wherein the inhibitory checkpoint molecule is a human inhibitory checkpoint molecule, preferably selected from the group consisting of hCLTA-4, such as hCTLA-4 having SEQ ID NO: 51, hPD-1, such as hPD-1 having SEQ ID NO: 52, hBTLA, hLAG3, hNOX2, hSIGLEC7, hSIGLEC9 and hTIM-3.

[0379] 12. The vector according to any one of embodiments 1 to 8, wherein the immunoinhibitory compound is a cytokine selected from the group consisting of IL-10, TGF-β1, TGF-β2, TGF-β3, IL-27, IL-2, GM-CSF, FLT3L, IFN-γ, IL-37 and IL-35.

[0380] 13. The vector of embodiment 12, wherein the cytokine is a human cytokine selected from the group consisting of hIL-10, such as hIL-10 having SEQ ID NO: 53, hTGF-β1, such as hTGF-β1 having SEQ ID NO: 54, hTGF-β2, hTGF-β3, hIL-27, hIL-2, such as hIL-2 having SEQ ID NO: 55, hGM-CSF, such as hGM-CSF having SEQ ID NO: 56, hFLT3L, hIFN-γ, such as hIFN-γ having SEQ ID NO: 57, hIL-37 and hIL-35.

[0381] 14. The vector according to any one of the preceding embodiments, comprising a plurality of further nucleic acid sequences encoding a plurality of immunoinhibitory compounds, such as 2, 3, 4, 5, 6, 7 or 8 immunoinhibitory compounds, such as 2, 3, 4, 5, 6, 7 or 8 different immunoinhibitory compounds.

[0382] 15. The vector of embodiment 14, wherein the multiple immunoinhibitory compounds are different immunoinhibitory compounds that generate or promote a tolerance-inducing environment at different levels.

[0383] 16. The vector of embodiment 14 or 15, wherein the different immunoinhibitory compounds induce tolerance, increase tolerance, maintain tolerance, or induce tolerance, increase tolerance, or induce tolerance and maintain tolerance.

[0384] 17. A vector according to any one of embodiments 1 to 16, comprising one or more co-expression elements.

[0385] 18. The vector of embodiment 17, wherein the one or more co-expression elements cause transcription of the first polypeptide and the one or more immunoinhibitory compounds on a single transcript and independent translation into a separate first polypeptide and a separate one or more immunoinhibitory compounds.

[0386] 19. The vector of embodiment 17 or 18, wherein the one or more co-expression elements is an IRES element or a nucleic acid sequence encoding a 2A self-cleaving peptide.

[0387] 20. The vector according to embodiment 17 or 18, comprising two or more co-expression elements, which are a nucleic acid sequence encoding an IRES element or a 2A self-cleaving peptide, or a nucleic acid sequence encoding an IRES element and a 2A self-cleaving peptide.

[0388] The vector according to any one of embodiments 17 to 20, wherein the 21.2A self-cleaving peptide is selected from the group consisting of a T2A peptide, a P2A peptide, an E2A peptide and an F2A peptide.

[0389] A vector described in any one of embodiments 17 to 21, wherein the 22.2A self-cleaving peptide is selected from the group consisting of a T2A peptide having an amino acid sequence having 80% to 100% sequence identity to the amino acid sequence of SEQ ID NO: 6, a P2A peptide having an amino acid sequence having 80% to 100% sequence identity to the amino acid sequence of SEQ ID NO: 7, an E2A peptide having an amino acid sequence having 80% to 100% sequence identity to the amino acid sequence of SEQ ID NO: 8, and an F2A peptide having an amino acid sequence having 80% to 100% sequence identity to the amino acid sequence of SEQ ID NO: 9.

[0390] A vector described in any one of embodiments 17 to 22, wherein the 23.2A self-cleaving peptide is selected from the group consisting of a T2A peptide having the amino acid sequence of SEQ ID NO: 6, a P2A peptide having the amino acid sequence of SEQ ID NO: 7, an E2A peptide having the amino acid sequence of SEQ ID NO: 8, and an F2A peptide having the amino acid sequence of SEQ ID NO: 9.

[0391] 24. The vector of embodiment 17, wherein the one or more coexpression elements cause transcription of the first polypeptide and the one or more immunoinhibitory compounds as separate transcripts.

[0392] 25. The vector of embodiment 24, wherein the one or more co-expression elements is a bidirectional promoter.

[0393] 26. The vector of embodiment 24, wherein the one or more coexpression elements are promoters, and the vector comprises a separate promoter for each of the nucleic acid sequences encoding the first polypeptide and the one or more immunoinhibitory compounds.

[0394] 27. The vector of embodiment 24, wherein the one or more co-expression elements is a bidirectional promoter and a promoter.

