CN117355326A

CN117355326A - Co-expression of constructs and immunosuppressive compounds

Info

Publication number: CN117355326A
Application number: CN202280034533.4A
Authority: CN
Inventors: 阿涅特·布伦斯维克·弗雷德里克森; A·T·H·伯萨斯; 斯蒂因·格拉纳姆; H·迈塞特
Original assignee: Necord Therapeutics Ltd
Current assignee: Necord Therapeutics Ltd
Priority date: 2021-05-10
Filing date: 2022-05-10
Publication date: 2024-01-05

Abstract

The present invention relates to vectors, such as DNA plasmids, comprising a plurality of nucleic acid sequences engineered to be co-expressed as separate molecules. Such separate molecules include a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerizing unit (e.g., a dimerization unit), and an antigenic unit comprising one or more T cell epitopes of an autoantigen, allergen, alloantigen, or xenogeneic antigen; and one or more immunosuppressive compounds.

Description

Co-expression of constructs and immunosuppressive compounds

Technical Field

The present invention relates to vectors, such as DNA plasmids, comprising a plurality of 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 diseases.

Background

An immune response is required to protect against diseases (e.g., diseases caused by pathogens such as viruses, bacteria, or parasites). However, undesired immune activation may cause a process that leads to damage or destruction of the own tissue. For example, undesired immune activation occurs in autoimmune diseases, where antibodies and/or T lymphocytes react with autoantigens, resulting in, for example, tissue damage and lesions. Undesired immune activation also occurs in allergic reactions, characterized by excessive immune reactions to substances that are generally harmless in the environment and possibly inflammatory reactions that lead to tissue destruction. In addition, undesired immune activation also occurs in graft rejection, such as rejection of transplanted organs or tissues, which is significantly mediated by alloreactive T cells present in the host, recognizing the donor's alloantigen or xenogeneic antigen and causing destruction of the transplanted organ or tissue.

Immune tolerance is the inability to obtain a specific immune response to a substance or tissue capable of eliciting an immune response in a given organism.

In general, in order to induce tolerance to a specific antigen, the antigen must be presented by Antigen Presenting Cells (APCs) to other immune cells in the absence of an activation signal, resulting in death or functional inactivation of antigen-specific lymphocytes or in the production of antigen-specific cells that maintain tolerance. This process generally accounts for tolerance to self-antigens or self-tolerance. Immunosuppressive drugs can be used to suppress or reduce unwanted immune responses, for example, for treating patients suffering from autoimmune diseases or having allografts. Conventional strategies for generating immunosuppression against adverse immune responses are immunosuppressive drugs based on broad-spectrum action. In addition, immunosuppressive drug therapy often lasts for a lifetime in order to maintain immunosuppression. Unfortunately, the use of broad-spectrum immunosuppressive drugs is associated with a risk of severe side effects (such as immunodeficiency) because most of these drugs act non-selectively, resulting in increased susceptibility to infection and decreased immune surveillance of cancer. Thus, novel compounds and compositions that induce antigen-specific tolerance are beneficial.

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

The present invention relates to constructs comprising an antigen unit comprising one or more T cell epitopes of self-antigens, allergens, alloantigens or xenogeneic antigens and a targeting unit that interacts with surface molecules on APCs in a non-inflammatory or tolerogenic manner resulting in presentation of the antigen in the absence of an inflammatory activation state.

A vaccine body (vaccinody) construct is a dimeric fusion protein consisting of two polypeptides, each comprising a targeting unit that targets antigen presenting cells, a dimerization unit, and an antigenic unit comprising one or more disease-associated antigens or portions thereof. In another embodiment, the vaccine construct is a multimeric fusion protein consisting of a plurality of polypeptides each comprising a targeting unit for targeting APCs, a multimerizing unit and an antigenic unit comprising one or more disease-associated antigens or parts thereof-see e.g. WO 2004/076489 A1, WO 2011/161244 A1, WO 2013/092875 A1 or WO 2017/118695A1. These constructs have been shown to be effective in generating an immune response against the antigen or portion thereof (e.g., epitope) contained in the antigenic unit.

The vaccine construct may be administered to a subject in the form of a polynucleotide encoding a polypeptide, for example a polynucleotide contained in a vector such as a DNA plasmid. Upon administration to a host cell, e.g., to a muscle cell of a subject (e.g., a human), the polypeptide expresses and forms a multimeric fusion protein, e.g., a dimeric protein, as a result of multimerization units, e.g., dimerization units.

The inventors surprisingly found that an improved vaccine body platform can deliver disease-associated antigens to APCs that internalize the construct and present antigens contained in the construct in a tolerogenic manner by binding to and signaling by selected surface receptors on the APCs in an optimal manner for inducing a selected antigen-specific tolerance response.

Summary of The Invention

The present invention provides vectors, such as DNA plasmids, for coexpression of a construct and one or more immunosuppressive compounds. The vectors and pharmaceutical compositions comprising such vectors are useful for the treatment of conditions involving undesired immune responses, such as for the prophylactic or therapeutic treatment of autoimmune diseases, allergic diseases and graft rejection.

Such constructs and immunosuppressive compounds will allow presentation of epitopes in the antigenic unit in a manner that induces tolerance after administration of the vector to a subject, and the vector of the invention is thus suitable for use as a prophylactic or therapeutic treatment of immune diseases such as autoimmune diseases, allergic diseases and graft rejection.

Since the constructs and immunosuppressive compounds down-regulate the immune system disease specific cells that contribute to the immune disease in question, they will not suppress the whole immune system. Thus, treatment of the immune disease in question with the vector of the invention does not result in increased susceptibility to infection and decreased immune surveillance of cancer.

The one or more immunosuppressive compounds help to create or promote an environment that facilitates presentation of epitopes in the antigenic unit in a manner that induces tolerance or by, for example, facilitating the induction of tolerance-maintaining cells or helping to maintain such cells.

In a first aspect, the present invention relates to a carrier comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerizing unit (e.g., a dimerization unit), and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, allergen, alloantigen, or xenogeneic antigen; and

(b) One or more other nucleic acid sequences encoding one or more immunosuppressive compounds,

wherein the vector allows co-expression of the first polypeptide and the one or more immunosuppressive compounds as separate molecules.

In one embodiment, the vector of the invention comprises a first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets but does not activate antigen presenting cells.

The vectors of the invention may be used for the prophylactic or therapeutic treatment of immune disorders, for example, by administering the vector/pharmaceutical composition to a subject in need of such prophylactic or therapeutic treatment in the form of a pharmaceutical composition, i.e., a composition comprising the vector and a pharmaceutically acceptable carrier (carrier) or diluent.

Brief Description of Drawings

Fig. 1: IRES co-expression elements for use in vectors of the invention are shown inserted between two coding regions. When mRNA is formed, two ribosomes (T) are able to initiate translation at two separate sites on the mRNA and form two proteins (a and B). A and B may, for example, be a first polypeptide and an immunosuppressive compound.

Fig. 2: the 2A self-cleaving peptide coexpression element inserted between two genes for use in the vector of the invention is shown. After transcription, one ribosome translates the mRNA and two proteins are formed (a and B). The top of the figure shows how the fusion protein is formed if the 2A sequence is not part of the coding sequence. A and B may, for example, be a first polypeptide and an immunosuppressive compound.

Fig. 3: a bi-directional promoter (P) co-expression element for use in the vectors of the invention is shown between two coding regions. One mRNA is formed and two ribosomes (T) are able to initiate translation at two different mrnas and form two proteins (a and B). A and B may, for example, be a first polypeptide and an immunosuppressive compound.

Fig. 4: two promoters (P), i.e.coexpression elements, are shown for use in the vectors of the invention, located before the two coding regions. Two mRNAs are formed and two ribosomes (T) are able to initiate translation at two different mRNAs and form two proteins (A and B). A and B may, for example, be a first polypeptide and an immunosuppressive compound.

Fig. 5: embodiments of constructs based on the first polypeptide encoded by the vectors of the invention are shown.

Fig. 6: ELISA results (protein expression and secretion) obtained after transient transfection of HEK293 cells with the DNA vector VB5049 (vector of the invention) or VB5052 are shown.

Fig. 7: results of western blot analysis of supernatants from Expi293F cells transfected with DNA vector VB5049 (vector of the invention) or VB5052 are shown.

Fig. 8: results of FluoroSpot assay (IL-10/IFN- γ ratio) of splenocytes obtained from mice after single administration of DNA vector VB5049 or VB5052 and re-stimulation with MOG (35-55) peptide are shown.

Fig. 9: the percentages of splenic IFN- γT cells (9A) and IL-17+T cells (9B) in the total CD4+ T cell population measured after 16 hours restimulation with MOG (35-55) peptide as assessed by flow cytometry are shown. The DNA vector VB5049 x, VB5051 x or VB5052 x was administered once intramuscularly to C57BL/6 mice and the spleens were harvested 7 days later. Data were generated from pools of 5 mice/group and the mouse spleens pooled prior to analysis.

Fig. 10: results of MOG tetramer staining and MOG-specific T cell detection are shown. The DNA vector VB5049, VB5051 or VB5052 was administered intramuscularly to C57BL/6 mice on day 0 followed by electroporation and spleen harvesting on day 7 post administration. The percentage of spleen Foxp3+ cells in the CD4+ MOG (35-55) tet+ T cell population was determined by the H-2IAb/MOG (35-55) tetramer. Tetramer staining was performed ex vivo and splenocytes were not restimulated with MOG (35-55) peptide. Data were generated from pools of 5 mice/group and the mouse spleens pooled prior to analysis.

Fig. 11: shows the protein expression and secretion levels 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), the supernatant from the transiently transfected Expi293F cells with the DNA vector. Negative control (Neg ctrl): supernatant from the pi293F cells treated with transfection reagent ex fectamine alone.