[0395] 28. A vector described in any one of embodiments 17 to 27, comprising one or more co-expression elements selected from the group consisting of an IRES element, a nucleic acid sequence encoding a 2A self-cleaving peptide, a bidirectional promoter and a promoter.

[0396] 29. A vector according to any one of embodiments 1 to 28, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen.

[0397] 30. The vector of embodiment 29, wherein the antigenic unit comprises multiple T cell epitopes of an autoantigen.

[0398] 31. The vector of embodiment 29, wherein the antigenic unit comprises multiple T cell epitopes of multiple different self-antigens.

[0399] 32. The vector according to any one of embodiments 29 to 31, wherein the autoantigen is involved in multiple sclerosis.

[0400] 33. The vector of any one of embodiments 29-32, wherein the one or more T cell epitopes are selected from the group consisting of MOG, such as MOG(35-55), MBP, such as MBP(84-104) and MBP(76-112), PLP, such as PLP(139-151), PLP(131-159) and PLP(178-191), MAG, MOBP, CNPase, S100β, and transaldolase.

[0401] 34. The vector according to any one of embodiments 29 to 33, 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).

[0402] 35. The vector according to any one of embodiments 29 to 31, wherein the autoantigen is involved in type 1 diabetes.

[0403] 36. The vector of embodiment 35, wherein the autoantigen is selected from the group consisting of glutamic acid decarboxylase 65 kilodalton isoform (GAD65), insulin, IA-2, ZnT8, IGRP, ChgA, IAPP, peripherin, tetraspanin-7, GRP78, urocortin-3, and insulin gene enhancer protein isl-1.

[0404] 37. The vector according to any one of embodiments 29 to 31, wherein the autoantigen is involved in celiac disease.

[0405] 38. The vector of embodiment 37, wherein the autoantigen is selected from the group consisting of α-gliadin, γ-gliadin, such as α-gliadin (76-95), ω-gliadin, low molecular weight glutenin, high molecular weight glutenin, hordein, secalin and avenin b.

[0406] 39. The vector according to any one of embodiments 29 to 31, wherein the autoantigen is involved in rheumatoid arthritis.

[0407] 40. The vector of embodiment 39, wherein the autoantigen is selected from the group consisting of collagen, heat shock protein 60 (HSP60), Band 3, small nuclear ribonucleoprotein D1 (SmD1), acetylcholine receptor (AChR) and myelin protein zero (P0).

[0408] 41. The autoantigen is involved in a disease selected from the group consisting of multiple sclerosis, type 1 diabetes, celiac disease, rheumatoid arthritis, chronic inflammatory demyelinating polyneuropathy, Hashimoto's thyroiditis, pemphigus foliaceus, pemphigus vulgaris, thyroid eye disease, Graves' disease, primary biliary cirrhosis, myasthenia gravis, insulin-resistant diabetes mellitus, hemolytic anemia, and psoriasis, and / or the antigenic unit is selected from the group consisting of neurofascin 155, thyroid peroxidase, thyroglobulin, desmosome-associated glycoprotein, desmoglein 3 calcium-binding protein (calsequestrin), thyroid-stimulating hormone receptor, antimitochondrial antibody (AMA), antinuclear antibody (ANA), 32. The vector of any one of embodiments 29 to 31, comprising one or more T cell epitopes of one or more autoantigens selected from the group consisting of: rim-like / membrane (RL / M), multinucleated dot (MND), acetylcholine receptor, insulin receptor, erythrocyte 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), keratin 17, citrullinated proteins, homocitrullinated proteins, and the Fc portion of IgG.

[0409] 42. A vector according to any one of embodiments 1 to 28, wherein the antigenic unit comprises one or more T cell epitopes of an allergen.

[0410] 43. The vector of embodiment 42, wherein the antigenic unit comprises multiple T cell epitopes of an allergen.

[0411] 44. The vector of embodiment 42, wherein the antigenic unit comprises multiple T cell epitopes of multiple different allergens.

[0412] 45. The vector of any one of embodiments 42 to 44, wherein the allergen is a food allergen.

[0413] 46. ​​The vector according to any one of embodiments 42 to 45, wherein the antigenic unit comprises one or more T cell epitopes of one or more allergens selected from the group consisting of shellfish allergen, milk allergen, egg allergen, fish allergen, fruit allergen, wheat allergen, peanut allergen, tree nut allergen, soybean allergen, seed allergen, buckwheat allergen, celery allergen, garlic allergen, gluten allergen, oat allergen, bean allergen, corn allergen, milk allergen, mustard allergen, nut allergen, poultry allergen, meat allergen, rice allergen and sesame allergen.

[0414] 47. A vector according to any one of embodiments 42 to 45, wherein the antigenic unit comprises one or more T cell epitopes of one or more crustacean allergens selected from the group consisting of tropomyosin, arginine kinase, myosin light chain, sarcoplasmic reticulum calcium binding protein, troponin C, triosephosphate isomerase and actin.