Fig. 12: the expression and secretion levels of the immunosuppressive compounds encoded by the DNA vectors shown are shown as detected by sandwich ELISA, with supernatants from Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): supernatant from the pi293F cells treated with transfection reagent ex fectamine alone. The following antibodies were used: (a) capture antibody: 1. Mu.g/ml rat anti-murine IL-10 antibody, 100. Mu.l/well, MAB417, R & D Systems. Detection of antibodies: 0.2. Mu.g/ml, goat anti-murine IL-10 biotinylated antibody, 100. Mu.l/well, BAF417, R & D Systems. (B) capture antibody: 2. Mu.g/ml TGF-. Beta.1 antibody, 100. Mu.l/well, MAB2402, RD Systems. Detection of antibodies: 0.8 μg/ml chicken anti-human TGF-beta 1 biotinylated antibody, 100 μl/well, BAF240, RD Systems. (C) capture antibody: goat anti-murine CTLA-4 antibody at 0.8. Mu.g/ml, 100. Mu.l/well, AF476, RD Systems. Detection of antibodies: goat anti-murine CTLA-4 biotinylated antibody at 0.8. Mu.g/ml, 100. Mu.l/well, BAF476, RD Systems. (D) capture antibody: 2. Mu.g/ml rat anti-murine IL-2 antibody, 100. Mu.l/well, 503701, bioLegend. Detection of antibodies: 2. Mu.g/ml rat anti-murine IL-2 biotinylated antibody, 100. Mu.l/well, 503803, biolegend. (E) capture antibody: 2. Mu.g/ml rat anti-murine IFN-. Gamma.antibody, 100. Mu.l/well, 505502, bioLegend. Detection of antibodies: 2. Mu.g/ml rat anti-murine IFN-. Gamma.biotinylated antibody, 100. Mu.l/well, 505704, bioLegend.

Fig. 13: shows the expression and secretion of the first polypeptide and the immunosuppressive compound, separately, encoded by the indicated DNA vector, detected by sandwich ELISA, the supernatant from an Expi293F cell transiently transfected with the DNA vector. Negative control (Neg ctrl): supernatant from the pi293F cells treated with transfection reagent ex fectamine alone. The following antibodies were used: capture antibody: mouse anti-MOG antibody, 0.25. Mu.g/ml, 100. Mu.l/well, sc-73330,Santa Cruz Biotechnology. Detection of antibodies: (A) Goat anti-mouse anti-IL-10 biotinylated antibody at 0.2. Mu.g/ml, 100. Mu.l/well, BAF417, R & D Systems. (B) 0.8 μg/ml chicken anti-human TGF-beta 1 biotinylated antibody, 100 μl/well, BAF240, RD Systems. (C) Goat anti-mouse CTLA-4 biotinylated antibody at 0.8. Mu.g/ml, 100. Mu.l/well, BAF476, RD Systems. (D) 2. Mu.g/ml rat anti-mouse IFN-. Gamma.biotinylated antibody, 100. Mu.l/well, 505704, bioLegend.

Fig. 14: results of western blot analysis of supernatants from Expi293F cells transfected with the indicated DNA vectors are shown. A: reduced supernatant samples (35 μl loaded). A first antibody: mice were anti-MOG (sc-73130). A second antibody: donkey is resistant to mice, dylight800 (SA 5-10172). Protein standards were detected in Chemidoc channel Dylight 650 (signal not shown). Chemidoc channel dyight 800. The black arrow indicates the first polypeptide expressed as a separate protein from the corresponding DNA vector and secreted from the transfected cell. B: non-reduced supernatant samples (35 μl loaded). A first antibody: mice were anti-MOG (sc-73130). A second antibody: donkey is resistant to mice, dylight800 (SA 5-10172). Chemidoc channels Dylight 650 (for protein standards) and 800. The black arrows indicate the dimeric proteins formed by the two first polypeptide molecules expressed from the corresponding DNA vectors and secreted from the transfected cells. C: reduced supernatant samples (35 μl loaded). A first antibody: rat anti-IL 10 (MAB 417). A second antibody: donkey is resistant to rats, dylight 488 (SA 5-10026). Chemidoc channels Dylight 650 (for protein standards) and 488. The black arrow indicates IL-10 expressed as an individual protein from the corresponding DNA vector and secreted from the transfected cells. D: reduced supernatant samples (35 μl loaded). A first antibody: goat anti-CTLA-4 (AF 476). A second antibody: donkey anti-goat, dylight800 (SA 5-10092). Chemidoc channels Dylight 650 (for protein standards) and 800. The black arrow indicates CLTA-4 expressed as an individual protein from the corresponding DNA vector and secreted from the transfected cells. E: reduced supernatant samples (35 μl loaded). A first antibody: rat anti-IL 2 (503702). A second antibody: donkey is resistant to rat, dylight 650 (SA 5-10029). Chemidoc channel dyight 650. The black arrow indicates IL-2 expressed as an individual protein from the corresponding DNA vector and secreted from the transfected cells.

Fig. 15: expression and secretion of MOG (27-63) encoded by DNA vector VB5051, detected by sandwich ELISA, was shown, supernatant from Expi293F cells transiently transfected with the DNA vector. Negative control (Neg ctrl): supernatant from the pi293F cells treated with transfection reagent ex fectamine alone. Detection of antibodies: mouse anti-MOG antibody, 3.3. Mu.g/ml, 100. Mu.l/well, sc-73330,Santa Cruz Biotechnology.

Fig. 16: the results of the bicolor IL-10/IFN-. Gamma.FluoSpot assay are shown. The indicated DNA vectors were administered intramuscularly four times (day 0, day 3, day 7 and day 10) to C57BL/6 mice, followed by electroporation and spleen harvesting at day 14 after the first administration. IL-10 secretion and IFN-gamma secretion (SFU/10) of splenocytes were tested in a two-color FluoSpot assay with (A) or MOG (35-55) peptides 44 hours after restimulation (B) without restimulation ⁶ Individual spleen cells). Individual mice and mean ± SEM,5 mice/group, × (p<0.05 A two-tailed Mann-Whitney test).

Fig. 17: the IL-10/IFN-gamma ratios calculated from the data shown in FIG. 16B are shown. Individual mice and mean ± SEM,5 mice/group, (< 0.05 p), two-tailed Mann-Whitney test are shown.

Fig. 18: the results of detection of foxp3+ producing cd4+ T cells by flow cytometry are shown. The indicated DNA vectors were administered to C57BL/6 mice on day 0, day 3, day 7 and day 10 followed by electroporation four times and spleens were harvested on day 14 after the first administration. The percentage of splenic CD4+Foxp3+ T cells was determined after 16 hours restimulation of MOG (35-55) peptide. Data were generated from pools of 5 mice/group and the mouse spleens pooled prior to analysis.

Fig. 19: results of IFN-gamma and IL-17 detection by flow cytometry are shown. The indicated DNA vectors were administered to C57BL/6 mice on day 0, day 3, day 7 and day 10 followed by electroporation four times and spleens were harvested on day 14 after the first administration. The percentage of (A) IFN-. Gamma.T cells and (B) IL-17+ T cells in the total CD4+ T cell population was determined after 16 hours of restimulation of MOG (35-55) peptide. Data were generated from pools of 5 mice/group and the mouse spleens pooled prior to analysis.

Fig. 20: the results of the bicolor IL-10/IFN-. Gamma.FluoSpot assay are shown. The indicated DNA vectors were administered intramuscularly to C57BL/6 mice on day 0, followed by electroporation, and spleens were harvested on day 7 post-administration. IL-10 secretion and IFN-gamma secretion (SFU/10) of splenocytes were tested with two-color FluoSpot after either no restimulation (A) or 44 hours of restimulation of MOG (35-55) peptide (B) ⁶ Individual spleen cells). Individual mice and mean ± SEM,5 mice/group, × (p<0.01 A two-tailed Mann-Whitney test).

Fig. 21: the IL-10/IFN-gamma ratios calculated from the data shown in FIG. 20B are shown. Individual mice and mean ± SEM,5 mice/group, (< 0.05 p), two-tailed Mann-Whitney test are shown.

Fig. 22: results of MOG tetramer staining and MOG-specific T cell detection are shown. The indicated DNA vectors were administered intramuscularly to C57BL/6 mice on day 0, followed by electroporation, and spleens were harvested on day 7 post-administration. The percentage of spleen Foxp3+ cells in the CD4+ MOG (38-49) tet+ T cell population was determined by the H-2IAb/MOG (38-49) tetramer. Tetramer staining was performed ex vivo and splenocytes were not restimulated with MOG (35-55) peptide. Data were generated from pools of 5 mice/group and the mouse spleens pooled prior to analysis.

Fig. 23: the results of the bicolor IL-10/IFN-. Gamma.FluoSpot assay are shown. The indicated DNA vectors were administered intramuscularly to C57BL/6 mice on day 0, followed by electroporation, and spleens were harvested on day 7 post-administration. IL-10 secretion and IFN-gamma secretion (SFU/10) of splenocytes were tested with two-color FluoSpot after either no restimulation (control, A) or 44 hours of restimulation of MOG (35-55) peptide (B) ⁶ Individual spleen cells). Individual mice and mean ± SEM,5 mice/group, × (p<0.01 A two-tailed Mann-Whitney test).

Fig. 24: the IL-10/IFN-gamma ratios calculated from the data shown in FIG. 23B are shown. Individual mice and mean ± SEM,5 mice/group, (< 0.01 p), two-tailed Mann-Whitney test are shown.

Fig. 25: results of MOG tetramer staining and MOG-specific T cell detection are shown. The indicated DNA vectors were administered intramuscularly to C57BL/6 mice on day 0, followed by electroporation, and spleens were harvested on day 7 post-administration. The percentage of spleen Foxp3+ cells in the CD4+ MOG (38-49) tet+ T cell population was determined by the H-2IAb/MOG (38-49) tetramer. Tetramer staining was performed ex vivo and splenocytes were not restimulated with MOG (35-55) peptide. Data were generated from pools of 5 mice/group and the mouse spleens pooled prior to analysis.

Fig. 26: shows the detection result of the proliferated Treg. The indicated DNA vectors were administered intramuscularly to C57BL/6 mice on day 0, followed by electroporation, and spleens were harvested on day 7 post-administration. The percentage of ki67+ cells in the Treg (cd4+cd25+foxp3+) cell population was isolated. Data were generated from pools of 5 mice/group and the mouse spleens pooled prior to analysis.

Fig. 27: the results of the bicolor IL-10/IFN-. Gamma.FluoSpot assay are shown. C57BL/6 mice were administered intramuscularly on day 0 with the indicated DNA vectors, followed by electroporation and spleen harvested on day 7 post administration. IL-10 secretion and IFN-gamma secretion (SFU/10) of splenocytes were tested with two-color FluoSpot after either no restimulation (control, A) or 44 hours of restimulation of MOG (35-55) peptide (B) ⁶ Individual spleen cells). Individual mice and mean ± SEM,5 mice/group, × (p<0.01 A two-tailed Mann-Whitney test).

Fig. 28: the IL-10/IFN-gamma ratios calculated from the data shown in FIG. 27B are shown. Individual mice and mean ± SEM,5 mice/group, (< 0.01 p), two-tailed Mann-Whitney test are shown.

Fig. 29: results of MOG tetramer staining and MOG-specific T cell detection are shown. C57BL/6 mice were administered intramuscularly on day 0 with the indicated DNA vectors, followed by electroporation and spleen harvested on day 7 post administration. The percentage of spleen Foxp3+ cells in the CD4+ MOG (38-49) tet+ T cell population was determined by the H-2IAb/MOG (38-49) tetramer. Tetramer staining was performed ex vivo and splenocytes were not restimulated with MOG (35-55) peptide. Data were generated from pools of 5 mice/group and the mouse spleens pooled prior to analysis.

Fig. 30: shows the detection result of the proliferated Treg. The indicated DNA vectors were administered intramuscularly to C57BL/6 mice on day 0, followed by electroporation, and spleens were harvested on day 7 post-administration. The percentage of ki67+ cells in Treg (cd4+cd25+foxp3+) cell bodies was isolated. Data were generated from pools of 5 mice/group and the mouse spleens pooled prior to analysis.

Fig. 31: expression and secretion of the first polypeptide encoded by the indicated DNA vector, as detected by sandwich ELISA, was shown, with supernatant from an Expi293F cell transiently transfected with the DNA vector. Negative control (Neg ctrl): supernatant from the pi293F cells treated with transfection reagent ex fectamine alone. Capture antibody: a murine anti-MOG antibody; detection of antibodies: anti-hIgG CH3 domain antibodies.

Fig. 32: the expression and secretion levels of the immunosuppressive compounds encoded by the DNA vectors shown are shown as detected by sandwich ELISA, with supernatants from Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): supernatant from the pi293F cells treated with transfection reagent ex fectamine alone. The following conditions and antibodies were used: a) The supernatant was diluted 1:100. Capture antibody: mouse IL-10 antibody, 0.4. Mu.g/ml, 100. Mu.l/well, MAB417, R & DSystems. Detection of antibodies: mouse IL-10 biotinylated antibody, 0.2 μg/ml, BAF417, R & DSystems. B) Capture antibody: TGF-beta 1 antibodies, 2. Mu.g/ml, 100. Mu.l/well, MAB2402, RD Systems. Detection of antibodies: chicken anti-tgfβ1 biotinylated antibody, 0.8 μg/ml,100 μl/well, BAF240, R & D Systems. C) Capture antibody: mouse GM-CSF antibody, 2. Mu.g/ml, 100. Mu.l/well, MAB415, R & D Systems. Detection of antibodies: anti-GM-CSF biotinylated antibody, 0.8 μg/ml, BAF415, R & D Systems.

Fig. 33: the individual expression and secretion of the proteins encoded by the indicated DNA vectors, as detected by sandwich ELISA, were shown, with supernatants from Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): supernatant from the pi293F cells treated with transfection reagent ex fectamine alone. Capture antibody: mouse anti-MOG antibody, 0.25. Mu.g/ml, 100. Mu.l/well, sc-73330,Santa Cruz Biotechnology. The following detection antibodies were used: a) Mouse anti-IL-10 biotinylated antibody, 0.2. Mu.g/ml, 100. Mu.l/well, BAF417, R & D Systems, B) chicken anti-TGF beta 1 biotinylated antibody, 0.8. Mu.g/ml, 100. Mu.l/well, BAF240, R & D Systems. C) anti-GM-CSF biotinylated antibody, 0.8 μg/ml, BAF415, R & D Systems.

Fig. 34: results of western blot analysis of supernatants from Expi293F cells transfected with the indicated DNA vectors are shown. A: reduced supernatant samples (35 μl loaded). A first antibody: mice were anti-MOG (sc-73130). A second antibody: donkey is resistant to mice, dylight 800 (SA 5-10172). Protein standards were detected in Chemidoc channel Dylight650 (signal not shown). Chemidoc channel dyight 800. The black arrow indicates the first polypeptide expressed as a separate protein from the corresponding DNA vector and secreted from the transfected cell. B: non-reduced supernatant samples (35 μl loaded). A first antibody: mice were anti-MOG (sc-73130). A second antibody: donkey is resistant to mice, dylight 800 (SA 5-10172). Chemidoc channels Dylight650 (for protein standards) and 800. The black arrow indicates the dimeric protein formed by the two first polypeptide molecules expressed from the indicated DNA vector and secreted from the transfected cells. C: reduced supernatant samples (35 μl loaded). A first antibody: rat anti-IL 10 (MAB 417). A second antibody: donkey is resistant to rats, dylight 488 (SA 5-10026). Chemidoc channels Dylight650 (for protein standards) and 488. The black arrow indicates IL-10 expressed as an individual protein from the corresponding DNA vector and secreted from the transfected cells. For VB5044 and VB5054, the increase in IL-10 size was due to fusion of the P2A tail with IL-10 by ribosome jump. D: reduced supernatant samples (35 μl loaded). A first antibody: rabbit anti-TGF- β1 antibody (USB 1042777-biotin). A second antibody: donkey is resistant to rabbit, dylight650 (SA 5-10041). Chemidoc channel dyight 650. The black arrows indicate TGF- β1 expressed as individual proteins from the corresponding DNA vectors and secreted from transfected cells. E: reduced supernatant samples (35 μl loaded). A first antibody: goat anti-murine GM-CSF (BAF 415). A second antibody: donkey is against goat, dylight 800 (SA 5-10092). Chemidoc channels Dylight650 (for protein standards) and 800. The black arrow indicates GM-CSF expressed as an individual protein from the corresponding DNA vector and secreted from transfected cells.

Fig. 35: the expression and secretion levels of the protein of the first polypeptide detected by sandwich ELISA (capture antibody: anti-MOG antibody; detection antibody: anti-hIgG CH3 domain antibody) were shown, and supernatants were from the Expi293F cells transiently transfected with the DNA vector VB5068, VB5069 or VB 5070. Negative control (Neg ctrl): supernatant from the pi293F cells treated with transfection reagent ex fectamine alone.

Fig. 36: the expression and secretion of the first polypeptide encoded by the indicated DNA vector, as detected by sandwich ELISA (capture antibody: anti-MOG antibody; detection antibody: anti-hIgG CH3 domain antibody), was shown, with supernatant from an Expi293F cell transiently transfected with the DNA vector. Negative control (Neg ctrl): supernatant from the pi293F cells treated with transfection reagent ex fectamine alone. The following detection antibodies were used: a) Anti-murine SCBG3A2 biotinylated antibody, 0.83. Mu.g/ml, 100. Mu.l/well, BAF3465, R & D Systems, B) anti-murine PD-1 biotinylated antibody, 0.72. Mu.g/ml, 100. Mu.l/ml, DY1021, R & D Systems.

Fig. 37: the expression and secretion levels of IL-10 encoded by the indicated DNA vectors were shown by sandwich ELISA, and supernatants were from Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): supernatant from the pi293F cells treated with transfection reagent ex fectamine alone. Capture antibody: mouse IL-10 antibody, 0.4. Mu.g/ml, 100. Mu.l/well, MAB417, R & D Systems, detection antibody: mouse anti-IL-10 biotinylated antibody, 0.2. Mu.g/ml, 100. Mu.l/well, BAF417, R & D Systems

Fig. 38: the separate expression and secretion of the immunosuppressive compounds encoded by the DNA vectors shown were shown to be detected by sandwich ELISA, with supernatants from Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): supernatant from the pi293F cells treated with transfection reagent ex fectamine alone. Supernatant dilution was 1:100. Capture antibody: mouse anti-MOG antibody, 0.25 μg/ml,100 μl/well, sc-73330,Santa Cruz Biotechnology; detection of antibodies: mouse anti-IL-10 biotinylated antibody, 0.2. Mu.g/ml, 100. Mu.l/well, BAF417, R & DSystems.