[0415] 48. The vector of embodiment 47, wherein the one or more T cell epitopes are selected from the group consisting of Pan b 1 T cell epitope(251-270), Met e 1 T cell epitope(241-260), Met e 1 T cell epitope(210-230), Met e 1 T cell epitope(136-155), Met e 1 T cell epitope(76-95), Met e 1 T cell epitope(46-65) and Met e 1 T cell epitope(16-35).

[0416] 49. The vector according to any one of embodiments 42 to 44, wherein the antigenic unit comprises one or more T cell epitopes of one or more allergens selected from the group consisting of bee venom allergens, hornet allergens, latex allergens, dust mite allergens, leopard mite allergens, storage dust mite allergens, cockroach allergens, mold allergens, fungal allergens, furry animal allergens such as dog allergens, cat allergens and horse allergens, pollen allergens such as grass pollen allergens, tree pollen allergens, weed pollen allergens, insect allergens, and drug allergens.

[0417] 50. A vector according to any one of embodiments 42 to 44 and 49, wherein the antigenic unit comprises one or more T cell epitopes of a drug allergen of a drug selected from the group consisting of factor VIII, insulin and therapeutic monoclonal antibodies.

[0418] 51. A vector according to any one of embodiments 1 to 28, wherein the antigenic unit comprises one or more T cell epitopes of an alloantigen.

[0419] 52. The vector of embodiment 51, wherein the antigenic unit comprises multiple T cell epitopes of an alloantigen.

[0420] 53. The vector of embodiment 51, wherein the antigenic unit comprises multiple T cell epitopes of multiple different alloantigens.

[0421] 54. A vector according to any one of embodiments 1 to 28, wherein the antigenic unit comprises one or more T cell epitopes of a heterologous antigen.

[0422] 55. The vector of embodiment 54, wherein the antigenic unit comprises multiple T cell epitopes of a heterologous antigen.

[0423] 56. The vector of embodiment 54, wherein the antigenic unit comprises multiple T cell epitopes of multiple different heterologous antigens.

[0424] 57. A vector according to any one of the preceding embodiments, wherein the antigenic unit comprises multiple T cell epitopes that are discrete T cell epitopes.

[0425] 58. A vector according to any one of the preceding embodiments, wherein the antigenic unit comprises multiple T cell epitopes that are minimal T cell epitopes contained in one or more hotspots.

[0426] 59. A vector described in any one of embodiments 1 to 56, wherein the antigenic unit comprises multiple T cell epitopes from the group consisting of discrete T cell epitopes and minimal T cell epitopes contained in one or more hotspots.

[0427] 60. The vector according to any one of embodiments 1 to 59, wherein the one or more T cell epitopes have a length of 7 to about 200 amino acids, such as 7 to 150 amino acids, preferably 7 to 100 amino acids, such as 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.

[0428] 61. The vector of embodiment 60, wherein the one or more T cell epitopes have a length suitable for presentation by MHC (major histocompatibility complex), e.g., a length of 7 to 11 amino acids for MHC class I presentation, or a length of about 15 amino acids for MHC class II presentation.

[0429] 62. The vector according to any one of embodiments 1 to 61, wherein the antigenic unit comprises up to 3500 amino acids, such as about 21 to about 2000 amino acids, or about 60 to 3500 amino acids, such as about 80 or about 100 or about 150 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.

[0430] 63. The antigenic unit comprises 1 to 10 T cell epitopes, such as 1, 2, 3, 4, 5, 6, 7, 8, or 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, 22, 23, 24, 25, 26, 27, 28, 29, or 30 T cell epitopes. 63. The vector according to any one of embodiments 1 to 62, comprising 21 to 30 T cell epitopes, such as 21, 32, 33, 34, 35, 36, 37, 38, 39 or 40 T cell epitopes, or 41 to 50 T cell epitopes, such as 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 T cell epitopes.

[0431] 64. A vector according to any one of embodiments 1 to 63, wherein the antigenic unit comprises multiple discrete T cell epitopes separated by T cell epitope linkers.

[0432] 65. A vector according to any one of embodiments 1 to 64, wherein the targeting unit is or comprises a moiety that interacts with a surface molecule on an antigen-presenting cell without activating the cell.

[0433] 66. A vector described in any one of embodiments 1 to 65, wherein the targeting unit is or comprises a moiety that interacts with a surface molecule on an antigen-presenting cell without inducing maturation of the cell.

[0434] 67. The surface molecule is a TGFβ receptor such as TGFβR1, TGFβR2, and TGFβR3, IL-10R such as IL-10RA and IL-10RB, IL-2R, IL-4R, IL-6R, IL-11R, IL-13R, IL-27R, IL-35R, IL-37R, GM-CSFR, FLT3, CCR7, CD11b, CD11c, CD103, CD14, CD36, CD205, CD109, VISTA, MA 67. The vector of embodiment 65 or 66, which is selected from the group consisting of RCO, MHCII, CD83, SIGLEC, Clec10A (MGL), ASGR (ASGR1 / ASGR2), CD80, CD86, Clec9A, Clec12A, Clec12B, DCIR2, Langerin, MR, DC-Sign, Treml4, Dectin-1, PDL1, PDL2, HVEM, CD163 and CD141.

[0435] 68. The surface molecule is a surface molecule present on a human antigen-presenting cell, and the surface molecule is selected from the group consisting of hTGFβ receptors such as hTGFβR1, hTGFβR2, and hTGFβR3, hIL-10R such as hIL-10RA and hIL-10RB, hIL-2R, hIL-4R, hIL-6R, hIL-11R, hIL-13R, hIL-27R, hIL-35R, hIL-37R, hGM-CSFR, hFLT3, hCCR7, hCD11b, hCD11c, hCD103, hCD14, hCD36, hCD205, 67. The vector of embodiment 65 or 66, which is selected from the group consisting of hCD109, hVISTA, hMARCO, hMHCII, hCD83, hSIGLEC, hClec10A (hMGL), hASGR (hASGR1 / hASGR2), hCD80, hCD86, hClec9A, hClec12A, hClec12B, hDCIR2, hLangerin, hMR, hDC-Sign, hTreml4, hDectin-1, hPDL1, hPDL2, hHVEM, hCD163 and hCD141.

[0436] 69. The vector according to any one of embodiments 65 to 68, wherein the moiety is a natural ligand, an antibody or part thereof, such as an scFv, or a synthetic ligand.

[0437] 70. The vector according to any one of embodiments 65 to 68, wherein the moiety is a natural ligand selected from the group consisting of TGFβ, such as TGF-β1, TGF-β2 and TGF-β3, IL-10, IL-2, IL-4, IL-6, IL-11, IL-13, IL-27, IL-35, IL-37, GM-CSF, FLT3L, CCL19, CCL21, ICAM-1 (intercellular adhesion molecule 1, also known as CD54), keratin, VSIG-3, preferably the extracellular domain of VSIG-3, SCGB3A2, CTLA-4, preferably the extracellular domain of CTLA-4, PD-1, preferably the extracellular domain of PD-1, and BTLA, preferably the extracellular domain of BTLA.

[0438] 71. The vector according to any one of embodiments 65 to 68, wherein the moiety is a human natural ligand selected from the group consisting of hTGFβ, hIL-10, hIL-2, such as hIL-2 having SEQ ID NO: 55, hIL-4, hIL-6, hIL-11, hIL-13, hIL-27, hIL-35, hIL-37, hGM-CSF, such as hGM-CSF having SEQ ID NO: 56, hFLT3L, hCCL19, hCCL21, hICAM-1 (intercellular adhesion molecule 1, also known as CD54), hKeratin, hVSIG-3, preferably the extracellular domain of hVSIG-3, hSCGB3A2, hCTLA-4, preferably the extracellular domain of hCTLA-4, such as the extracellular domain of hCTLA4 having SEQ ID NO: 51, hPD-1, preferably the extracellular domain of PD-1, such as the extracellular domain of hPD-1 having SEQ ID NO: 52, and hBTLA, preferably the extracellular domain of hBTLA.

[0439] 72. A vector according to any one of embodiments 65 to 70, wherein the targeting unit is or comprises the extracellular domain of IL1-10, TGFβ, such as TGFβ-1, TGFβ-2 and TGFβ-3, SCGB3A2 or VSIG-3, preferably VSIG-3.

[0440] 73. A vector according to any one of embodiments 65 to 70, wherein the targeting unit is or comprises the extracellular domain of hTGFβ, such as hIL1-10, hTGFβ-1, hTGFβ-2 and hTGFβ-3, hSCGB3A2 or hVSIG-3, preferably hVSIG-3.

[0441] 74. The vector according to any one of embodiments 1 to 73, wherein the multimerization unit is selected from the group consisting of a dimerization unit, 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 or the C-terminal domain of T4 fibritin, and a tetramerization unit, such as a domain derived from p53, wherein the multimerization unit optionally comprises a hinge region, such as hinge exon h1 and hinge exon h4, preferably hinge exon h1 and hinge exon h4 of IgG3.

[0442] 75. The vector of embodiment 74, wherein the vector comprises a hinge region capable of forming one or more covalent bonds.

[0443] 76. The vector of embodiment 74 or 75, wherein the hinge region is derived from Ig.

[0444] 77. A vector according to any one of embodiments 74 to 76, wherein the multimerization unit is a dimerization unit, which further comprises another domain that promotes dimerization.

[0445] 78. The vector according to embodiment 77, wherein the other domain is an immunoglobulin domain, preferably an immunoglobulin constant domain.