Fig. 39: results of western blot analysis of supernatants from Expi293F cells transfected with the indicated DNA vectors are shown. A: reduced supernatant samples (35 μl loaded). A first antibody: mice were anti-MOG (sc-73130). A second antibody: donkey is resistant to mice, dylight 800 (SA 5-10172). Chemidoc channel dyight 800. The black arrow indicates the intact first polypeptide expressed as individual proteins from the corresponding DNA vector and secreted from the transfected cells. B: reduced supernatant samples (35 μl loaded). A first antibody: rat anti-IL 10 (MAB 417). A second antibody: donkey is resistant to rats, dylight488 (SA 5-10026). Chemidoc channels Dylight 650 (for protein standards) and 488. The black arrow indicates IL-10 expressed as an individual protein from the corresponding DNA vector and secreted from the transfected cells.

Fig. 40: the results of the bicolor IL-10/IFN-. Gamma.FluoSpot assay are shown. C57BL/6 mice were administered intramuscularly on day 0 with the indicated DNA vectors, followed by electroporation and spleen harvested on day 7 post administration. IL-10 secretion and IFN-gamma secretion (SFU/10) of splenocytes were tested with two-color FluoSpot after either no restimulation (control, A) or 44 hours of restimulation of MOG (35-55) peptide (B) ⁶ Individual spleen cells). Individual mice and mean ± SEM,5 mice/group are shown.

Fig. 41: results of MOG tetramer staining and MOG-specific T cell detection are shown. C57BL/6 mice were administered intramuscularly on day 0 with the indicated DNA vectors, followed by electroporation and spleen harvested on day 7 post administration. The percentage of Foxp3+ splenocytes in CD4+ MOG (38-49) tet+ splenocytes was determined by the H-2IAb/MOG (38-49) tetramer. Tetramer staining was performed ex vivo and splenocytes were not restimulated with MOG (35-55) peptide. Data were generated from pools of 5 mice/group and the mouse spleens pooled prior to analysis.

Fig. 42: results of MOG tetramer staining and MOG-specific T cell detection are shown. C57BL/6 mice were administered intramuscularly on day 0 with the indicated DNA vectors, followed by electroporation and spleen harvested on day 7 post administration. The percentage of MOG (38-49) tet+ splenocytes in CD4+CD25+Foxp3+ splenocytes was determined by the H-2IAb/MOG (38-49) tetramer. Tetramer staining was performed ex vivo and splenocytes were not restimulated with MOG (35-55) peptide. Data were generated from pools of 5 mice/group and the mouse spleens pooled prior to analysis.

Fig. 43: shows the detection result of the proliferated Treg. The indicated DNA vectors were administered intramuscularly to C57BL/6 mice on day 0, followed by electroporation, and spleens were harvested on day 7 post-administration. The percentage of ki67+ splenocytes in the Treg (cd4+cd25+foxp3+) cell population was isolated. Data were generated from pools of 5 mice/group and the mouse spleens pooled prior to analysis.

Fig. 44: the results of detection of cd4+cd25+foxp3+ by flow cytometry are shown. The indicated DNA vectors were administered to C57BL/6 mice on day 0, followed by electroporation, and spleens were harvested on day 7 post-administration. The percentage of CD4+CD25+Foxp3+ splenocytes was determined after 16 hours of restimulation of MOG (35-55) peptide. Data were generated from pools of 5 mice/group and the mouse spleens pooled prior to analysis.

Fig. 45: shows that the detection of antibodies by sandwich ELISA (capture antibodies: mouse anti-human IgG (CH 3 domain), 1ug/ml,100 ul/well, MCA878G, bioRad; detection antibodies: captureStylelect) ^TM Biotin anti-IgG-Fc (human) conjugate, 1 μg/ml,100 μl/well, 7103262100,Thermo Fisher) detected expression and secretion of the first polypeptide encoded by the indicated DNA vector, supernatant from an Expi293F cell transiently transfected with the DNA vector. Negative control (Neg ctrl): supernatant from the pi293F cells treated with transfection reagent ex fectamine alone.

Fig. 46: the expression and secretion levels of IL-10 encoded by the indicated DNA vectors were shown by sandwich ELISA, and supernatants were from Expi293F cells transiently transfected with the DNA vectors. Negative control (Neg ctrl): supernatant from the pi293F cells treated with transfection reagent ex fectamine alone. Capture antibody: mouse IL-10 antibody, 0.4. Mu.g/ml, 100. Mu.l/well, MAB417, R & D Systems. Detection of antibodies: mouse IL-10 biotinylated antibody, 0.2 μg/ml, BAF417, R & D Systems.

Fig. 47: results of western blot analysis of supernatants from Expi293F cells transfected with the indicated DNA vectors are shown. Reduced supernatant samples (35 μl loaded). A first antibody: rat anti-IL-10 (MAB 417). A second antibody: donkey is resistant to rat, dylight 650 (SA 5-10029). Chemidoc channel dyight 650. The black arrow indicates IL-10 expressed as a separate protein from the indicated DNA vector and secreted from the transfected cells.

Detailed Description

The first polypeptide and/or multimeric protein will also be referred to herein as a construct.

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

A "tolerance-inducing construct" is one that does not stimulate an inflammatory immune response against T cell epitopes contained in antigenic units, but rather induces tolerance, when administered to a subject in a form suitable for administration and in an amount effective to induce tolerance (i.e., an effective amount).

The term "tolerability (or tolerance)" as used herein refers to a reduced level of an inflammatory immune response to an antigen, such as a self-antigen, allergen or alloantigen or xenogeneic antigen, a delayed onset or progression of an inflammatory immune response, and/or a reduced risk of onset or progression of an inflammatory immune response.

A "subject" is an animal, such as a mouse or a human, preferably a human. The terms "mouse", "mouse" and "m" are used interchangeably herein to refer to or mean a mouse. The terms human and "h" are used interchangeably herein to refer to or represent a person. The subject may be a patient in need of therapeutic treatment, i.e. a person suffering from an immune disease such as autoimmune disease, allergy or graft rejection, or it may be a subject in need of prophylactic treatment or a subject suspected of suffering from an immune disease. The terms "subject" and "individual" are used interchangeably herein.

A "disease" is an abnormal medical condition that is generally associated with specific signs and symptoms in a subject suffering from the disease. As used herein, "immune disease" refers to a condition involving an unwanted immune response, which includes autoimmune disease, allergy or graft rejection, i.e., rejection of allografts or xenografts, such as host rejection of cells, tissues or organs from the same species (allograft, allo) or a different species (xeno) that are transplanted into a host.

"treatment" is prophylactic or therapeutic treatment.

A "prophylactic treatment" is one which is administered to a subject who does not exhibit signs or symptoms of an immune disease or exhibits only early signs or symptoms thereof, the purpose of which is to prevent or reduce the risk of developing an immune disease. The prophylactic treatment may be effected as a prophylactic treatment against an immune disorder or as a treatment to inhibit or reduce further development or enhancement of an immune disorder and/or a symptom associated therewith. The terms prophylactic treatment, prevention and prevention are used interchangeably herein.

A "therapeutic treatment (therapeutic treatment)" is a treatment administered to a subject exhibiting symptoms or signs of an immune disease, in which treatment the subject is administered to the subject with the aim of weakening or eliminating these signs or symptoms or delaying or preventing the progression of the condition.

"T cell epitope" as used herein refers to a discrete single T cell epitope or a portion or region of an antigen containing multiple T cell epitopes (e.g., multiple minimal T cell epitopes), such as a hot spot (hotspot).

A "nucleotide sequence" is a sequence consisting of nucleotides. The terms "nucleotide sequence" and "nucleic acid sequence" are used interchangeably herein.

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

Carrier body

The vector of the invention may be any molecule suitable for carrying a foreign nucleic acid sequence (e.g.DNA or RNA) into a cell in which the molecule may be expressed, i.e.an expression vector.

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.

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 alphaviruses, lentiviruses, moloney murine leukemia virus, and rhabdoviruses (rhabdoviruses).

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

DNA plasmid

Plasmids are small extrachromosomal DNA molecules that are physically separated from chromosomal DNA inside a cell and that can replicate independently. Plasmids are mostly present in bacteria as small circular double stranded DNA molecules; however, plasmids are sometimes present in archaebacteria and eukaryotes. Artificial plasmids are widely used as vectors for molecular cloning, serving to deliver recombinant DNA sequences and ensure their high expression within host organisms. Plasmids contain several important features, including the characteristics of the cell selected to contain the plasmid (e.g., antibiotic resistance genes), the origin of replication, the Multiple Cloning Site (MCS), and the promoter that drives expression of the inserted gene of interest.

Typically, a promoter is a sequence capable of attracting a promoter factor and a polymerase to the promoter, thereby transcribing the gene. The promoter is located upstream of the transcription initiation site of the gene on the DNA. Promoters may be about 100-1000 base pairs long. The nature of the promoter generally depends on the gene and transcription product, and the type or class of RNA polymerase recruited to the site. When the RNA polymerase reads the DNA of the plasmid, the RNA molecule is transcribed. After processing, where the ribosome is translating the mRNA into a protein, the mRNA will be able to be translated many times and thus produce many copies of the protein encoded by the gene of interest. Typically, ribosomes facilitate the decoding process by inducing the binding of complementary tRNA anticodon sequences to mRNA codons. tRNA carries specific amino acids that are linked together into a polypeptide when mRNA passes through and is "read" by the ribosome. Translation is performed in three stages: initiation, extension, and termination. After the translation process, the polypeptide folds into an active protein and performs its function in the cell or exports the cell and performs its function elsewhere, sometimes after a considerable number of translational modifications.

When a protein is destined to be exported from a cell, the signal peptide directs the protein into the endoplasmic reticulum, where the signal peptide is cleaved off and the protein is transferred to the cell perimeter after translation has been terminated.

The DNA plasmid is not limited to any particular plasmid, and one of ordinary skill in the art will appreciate that any plasmid having a suitable backbone can be selected and engineered to contain the elements and units of the present disclosure using methods known in the art.

Co-expression

The vectors of the present disclosure co-express several proteins. Such vectors (and plasmids) are also known as polycistronic (or polycistronic) vectors (and polycistronic plasmids). One of ordinary skill in the art knows how to engineer vectors to include sequences encoding these proteins and can choose different techniques so that these proteins are co-expressed as separate proteins from one vector.

Thus, one of ordinary skill in the art can construct vectors of the invention that co-express different proteins (i.e., the first polypeptide and the one or more immunosuppressive compounds).

In a preferred embodiment, the vector of the invention comprises one or more coexpression elements, i.e. nucleic acid sequences allowing the co-expression of the first polypeptide and the one or more immunosuppressive compounds from the same vector.

In one embodiment of the disclosure, the vector comprises a co-expression element (or more than one co-expression element) that causes transcription of the first polypeptide and the one or more immunosuppressive compounds on a single transcript, but independent translation into the first polypeptide and the one or more immunosuppressive compounds. Thus, the presence of the coexpression element results in the final production of an individual translation product.

IRES

In one embodiment of the present disclosure, the co-expression element is an IRES element, the concept of which is shown in fig. 1. An internal ribosome entry site (abbreviated IRES) is an RNA element that allows translation to be initiated in a cap-independent manner as part of a larger protein synthesis process. In eukaryotic translation, initiation generally occurs at the 5 'end of the mRNA molecule, as 5' cap recognition is required for assembly of the initiation complex. By placing an IRES element between two coding regions, the initiation complex can assemble at this site and allow translation of the downstream coding region. Thus, in one embodiment of the present disclosure, the vector comprises an IRES and one transcript is produced from the vector, which is subsequently translated into a separate protein.

The IRES element allows co-expression of the first polypeptide and the one or more immunosuppressive compounds under the control of the same promoter. The promoter directs transcription of a single mRNA comprising a nucleic acid sequence encoding a first polypeptide and a coding region for a nucleic acid sequence encoding the one or more immunosuppressive compounds. If more than one immunosuppressive compound is expressed from the vector of the invention, it is desirable that an IRES element is present in the vector of the invention upstream of each nucleic acid sequence encoding an immunosuppressive compound. Alternatively, if more than one immunosuppressive compound is expressed from the vector of the invention, another type of co-expression element may be used.

IRES elements used in the vectors of the invention may be derived from the viral genome or from cellular mRNA. Vectors such as DNA plasmids comprising IRES elements are commercially available.

2A self-cleaving peptides

In another embodiment of the present disclosure, the coexpression element is a nucleic acid sequence encoding a 2A self-cleaving peptide (or simply "2A peptide"), the concept of which is shown in fig. 2.

In this application, the terms "2A self-cleaving peptide" and "2A peptide" are used for peptides encoded by nucleic acid sequences that when located between two coding regions cause the transcription of the two coding regions into a single transcript, but the transcript translates into two separate peptide chains. Typically, when the mRNA is translated by the ribosome, the amino acids are covalently bound in an N-terminal to C-terminal fashion. The presence of a nucleic acid sequence encoding a 2A self-cleaving peptide results in two separate peptide chains, as the ribosome skips peptide bond synthesis at the C-terminus of the 2A peptide. 2A self-cleaving peptide is typically 18-22 amino acids in length and typically comprises the consensus sequence DXEXNPGP (SEQ ID NO: 68), wherein X may be any amino acid.

In one embodiment of the invention, the ribosome skips the peptide bond between glycine and proline residues present on the C-terminus of the 2A self-cleaving peptide, which means that the upstream gene product will have some additional amino acid residues added to the terminus, while the downstream gene product will start with proline.

In one embodiment, the 2A self-cleaving peptide is a 18-22 amino acid long sequence comprising a consensus sequence DXEXNPGP (SEQ ID NO: 68), wherein X may be any amino acid.

Thus, the 2A self-cleaving peptide also allows for co-expression of the first polypeptide and the one or more immunosuppressive compounds under the control of the same promoter. As with IRES elements, if more than one immunosuppressive compound is expressed from the vector of the invention, it is desirable that a nucleic acid sequence encoding a 2A peptide is present in the vector upstream of each nucleic acid sequence encoding an immunosuppressive compound. As one example, the vector comprises a first nucleic acid sequence encoding a first polypeptide, a second nucleic acid sequence encoding a first immunosuppressive compound, and a third nucleic acid sequence encoding a second immunosuppressive 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, if more than one immunosuppressive compound is expressed from the vector of the invention, another type of co-expression element may be used.

In yet another 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.

In one embodiment, the T2A peptide has the same amino acid sequence as the T2A sequence listed in table 1 or 2. In yet another 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, such as 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 of table 1. In another embodiment, the T2A peptide has the amino acid sequence of SEQ ID NO. 6.

In one embodiment, the P2A peptide has the same amino acid sequence as the P2A sequence listed in table 1 or 2. In yet another embodiment, the amino acid sequence DVEENPGP (SEQ ID NO: 69) is present, but the remainder of the P2A amino acid sequence has 80% to 100% sequence identity, such as 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.

In one embodiment, the E2A peptide has the same amino acid sequence as the E2A sequence listed in table 1 or 2. In yet another 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, such as 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 of Table 1. In another embodiment, the E2A peptide has the amino acid sequence of SEQ ID NO. 8.

In one embodiment, the F2A peptide has the same amino acid sequence as the F2A sequence listed in table 1 or 2. In yet another embodiment, the amino acid sequence DVESNPGP (SEQ ID NO: 70) is present, but the remainder of the F2A amino acid sequence has 80% to 100% sequence identity, such as 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 of table 1. In another embodiment, the F2A peptide has the amino acid sequence of SEQ ID NO. 9.

Table 1:2A sequence of self-cleaving peptide

Name of the name	Sequence(s)	SEQ ID NO.
			T2A	EGRGSLLTCGDVEENPGP	SEQ ID NO:6
P2A	ATNFSLLKQAGDVEENPGP	SEQ ID NO:7
			E2A	QCTNYALLKLAGDVESNPGP	SEQ ID NO:8
F2A	VKQTLNFDLLKLAGDVESNPGP	SEQ ID NO:9

It is generally known that the efficiency of 2A peptides can be regulated to increase their cleavage and expression efficiency, for example by inserting GSG sequences before the N-terminus of the wild-type sequence, as shown in table 2.

Table 2: other sequences of 2A self-cleaving peptides

Name of the name	Sequence(s)	SEQ ID NO.
			T2A	GSGEGRGSLLTCGDVEENPGP	SEQ ID NO:71
P2A	GSGATNFSLLKQAGDVEENPGP	SEQ ID NO:72
			E2A	GSGQCTNYALLKLAGDVESNPGP	SEQ ID NO:73
F2A	GSGVKQTLNFDLLKLAGDVESNPGP	SEQ ID NO:74

In another embodiment, the vector of the invention contains both an IRES element and a nucleic acid sequence encoding a 2A peptide. As one example, the vector comprises a first nucleic acid sequence encoding a first polypeptide, a second nucleic acid sequence encoding a first immunosuppressive compound, and a third nucleic acid sequence encoding a second immunosuppressive 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. Other nucleic acid sequences encoding other immunosuppressive compounds can be contained in the vector in the same manner.

In another embodiment, the vector of the invention comprises a nucleic acid sequence encoding two 2A peptides, i.e. a contiguous sequence consisting of two 2A peptides. As one example, the vector comprises a first nucleic acid sequence encoding a first polypeptide and a second nucleic acid encoding an immunosuppressive compound. The vector may comprise a nucleic acid sequence encoding two 2A peptides as a contiguous sequence between the first and second nucleic acid sequences.

Bidirectional promoter

In one embodiment of the disclosure, the vector comprises a co-expression element (or more than one co-expression element) that causes transcription of the first polypeptide and the one or more immunosuppressive compounds as separate transcripts, which produce separate transcripts and thus separate proteins.

In one embodiment of the present disclosure, the coexpression element is a bi-directional promoter, the concept of which is shown in fig. 3. A bi-directional promoter is a generally short (e.g., <1 kbp) intergenic DNA region between the 5' ends of the genes in a bi-directional gene pair. "bidirectional gene pair" refers to two adjacent genes encoded on opposite strands, wherein their 5' ends are opposite each other.

In One embodiment of the disclosure, the bi-directional promoter is a CAG promoter in back-to-back arrangement with four CMV enhancers (Sladitschek HL, neveu PA et al, PLoS One 11 (5), e0155177,2016).

In one embodiment of the disclosure, the bi-directional promoter is RPBSA (Kevin He et al, int.j.mo.l sci.21 (23), 9256,2020).

In one embodiment of the present disclosure, the bi-directional promoters are a back-to-back arrangement of mouse Pgk and human eukaryotic translation elongation factor 1α1 promoters (Golding and Mann, gene Therapy 18,817-826,2011).

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 immunosuppressive compound as a bidirectional gene pair comprising a bidirectional promoter between its 5' ends.

Multiple promoters

In another embodiment of the disclosure, the co-expression element is a variety of promoters, i.e., the vector, e.g., plasmid, comprises a separate promoter for each of the nucleic acid sequences encoding the first polypeptide and the one or more immunosuppressive compounds, i.e., for separately transcribing each of the first polypeptide and the one or more immunosuppressive compounds.

In one embodiment, the nucleic acid sequences will each have a different promoter, the concept of which is shown in fig. 4. In one embodiment, all 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; that is, nucleic acid sequences with stronger promoters may be expressed at a higher level than other nucleic acid sequences.