[0446] 79. The vector according to embodiment 77 or 78, wherein the other domain is the carboxy-terminal C domain derived from IgG, preferably IgG3.

[0447] 80. A vector according to any one of embodiments 77 to 79, wherein the dimerization unit further comprises a dimerization unit linker, such as a glycine-serine rich linker, such as GGGSSGGGSG.

[0448] 81. The vector of embodiment 80, wherein a dimerization unit linker connects the hinge region and other domains that promote dimerization.

[0449] 82. The vector according to any one of embodiments 77 to 81, wherein the dimerization unit comprises hinge exon h1 and hinge exon h4, a dimerization unit linker, and a CH3 domain derived from human IgG3.

[0450] 83. The vector according to embodiment 82, 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.

[0451] 84. The vector according to embodiment 83, 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.

[0452] 85. The vector according to embodiment 84, wherein the dimerization unit consists of the amino acid sequence of SEQ ID NO:1.

[0453] 86. The vector according to any one of embodiments 1 to 85, wherein the first nucleic acid sequence encodes a first polypeptide further comprising a unit linker that connects the antigenic unit to the multimerization unit, and the unit linker is a non-immunogenic linker and / or a flexible linker or a rigid linker.

[0454] 87. The vector according to any one of the preceding embodiments, wherein the first nucleic acid sequence encodes a first polypeptide further comprising a signal peptide.

[0455] 88. The vector of embodiment 87, wherein the signal peptide is the natural leader sequence of the protein that is the targeting unit.

[0456] 89. The vector according to embodiment 87 or 88, wherein the signal peptide is selected from the group consisting of human Ig VH signal peptide, the signal peptide of hTGF-β1, the signal peptide of hTGF-β2, the signal peptide of hTGF-β3, the signal peptide of hIL-10, the signal peptide of hIL-2, the signal peptide of hIL-4, the signal peptide of hIL-6, the signal peptide of hIL-11, the signal peptide of hIL-13, the signal peptide of hIL-27, the signal peptide of hIL-35, the signal peptide of hIL-37, the signal peptide of hGM-CSF, the signal peptide of hFLT3L, the signal peptide of hCCL19, the signal peptide of hCCL21, the signal peptide of hICAM-1, the signal peptide of hKeratin, the signal peptide of hVSIG-3, the signal peptide of hSCGB3A2, the signal peptide of hCTLA-4, the signal peptide of hPD-1, and the signal peptide of hBTLA.

[0457] 90. The vector according to any one of the preceding embodiments, wherein the one or more further nucleic acid sequences encode one or more immunoinhibitory compounds further comprising a signal peptide.

[0458] 91. The vector of embodiment 90, wherein the signal peptide is the natural leader sequence of the immunoinhibitory compound.

[0459] 92. The vector according to any one of embodiments 90 to 91, wherein the signal peptide is selected from the group consisting of signal peptides selected from the group consisting of the signal peptide of hCLTA-4, the signal peptide of hPD-1, the signal peptide of hBTLA, the signal peptide of hLAG3, the signal peptide of hNOX2, the signal peptide of hSIGLEC7, the signal peptide of hSIGLEC9, the signal peptide of hTIM-3, the signal peptide of hIL-10, the signal peptide of hTGF-β1, the signal peptide of hTGF-β2, the signal peptide of hTGF-β3, the signal peptide of hIL-27, the signal peptide of hIL-2, the signal peptide of hGM-CSF, the signal peptide of hFLT3L, the signal peptide of hIFN-γ, and the signal peptide of hIL-37 and the signal peptide of hIL-35.

[0460] 93. The vector according to any one of the preceding embodiments, wherein the vector is a viral vector, such as an RNA viral vector or a DNA viral vector, or a plasmid, such as an RNA plasmid or a DNA plasmid.

[0461] 94. The vector according to any one of the preceding embodiments, wherein the vector is a DNA viral vector or a DNA plasmid, preferably a DNA plasmid.

[0462] 95. A method for producing a vector according to any one of embodiments 1 to 94, comprising: a) transfecting a cell in vitro with a vector; b) culturing the cells; and c) optionally lysing the cells to release the vectors from the cells; d) recovering and optionally purifying the vector.

[0463] 96. A host cell comprising a vector according to any one of embodiments 1 to 94, such as a host cell selected from the group consisting of prokaryotic cells, yeast cells, insect cells, higher eukaryotic cells such as cells of animal or human origin.

[0464] 97. A vector according to any one of embodiments 1 to 94 for use as a medicament.

[0465] 98. A pharmaceutical composition comprising a vector according to any one of embodiments 1 to 94 and a pharma- ceutically acceptable carrier or diluent.

[0466] 99. The pharmaceutical composition of embodiment 98, wherein the pharma- ceutically acceptable carrier or diluent is selected from the group consisting of saline, buffered saline such as PBS, dextrose, water, glycerol, ethanol, isotonic aqueous buffer and Tyrode's buffer, and combinations thereof.