Numerous promoters are known in the art and are suitable for incorporation into the plasmids of the present invention. In one embodiment of the disclosure, the promoter is derived from a cytomegalovirus, such as a CMV promoter.

Combinations of different co-expression units

In one embodiment, the vector of the invention comprises one or more co-expression elements, preferably selected from the group consisting of IRES elements, 2A peptides, bi-directional promoters and promoters.

The vectors of the invention may comprise all kinds of co-expression element combinations.

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 immunosuppressive compound, and a third nucleic acid sequence encoding a second immunosuppressive compound. In one embodiment, the DNA plasmid comprises an IRES and a 2A peptide that allows for co-expression of the first polypeptide (under the control of a promoter) and the first and second immunosuppressive compounds. In another embodiment, the DNA plasmid comprises a bi-directional promoter and another promoter.

The skilled artisan will appreciate that the terms first, second and third nucleic acid sequences as in the examples above do not denote that the plasmids of the invention comprise nucleic acid sequences in the order of the first, second and third nucleic acid sequences. The second nucleic acid sequence may be downstream or upstream of the first or third nucleic acid sequence, the third nucleic acid sequence may be downstream or upstream of the first or second nucleic acid sequence, and the first nucleic acid sequence may be upstream or downstream of the second or third nucleic acid sequence. In another embodiment, the first nucleic acid sequence and the second nucleic acid sequence may be in opposite orientations on the same DNA strand, as may the first nucleic acid sequence and the third nucleic acid sequence or the second nucleic acid sequence and the third nucleic acid sequence. In other embodiments, the nucleic acid sequences encoding the first polypeptide and the immunosuppressive compound may be on opposite DNA strands.

Immunosuppressive compounds

The vectors of the invention comprise one or more nucleic acid sequences encoding one or more immunosuppressive compounds.

In one embodiment of the present disclosure, an immunosuppressive compound is a compound that induces, increases, or maintains immune tolerance. In another embodiment of the present disclosure, the immunosuppressive compound is a compound known to induce, increase, or maintain immune tolerance.

In yet another embodiment, an immunosuppressive compound is a compound that facilitates presentation of epitopes in antigenic units in a tolerogenic manner and/or facilitates induction of tolerogenic maintenance cells (regulatory T cells, not just anergy or apoptosis of effector T cells) and/or facilitates maintenance of such tolerogenic maintenance cells.

In yet another embodiment, an immunosuppressive compound is a compound that promotes and/or supports presentation of epitopes in antigenic units in a tolerogenic manner, and/or promotes and/or supports induction of tolerogenic maintenance cells (regulatory T cells, not just anergy or apoptosis of effector T cells) and/or helps maintain such tolerogenic maintenance cells.

In one embodiment of the present disclosure, the immunosuppressive 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 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.

In a preferred embodiment, the immunosuppressive compound is an extracellular portion (e.g., an extracellular domain) of a human (h) inhibitory checkpoint molecule, such as an extracellular portion (e.g., 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, e.g., hCLA-4 of SEQ ID NO. 51. In one embodiment, the inhibitory checkpoint molecule is hPD-1, as shown in hPD-1 of 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 present disclosure, the immunosuppressive 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-gamma. In one embodiment, the cytokine is IL-37. In one embodiment, the cytokine is IL-35.

In a preferred embodiment, the immunosuppressive 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, e.g., hIL-10 of SEQ ID NO. 53. In one embodiment, the cytokine is hTGF- β1, e.g., hTGF- β1 of SEQ ID NO. 54. In one embodiment, the cytokine is hTGF-. Beta.2, e.g., hTGF-. Beta.2 of SEQ ID NO. 58. In one embodiment, the cytokine is hTGF-beta 3, e.g., hTGF-beta 3 of SEQ ID NO: 59. In one embodiment, the cytokine is hIL-27. In one embodiment, the cytokine is hIL-2, e.g., hIL-2 of SEQ ID NO. 55. In one embodiment, the cytokine is hGM-CSF as shown in SEQ ID NO: 56. In one embodiment, the cytokine is hFLT3L. In one embodiment, the cytokine is hIFN-gamma, e.g., hIFN-gamma of SEQ ID NO: 57. In one embodiment, the cytokine is hIL-37. In one embodiment, the cytokine is hIL-35.

Vectors comprising a plurality of nucleic acid sequences encoding a plurality of immunosuppressive compounds

In one embodiment of the disclosure, the vector comprises a nucleic acid sequence encoding 2, 3, 4, 5, 6, 7 or 8 immunosuppressive compounds. In another embodiment, the vector comprises a nucleic acid sequence encoding 2 to 6 immunosuppressive compounds, i.e., 2 or 3 or 4 or 5 or 6 immunosuppressive compounds. The immunosuppressive compounds may be the same or different, preferably different.

In a preferred embodiment, the different immunosuppressive compounds are produced at a number of different levels or promote a tolerance-inducing environment. For example, the vectors of the invention comprise nucleic acid sequences encoding 3 different immunosuppressive compounds, the first of which induces tolerance, the second of which increases tolerance, and the third of which maintains tolerance.

First nucleic acid sequence

The vectors of the present disclosure comprise a first nucleic acid sequence, i.e., DNA or RNA, including genomic DNA, cDNA and mRNA, that encodes a double or single strand of a first polypeptide. In one embodiment, the first nucleic acid sequence is DNA. In another embodiment, the first nucleic acid sequence is optimized for the species of subject to which the nucleic acid sequence is administered. For administration to humans, in one embodiment, the first nucleic acid sequence is subjected to human codon optimization.

The first nucleic acid sequence encodes a first polypeptide comprising a targeting unit that targets an APC, a multimerization unit (e.g., a dimerization unit), and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, allergen, alloantigen, or xenogeneic antigen. Upon administration to a subject, the first polypeptide expresses and forms a multimeric protein due to the presence of multimerization units, which elicits a tolerogenic response to one or more T cell epitopes contained in the antigenic unit (tolerogenic response).

Constructs

Constructs can be described as polypeptides having an N-terminal start end and a C-terminal end (shown in fig. 5). The elements and units of the first polypeptide-Targeting Units (TUs), multimerizing units (in fig. 5, e.g. dimerizing units (DimU)), and antigenic units-may be arranged in the polypeptide such that the antigenic units are located at the C-terminal end of the polypeptide (fig. 5 a) or at the N-terminal beginning of the polypeptide (fig. 5 b). Preferably, the antigenic unit is located at the C-terminal end of the polypeptide.

The antigenic unit comprises one or more T cell epitopes and, if multiple T cell epitopes are present, may comprise one or more T cell epitope linkers separating the T cell epitopes. The Unit Linker (UL) may connect the multimerization unit (e.g., dimerization unit) and the antigenic unit. Figure 5 shows an antigenic unit with 2T 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.

Hereinafter, various units and elements of the first polypeptide/construct will be discussed in detail. They are present in the first nucleic acid sequence as nucleic acid sequences encoding these units/elements, which are present as amino acid sequences in the first polypeptide or multimeric protein. For ease of reading, in the following the units/elements are mainly explained with respect to the first polypeptide/multimeric protein, i.e. based on their amino acid sequence.

Targeting unit

The first polypeptide encoded by the first nucleic acid comprised in the vector of the invention comprises a targeting unit that targets APCs.

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

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

APCs internalize the construct and present on their surface T cell surfaces contained in antigenic units in an anti-inflammatory tolerogenic manner on MHC, e.g., by not up-regulating costimulatory signals and/or by up-regulating inhibitory surface molecules and/or by promoting secretion of inhibitory cytokines.

In one embodiment, the targeting unit comprises or consists of a moiety that binds to a surface molecule on an APC selected from the group consisting of: TGF-beta receptors (including TGF-beta R1, TGF-beta R2, and TGF-beta R3), 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 (ASGR 1/ASGR 2), CD80, CD86, clec9A, clec12A, clec12B, DCIR2, langerin, MR, DC-Sign, treml4, dectin-1, PDL2, HVEM, CD163, and CD141.

In a preferred embodiment, the targeting unit comprises or consists of a moiety that binds to a surface molecule on a human (h) APC selected from the group consisting of: hTGFbeta receptors (including hTGFbeta R1, hTGFbeta R2, hTGFbeta R3), hIL-10R such as hIL-10RA and hIL-10RB, hIL-2R, hIL-4R, hIL-6R, hIL-11R, hIL-13R, hIL-27R, hIL-35R, hIL-37-R, hGM-CSFR, hFLT3, hCR 7, hCD11b, hCD11c, hCD103, hCD14, hCD36, hCD205, hCD109, hVISTA, hMARCO, hMHCII, hCD, hSIGLEC, hClec A (hMGL), hASGR (hASGR 1/hASGR 2), hCD80, hCD86, hlec 9A, hClec12A, hClec12B, hDCIR, langerin, hMR, hDC-Sign, hTreml4, hDectin-1, hPDL2, hHVEM, hCD163, and hCD141.

The moiety may be a natural ligand, an antibody or a portion thereof (e.g. scFv) or a synthetic ligand.

In one embodiment, the moiety is an antibody or portion thereof specific for any of the foregoing surface molecules, e.g., an scFv, whose binding to the surface molecule results in presentation of the T cell epitope contained in the antigenic unit in an anti-inflammatory tolerogenic manner.

In one embodiment, the moiety is a synthetic ligand specific for any of the foregoing surface molecules, the binding of which to the surface molecule results in presentation of the T cell epitope contained in the antigenic unit in an anti-inflammatory tolerogenic manner. Protein modeling can be used to design such synthetic ligands.

In yet another embodiment, the moiety is a natural ligand. In one embodiment, the natural ligand is selected from the group consisting of TGF beta, 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 CD 54), 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).