[0467] 100. The pharmaceutical composition according to embodiment 98 or 99, further comprising a transfection agent.

[0468] 101. The composition of any one of embodiments 98 to 100, further comprising a pharma- ceutically acceptable amphiphilic block copolymer comprising blocks of poly(ethylene oxide) and poly(propylene oxide), e.g., a pharma- ceutically acceptable amphiphilic block copolymer comprising blocks of poly(ethylene oxide) and poly(propylene oxide), in an amount of 0.2% w / v to 20% w / v.

[0469] 102. The composition of any one of embodiments 98 to 101, further comprising an adjuvant, for example an adjuvant selected from the group consisting of dexamethasone, the B subunit of the enterotoxin cholera toxin (CTB), a TLR2 ligand, an excretory / secretory (ES) product from a helminth, a rapamycin vitamin D3 analogue, and an aryl hydrocarbon receptor ligand.

[0470] 103. A pharmaceutical composition according to any one of embodiments 98 to 102, comprising said vector, such as said DNA plasmid, in the range of 0.1 to 10 mg.

[0471] 104. A method for treating a subject having an immune disease from the group consisting of an autoimmune disease, an allergic disease and a transplant rejection reaction, or a subject in need of its prevention, comprising administering to the subject a vector according to any one of embodiments 1 to 94 or a pharmaceutical composition according to any one of embodiments 98 to 103.

[0472] 105. The method of embodiment 104, wherein the vector or pharmaceutical composition is administered in a therapeutically or prophylactically effective amount.

[0473] 106. The method according to embodiment 104 or 105, wherein the vector or pharmaceutical composition is administered by intradermal, intramuscular or subcutaneous injection, or by mucosal or epidermal application, such as intranasal or oral.

[0474] 107. A method for treating a subject having an autoimmune disease or a subject in need of its prevention, comprising administering to the subject a vector comprising a vector according to any one of embodiments 1 to 41, or a pharmaceutical composition comprising such a vector according to any one of embodiments 98 to 103.

[0475] 108. The method of embodiment 107, wherein the vector or pharmaceutical composition is administered in a therapeutically effective amount.

[0476] 109. The method according to embodiment 107 or 108, wherein the vector or pharmaceutical composition is administered by intradermal, intramuscular or subcutaneous injection, or by mucosal or epidermal application, such as intranasal or oral.

[0477] 110. A method for treating a subject having an allergic disease or a subject in need of its prevention, comprising administering to the subject a vector comprising a vector according to any one of embodiments 1 to 28 or 42 to 50, or a pharmaceutical composition comprising such a vector according to any one of embodiments 98 to 103.

[0478] 111. The method of embodiment 110, wherein the vector or pharmaceutical composition is administered in a therapeutically effective amount.

[0479] 112. The method according to embodiment 110 or 111, wherein the vector or pharmaceutical composition is administered by intradermal, intramuscular or subcutaneous injection, or by mucosal or epidermal application, such as intranasal or oral.

[0480] 113. A method for treating a subject having or in need of prevention of transplant rejection, comprising administering to the subject a vector comprising a vector according to any one of embodiments 1-28 or 51-56, or a pharmaceutical composition comprising such a vector according to any one of embodiments 98-103.

[0481] 114. The method of embodiment 113, wherein the vector or pharmaceutical composition is administered in a therapeutically effective amount.

[0482] 115. The method according to embodiment 113 or 114, wherein the vector or pharmaceutical composition is administered by intradermal, intramuscular or subcutaneous injection, or by mucosal or epidermal application, such as intranasal or oral.

Claims

1. (a) a first nucleic acid sequence encoding a first polypeptide, the first polypeptide comprising a targeting unit for targeting an antigen-presenting cell, a multimerization unit, such as 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; (b) one or more additional nucleic acid sequences encoding one or more immunoinhibitory compounds, A vector allowing for the co-expression of said first polypeptide and said one or more immunoinhibitory compounds as separate molecules.

2. The vector of claim 1 , wherein the one or more immunoinhibitory compounds induce immune tolerance and / or increase immune tolerance and / or maintain immune tolerance.

3. wherein the one or more immune inhibitory compounds are an extracellular portion, such as an extracellular domain, of an inhibitory checkpoint molecule, preferably a human inhibitory checkpoint molecule, more preferably the inhibitory checkpoint molecule is selected from the group consisting of CLTA-4, PD-1, BTLA, LAG3, NOX2, SIGLEC7, SIGLEC9 and TIM-3, most preferably selected from the group consisting of hCLTA-4, hPD-1, hBTLA, hLAG3, hNOX2, hSIGLEC7, hSIGLEC9 and hTIM-3, and 3. The vector according to claim 1 or 2, wherein the one or more immune inhibitory compounds are selected from the group consisting of IL-10, TGF-β1, TGF-β2, TGF-β3, IL-27, IL-2, GM-CSF, FLT3L, IFN-γ, IL-37 and IL-35, preferably a cytokine, preferably a human cytokine, selected from the group consisting of hIL-10, hTGF-β1, hTGF-β2, hTGF-β3, hIL-27, hIL-2, hGM-CSF, hFLT3L, hIFN-γ, hIL-37 and hIL-35.

4. The vector of claim 1 or 2, wherein the vector comprises a plurality of further nucleic acid sequences encoding two or more immunoinhibitory compounds, such as 2, 3, 4, 5, 6, 7 or 8 immunoinhibitory compounds, such as 2, 3, 4, 5, 6, 7 or 8 different immunoinhibitory compounds.

5. The vector described in claim 1 or 2, wherein the vector comprises one or more co-expression elements, which a) cause transcription of the first polypeptide and the one or more immunoinhibitory compounds on a single transcript and independent translation into separate first polypeptides and separate one or more immunoinhibitory compounds, preferably an IRES element or a nucleic acid sequence encoding a 2A self-cleaving peptide, and / or b) cause transcription of the first polypeptide and the one or more immunoinhibitory compounds as separate transcripts, preferably a bidirectional promoter, or a promoter, and wherein the vector comprises a separate promoter for each of the nucleic acid sequences encoding the first polypeptide and the one or more immunoinhibitory compounds.

6. 3. The vector according to claim 1 or 2, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, e.g. a plurality of T cell epitopes of one or more autoantigens, optionally the one or more autoantigens are selected from the group consisting of an autoantigen involved in multiple sclerosis, an autoantigen involved in type 1 diabetes, an autoantigen involved in celiac disease, an autoantigen involved in rheumatoid arthritis, an autoantigen involved in chronic inflammatory demyelinating polyneuropathy, an autoantigen involved in Hashimoto's thyroiditis, an autoantigen involved in pemphigus foliaceus, an autoantigen involved in pemphigus vulgaris, an autoantigen involved in thyroid eye disease, an autoantigen involved in Graves' disease, an autoantigen involved in primary biliary cirrhosis, an autoantigen involved in myasthenia gravis, an autoantigen involved in insulin-resistant diabetes, an autoantigen involved in hemolytic anemia, and an autoantigen involved in psoriasis.

7. The antigenic unit comprises one or more T cell epitopes of an allergen, e.g. a plurality of T cell epitopes of one or more allergens, optionally the one or more allergens being selected from the group consisting of: shellfish allergen, milk allergen, egg allergen, fish allergen, fruit allergen, wheat allergen, peanut allergen, tree nut allergen, soy allergen, seed allergen, buckwheat allergen, celeriac allergen, garlic allergen, gluten allergen, oat allergen, bean allergen, corn allergen, 3. The vector according to claim 1 or 2, wherein the allergen is selected from the group consisting of milk allergen, mustard allergen, nut allergen, poultry allergen, meat allergen, rice allergen, sesame allergen, bee venom allergen, wasp allergen, latex allergen, dust mite allergen, insect allergen, mould allergen, fungal allergen, fur animal allergen, pollen allergen and drug allergen, such as a drug allergen of a drug selected from the group consisting of factor VIII, insulin and therapeutic monoclonal antibodies.

8. The vector according to claim 1 or 2, wherein the antigenic unit comprises one or more T cell epitopes of an alloantigen or a heterologous antigen, such as multiple T cell epitopes of one or more alloantigens or multiple T cell epitopes of one or more heterologous antigens.

9. 3. The vector of claim 1 or 2, wherein the antigenic unit comprises multiple discrete T cell epitopes separated by T cell epitope linkers and / or the antigenic unit comprises multiple T cell epitopes that are minimal T cell epitopes contained in one or more hotspots.

10. The targeting unit is or comprises a moiety that interacts with a surface molecule on the antigen presenting cell without activating the cell, e.g. without inducing maturation of the cell, preferably the surface molecule is a TGFβ receptor such as TGFβR1, TGFβR2 and TGFβR3, IL-10R such as IL-10RA and IL-10RB, IL-2R, IL-4R, IL-6R, IL-11R, IL-13R, IL-27R, IL-35R, IL-37R, GM-CSFR, FL from the group consisting of T3, CCR7, CD11b, CD11c, CD103, CD14, CD36, CD205, CD109, VISTA, MARCO, MHCII, CD83, SIGLEC, Clec10A (MGL), ASGR (ASGR1 / ASGR2), CD80, CD86, Clec9A, Clec12A, Clec12B, DCIR2, Langerin, MR, DC-Sign, Treml4, Dectin-1, PDL1, PDL2, HVEM, CD163, and CD141 and more preferably, the surface molecule is selected from hTGFβ receptors such as hTGFβR1, hTGFβR2, and hTGFβR3, hIL-10R such as hIL-10RA and hIL-10RB, hIL-2R, hIL-4R, hIL-6R, hIL-11R, hIL-13R, hIL-27R, hIL-35R, hIL-37R, hGM-CSFR, hFLT3, hCCR7, hCD11b, hCD11c, hCD103, hCD14, hCD36, hCD205, hCD109, 3. The vector of claim 1 or 2, which is selected from the group consisting of hVISTA, hMARCO, hMHCII, hCD83, hSIGLEC, hClec10A (hMGL), hASGR (hASGR1 / hASGR2), hCD80, hCD86, hClec9A, hClec12A, hClec12B, hDCIR2, hLangerin, hMR, hDC-Sign, hTrem14, hDectin-1, hPDL1, hPDL2, hHVEM, hCD163 and hCD141.