In a preferred embodiment, the moiety is a human (h) natural ligand selected from the group consisting of: hTGF beta, hIL-10, hIL-2 as shown in SEQ ID NO:55 hIL-2, hIL-4, hIL-6, hIL-11, hIL-13, hIL-27, hIL-35, hIL-37, hGM-CSF as shown in SEQ ID NO:56 hGM-CSF, hFLT3L, hCCL, hCL 21, hICAM-1 (intercellular adhesion molecule 1, also referred to as CD 54), human keratin, hVSIG-3 (preferably the extracellular domain of hVSIG-3), hSCGB3A2, hCDLA-4 (preferably the extracellular domain of hCDLA-4, such as the extracellular domain of hCDLA 4 of SEQ ID NO: 51), hPD-1 (preferably the extracellular domain of PD-1, such as the extracellular domain of hPD-1 of SEQ ID NO: 52), and hBTLA (preferably the extracellular domain of hBTLA).

In another embodiment, the targeting unit is or comprises IL10 or TGF-beta, preferably human IL-10 or human TGF-beta, including its isoforms TGF-beta-1, TGF-beta-2 and TGF-beta-3.

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.

In yet another preferred 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 a 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.

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 a 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.

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 up to 80 amino acids, such as up to 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 acids 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 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 with 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 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 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 comprises the 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 with 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 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 a further 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.

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 yet another preferred embodiment, the targeting unit comprises the amino acid sequence of SEQ ID NO. 53.

In yet another preferred 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 a 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 have been substituted, deleted or inserted, 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.

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 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 the nucleic acid sequence of SEQ ID NO. 63.

In yet another 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 the nucleic acid sequence of SEQ ID NO. 63. In yet another 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 with the nucleic acid sequence of SEQ ID NO. 63.

In yet another 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.

In one embodiment, the targeting unit is or comprises an extracellular portion, such as an extracellular domain, of SCGB3A2 or VSIG-3, preferably human VSIG-3 or human SCGB3A 2.

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 SCGB3 A2. 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 80% sequence identity to the amino acid sequence of SEQ ID NO. 64.

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 yet another preferred embodiment, the targeting unit comprises the amino acid sequence of SEQ ID NO. 64.

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 a further preferred embodiment, the targeting unit consists of the amino acid sequence of SEQ ID NO. 64.

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 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 the nucleic acid sequence of SEQ ID NO. 65.

In yet another 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 the nucleic acid sequence of SEQ ID NO. 65. In yet another preferred embodiment, the targeting unit comprises the amino 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 with the nucleic acid sequence of SEQ ID NO. 65.

In yet another 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.

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 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.

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 yet another 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 a further 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 have been substituted, deleted or inserted, 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 acids.

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 have been substituted, deleted or inserted, 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 acids.

In a preferred embodiment, the targeting unit comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO. 67.

In yet another 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 the nucleic acid sequence of SEQ ID NO. 67. In yet another 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 with the nucleic acid sequence of SEQ ID NO. 67.

In yet another 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.

In yet another embodiment, the targeting unit is or comprises an antibody or portion thereof, e.g., scFv, specific for CD205 (e.g., anti-human CD 205).

Multimerization unit/dimerization unit

The first polypeptide encoded by the first nucleic acid sequence comprised in the vector of the invention comprises multimerization units, such as dimerization units.

The term "multimerization unit" as used herein refers to a nucleotide or amino acid sequence between an antigenic unit and a targeting unit. In addition to linking the antigenic unit and the targeting unit, the multimerization unit also facilitates multimerization/linking of multiple polypeptides (e.g., two, three, four, or more polypeptides) into a multimeric protein, such as a dimeric, trimeric, or tetrameric protein. In addition, the multimerization units also provide flexibility in multimeric proteins to allow optimal binding of the targeting units to molecules on the upper surface of the APC, even if the distance between them is varied. The multimerization unit may be any unit that meets one or more of these requirements.

Multimerization units that promote multimerization/ligation of more than two 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 the human collagen-derived XVIII trimerization domain (see, e.g., A. Alvarez-Cienfunos et al, sci Rep 6,28643 (2016)) or the human collagen XV trimerization domain. Thus, in one embodiment, the multimerization unit is a trimerization unit comprising or consisting of the nucleic acid sequence of 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 secondary fibrin (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 the nucleic acid sequence of SEQ ID NO. 113 or an amino acid sequence encoded by the nucleic acid sequence, optionally further comprising a hinge region as described below.

Dimerization unit

The term "dimerization unit" as used herein refers to a nucleotide or amino acid sequence between an antigenic unit and a targeting unit. In addition to linking the antigenic unit and the targeting unit, the dimerization unit also facilitates dimerization/linking of the two monomeric polypeptides into a dimeric protein. In addition, the dimerization unit also provides flexibility in the dimeric protein to allow optimal binding of the targeting unit to the surface molecules on the APC even though they are at different distances. The dimerization unit may be any unit that meets these requirements.

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 dimerization promoting domain, wherein the dimerization unit linker connects the hinge region and the another dimerization promoting domain. 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 and preferably a dimerization unit linker GGGSSGGGSG (SEQ ID NO: 118).

The term "hinge region" refers to an amino acid sequence contained in a dimerization unit that facilitates the attachment of two polypeptides, i.e., facilitates the formation of a dimeric protein. In the case of multimerization units that promote multimerization/ligation of more than two polypeptides, the term "hinge region" refers to an amino acid sequence contained in such multimerization units that promotes ligation of more than two polypeptides, e.g., three or four polypeptides, and/or serves as a flexible spacer, allowing two targeting units of a multimeric protein to bind to multiple surface molecules on an APC at the same time, even if their distance is varied. The hinge region may be Ig derived, such as derived from IgG, e.g., igG1, igG2, or IgG3. In one embodiment, the hinge region is derived from or consists of an IgM, e.g.comprising or encoded by the nucleotide sequence of SEQ ID NO: 119.

The hinge region may promote dimerization by forming covalent bonds, such as disulfide bonds, between cysteines. Thus, in one embodiment, the hinge region has the ability to form one or more covalent bonds. Preferably, the covalent bond is a disulfide bond.

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

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

In a preferred embodiment, the dimerization unit comprises or consists of hinge exon h1 and hinge exon h4, which have the amino acid sequence of 1 to 27 of SEQ ID NO. 1.

In a preferred embodiment, the dimerization unit comprises or consists of the amino acid sequence of 1 to 27 of SEQ ID NO. 1, except that at most four amino acids, such as at most three amino acids, such as at most two amino acids or such as at most one 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 with the nucleic acid sequence of SEQ ID NO. 10.

In yet another preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 85% sequence identity with the nucleic acid sequence of 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 a further preferred embodiment, the dimerization unit comprises or consists of the nucleic acid sequence of SEQ ID NO. 10.

In another embodiment, the dimerization unit comprises another dimerization promoting domain, the other domain being an immunoglobulin domain, such as an immunoglobulin constant domain (C domain), such as a CH1 domain, a CH2 domain, or a carboxy-terminal C domain (i.e., a CH3 domain), or a sequence substantially identical to such a C domain or variant thereof. Preferably, the further dimerization promoting domain is a carboxy terminal C domain derived from IgG. More preferably, the further dimerization promoting domain is a carboxy terminal C domain derived from IgG 3.

In one embodiment, the dimerization unit comprises or is derived from the carboxy-terminal C domain of IgG3, which has an amino acid sequence having at least 80% sequence identity to the amino acid sequence of 38-144 of SEQ ID NO. 1.

In a preferred embodiment, the dimerization unit comprises or consists of a carboxy-terminal C domain derived from IgG3, which has an amino acid sequence having at least 85% sequence identity, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity with the amino acid sequence of 38-144 of SEQ ID NO. 1.

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

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 acids 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 with the nucleic acid sequence of SEQ ID NO. 11.

In yet another preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 85% sequence identity with the nucleic acid sequence of SEQ ID NO. 11, 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 a further preferred embodiment, the dimerization unit comprises or consists of the nucleic acid sequence of SEQ ID NO. 11.

Immunoglobulin domains promote dimerization through non-covalent interactions, such as hydrophobic interactions. Thus, in one embodiment, the immunoglobulin domain has the ability to form dimers through non-covalent interactions. Preferably, the non-covalent interactions are hydrophobic interactions.

Preferably, if the dimerization unit comprises a CH3 domain, it does not comprise a CH2 domain and vice versa.

In a preferred embodiment, the dimerization unit comprises hinge exon h1, hinge exon h4, dimerization unit linker and the CH3 domain of human IgG 3. In a preferred embodiment, the dimerization unit comprises a polypeptide consisting of hinge exon h1, hinge exon h4, dimerization unit linker and the CH3 domain of human IgG 3. In another preferred embodiment, the dimerization unit consists of a polypeptide consisting of hinge exon h1, hinge exon h4, dimerization unit linker and the CH3 domain of human IgG 3.

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.

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

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

In a more preferred embodiment, the dimerization unit consists of an amino acid sequence having at least 80% sequence identity, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity to the amino acid sequence of SEQ ID NO. 1.

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

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 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 with the nucleic acid sequence of SEQ ID NO. 12.

In yet another preferred embodiment, the dimerization unit comprises or consists of a nucleic acid sequence having at least 85% sequence identity with the nucleic acid sequence of 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 yet another preferred embodiment, the dimerization unit comprises or consists of the nucleic acid sequence of SEQ ID NO. 12.