11. The moiety is a natural ligand, an antibody or part thereof, e.g. an scFv, or a synthetic ligand, preferably the moiety is selected from the group consisting of TGFβ, such as TGF-β1, TGF-β2 and TGF-β3, IL-10, IL-2, IL-4, IL-6, IL-11, IL-13, IL-27, IL-35, IL-37, GM-CSF, FLT3L, CCL19, CCL21, ICAM-1, keratin, VSIG-3, preferably the extracellular domain of VSIG-3, SCGB3A2, CTLA-4, preferably the extracellular domain of CTLA-4, PD-1, preferably the extracellular domain of PD-1, and BTLA, preferably the extracellular domain of BTLA.

11. The vector according to claim 10, wherein the moiety is a human natural ligand selected from the group consisting of hTGFβ, hIL-10, hIL-2, hIL-4, hIL-6, hIL-11, hIL-13, hIL-27, hIL-35, hIL-37, hGM-CSF, hFLT3L, hCCL19, hCCL21, hICAM-1, hKeratin, hVSIG-3, preferably the extracellular domain of hVSIG-3, hSCGB3A2, hCTLA-4, preferably the extracellular domain of hCTLA-4, hPD-1, preferably the extracellular domain of PD-1, and hBTLA, preferably the extracellular domain of hBTLA.

12. 3. The vector of claim 1 or 2, wherein the multimerization unit is selected from the group consisting of a dimerization unit, 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 or the C-terminal domain of T4 fibritin, and a tetramerization unit, such as a domain derived from p53, wherein the multimerization unit optionally comprises a hinge region, such as hinge exon h1 and hinge exon h4.

13. The vector of claim 12, wherein the multimerization unit is a dimerization unit having the ability to form one or more covalent bonds, comprising a hinge region, preferably derived from Ig, preferably further comprising another domain which promotes dimerization, preferably the other domain being an immunoglobulin domain, more preferably a carboxy-terminal C domain derived from an immunoglobulin constant domain, such as an IgG, preferably IgG3, preferably the dimerization unit further comprising a dimerization unit linker, such as a glycine-serine rich linker such as GGGSSGGGSG, preferably the dimerization unit linker connecting the hinge region and the other domain which promotes dimerization.

14. 3. The vector of claim 1 or 2, wherein the first nucleic acid sequence encodes a first polypeptide further comprising a unit linker connecting the antigenic unit to the multimerization unit, the unit linker being a non-immunogenic linker and / or a flexible or rigid linker, and / or the first nucleic acid sequence encodes a first polypeptide further comprising a signal peptide, preferably the one or more further nucleic acid sequences also further encode a signal peptide.

15. The vector according to claim 1 or 2, wherein the vector is a viral vector, such as an RNA viral vector or a DNA viral vector, or a plasmid, such as an RNA plasmid or a DNA plasmid.

16. A method for producing the vector of claim 1 or 2, comprising: a) transfecting a cell in vitro with said vector; b) culturing the cells; and c) optionally lysing the cells to release the vectors from the cells; d) recovering and optionally purifying said vector.

17. A host cell comprising the vector of claim 1 or 2, such as a host cell selected from the group consisting of prokaryotic cells, yeast cells, insect cells, higher eukaryotic cells such as cells of animal or human origin.

18. A vector according to claim 1 or 2 for use as a medicament.

19. 3. A pharmaceutical composition comprising the vector of claim 1 or 2 and a pharma- ceutically acceptable carrier or diluent, and optionally further comprising a transfection agent and / or an adjuvant, such as an adjuvant selected from the group consisting of dexamethasone, the B subunit of the enterotoxin cholera toxin (CTB), a TLR2 ligand, an excretory / secretory (ES) product from a helminth, a rapamycin vitamin D3 analog, and an aryl hydrocarbon receptor ligand.

20. A pharmaceutical composition for use in the preventive or therapeutic treatment of an immune disease selected from the group consisting of an autoimmune disease, an allergic disease and a transplant rejection reaction, comprising the vector described in claim 1 or 2 and a pharma- ceutical acceptable carrier or diluent.

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