In the first polypeptide encoded by the first nucleic acid sequence, the multimerization units, e.g., dimerization units, may have any orientation relative to the antigenic units and targeting units. In one embodiment, the antigenic unit is linked (e.g., via a unit linker) to the C-terminus of the multimerization/dimerization unit and the targeting unit is linked to the N-terminus of the multimerization/dimerization unit. In another embodiment, the antigenic unit is linked (e.g., via a unit linker) to the N-terminus of the multimerization/dimerization unit and the targeting unit is linked to the C-terminus of the multimerization/dimerization unit. Preferably, the antigenic unit is linked to the C-terminus of the multimerization/dimerization unit (e.g. via a linker, preferably via a unit linker), and the targeting unit is linked to the N-terminus of the multimerization/dimerization unit.

Antigenic unit

The first polypeptide encoded by the first nucleic acid sequence comprised in the vector of the invention comprises an antigenic unit comprising one or more T cell epitopes of a self antigen, such as one or more epitopes of regulatory T cells (tregs) or one or more inhibitory neoantigens, allergens, alloantigens or xenogeneic antigens.

T cell epitopes suitable for inclusion in antigenic units may be known in the art, i.e. they have been studied, proposed and/or validated for the involvement of certain immune diseases and for the significance of these diseases and are published, for example, in the scientific literature.

In one embodiment, the antigenic unit comprises one or more T cell epitopes of the self antigen, i.e. one T cell epitope of the self antigen or more than one T cell epitope of the self antigen, i.e. a plurality of T cell epitopes of the self antigen. In one embodiment, the plurality of T cell epitopes belong to the same autoantigen, i.e. are comprised in the same autoantigen. In another embodiment, the plurality of T cell epitopes belong to, i.e. are comprised in, a plurality of different autoantigens.

The terms "plurality," plurality, "and" a plurality "are used interchangeably herein with" more than one.

For example, myelin Basic Protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), myelin Oligodendrocyte Glycoprotein (MOG) and myelin-associated basic oligodendrocyte protein (MOBP) have all been studied and proposed to be autoantigens involved in Multiple Sclerosis (MS), and antigenic units may for example comprise one or more T cell epitopes of MBP, i.e. one T cell epitope of MBP or a plurality of T cell epitopes of MBP. In addition, the antigenic unit may comprise a plurality of T cell epitopes, e.g. MOG and PLP, e.g. one or more T cell epitopes of MOG and one or more T cell epitopes of PLP.

In one embodiment, the antigenic unit comprises one or more T cell epitopes of the allergen, i.e. one T cell epitope of the allergen or more than one T cell epitope of the allergen, i.e. a plurality of T cell epitopes of the allergen. In one embodiment, the plurality of T cell epitopes belong to the same allergen, i.e. are comprised in the same allergen. In another embodiment, the plurality of T cell epitopes belong to a plurality of different allergens, i.e. are comprised in different allergens.

For example, fel d 1, fel d 4 and Fel d 7 are the three most prominent cat allergens, responsible for cat allergy in most humans, and an antigenic unit may comprise, for example, one or more T cell epitopes of Fel d 1, i.e. one T cell epitope of Fel d 1 or a plurality of T cell epitopes of Fel d 1. In addition, the antigenic unit may comprise a plurality of T cell epitopes, e.g. 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.

In one embodiment, the antigenic unit comprises one or more T cell epitopes of an alloantigen/xenogeneic antigen, i.e. one T cell epitope of an alloantigen/xenogeneic antigen, or more than one T cell epitope of an alloantigen/xenogeneic antigen, i.e. a plurality of T cell epitopes of an alloantigen/xenogeneic antigen. In one embodiment, the plurality of T cell epitopes belong to the same alloantigen/alloantigen, i.e. are comprised in the same alloantigen/alloantigen. In another embodiment, the plurality of T cell epitopes belong to, i.e. are comprised in, a plurality of different alloantigens/xenogeneic antigens.

In one embodiment, the antigenic unit comprises a T cell epitope. In another embodiment, the antigenic unit comprises more than one T cell epitope, i.e. a plurality of T cell epitopes.

In one embodiment, the vectors of the invention/constructs encoded by such vectors are used in personalized therapy, i.e. specifically designed for a specific subject/patient. In another embodiment, the vectors of/constructs encoded by such vectors are used universally in a patient population or patient, i.e., in-situ treatment.

Individualised constructs

For an individualized construct, T cell epitopes are selected to incorporate antigenic units that are optimized for the patient to be treated with the vector encoding such construct. This will increase the therapeutic effect compared to spot-product treatments comprising the construct.

Taking MS patients as an example, the antigenic units of the personalized constructs can be designed as follows:

1) Determination of HLA class I and/or HLA class II alleles in a patient

2) Identification of T cell epitopes comprised in one or more autoantigens (e.g., autoantigens that have been studied, proposed and/or validated as autoantigens involved in MS)

3) Selecting T cell epitopes based on predicted binding to HLA class I and/or HLA class II alleles of a patient

4) One or more test constructs are designed and generated, and the T cell epitopes are optionally arranged in the antigenic units of the constructs described herein.

The T cell epitopes selected in the above method are based on their predicted ability to bind to the patient's HLA class I/II alleles, i.e. by computer selection of predictive HLA binding algorithms. After identifying relevant epitopes, the epitopes are ranked according to their ability to bind to the patient's HLA class I/II alleles and the epitope predicted to bind optimally is selected for inclusion in the antigenic unit of the test construct.

Any suitable HLA binding algorithm may be used, such as one of the following:

the available peptide-MHC binding software assays (IEDB, netMHCpan and NetMHCIIpan) can be downloaded from the following websites or used online:

www.iedb.org/

services.healthtech.dtu.dk/service.phpNetMHCpan-4.0

services.healthtech.dtu.dk/service.phpNetMHCIIpan-3.2

tolerance-inducing constructs for off-the-shelf products

The antigenic units of the off-the-shelf constructs encoded by the vectors of the invention may comprise discrete T cell epitopes, hot spots of minimal T cell epitopes, or both. Such antigenic units preferably comprise a hot spot of a minimal T cell epitope, i.e. one or more regions of an antigen containing multiple minimal T cell epitopes (e.g. 7-15 amino acids in length) predicted to be presented by different HLA alleles so as to cover a range of numerous subjects (e.g. ethnic groups or even worldwide groups). By including such hot spots, the chance that the construct will induce tolerance in a range of numerous subjects is maximized.

Other embodiments of the antigenic units

The T cell epitope comprised in the antigenic unit of the first polypeptide encoded by the vector of the invention has a length of 7 to about 200 amino acids, wherein a longer T cell epitope may comprise the hot spot of the smallest T cell epitope.

In one embodiment, the antigenic unit comprises one or more T cell epitopes of 7 to 150 amino acids, preferably 7 to 100 amino acids in length, 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 or 15 to 30 or 20 to 75 amino acids or 25 to 50 amino acids.

T cell epitopes of about 60 to 200 amino acids in length can be split into shorter sequences and incorporated into adaptor-separated antigenic units such as those described herein. For example, a T cell epitope of 150 amino acids in length can be split into 3 sequences of 50 amino acids each and incorporated into an antigenic unit, with a linker separating the 3 sequences from each other.

In one embodiment, the length of a T cell epitope is adjusted such that the protein comprising the T cell epitope is not folded correctly. For example, the most prominent cat allergen Fel d 1 is a protein formed by two heterodimers, each consisting of two chains: chain 1 comprises 70 amino acid residues and chain 2 comprises 90 or 92 residues. Incorporation of the long T cell epitope of both chains into the antigenic unit may result in correct folding of the protein and may trigger an allergic reaction if more than one IgE on the subject's mast cells and basophils binds to the antigenic unit of the construct comprising the T cell epitope.

Thus, if a longer T cell epitope is incorporated into an antigenic unit, protein folding can be tested in vitro by, for example, ELISA, by using an antibody against the protein (e.g., cat allergen) and determining if the antibody binds to the T cell epitope. If the antibody binds to a T cell epitope, its length may be adjusted or it may be divided into shorter sequences as described herein.

In one embodiment, the T cell epitope has a length suitable for presentation by MHC (major histocompatibility complex). There are two main 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 of 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 to 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.

Arrangement and details of T cell epitopes

The number of T cell epitopes in an antigenic unit can vary and depends on the length and number of other elements contained in the antigenic unit (e.g., T cell epitopes as described herein).

In one embodiment, the antigenic unit comprises up to 3500 amino acids, such as from 60 to 3500 amino acids, for example from about 80 or about 100 or about 150 amino acids to about 3000 amino acids, such as from about 200 to about 2500 amino acids, such as from about 300 to about 2000 amino acids, or from about 400 to about 1500 amino acids, or from about 500 to about 1000 amino acids.

In one embodiment, the antigenic unit comprises 1 to 10T cell epitopes, such as 1, 2, 3, 4, 5, 6, 7, 8 or 9 or 10T cell epitopes, or 11 to 20T cell epitopes, such as 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20T cell epitopes, or 21 to 30T cell epitopes, such as 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30T cell epitopes, or 31 to 40T cell epitopes, such as 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40T cell epitopes, or 41 to 50T cell epitopes, such as 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50T cell epitopes.

In one embodiment, the T cell epitopes are randomly arranged in the antigenic unit. In another embodiment, one or more of the following methods of arranging these epitopes in antigenic units may be used.

In one embodiment, the T cell epitopes are arranged in the order of greater antigenicity to lesser antigenicity, in a direction from the multimerization unit (e.g., dimerization unit) to the end of the antigenic unit (see fig. 5). Alternatively, especially where hydrophilicity/hydrophobicity varies greatly between T cell epitopes, then the most hydrophobic T cell epitope may be located substantially at the midpoint of the antigenic unit and the most hydrophilic T cell epitope is closest to the multimerization unit or at the end of the antigenic unit.

Since it is actually possible to center an antigenic unit only if the antigenic unit comprises an odd number of T cell epitopes, the term "substantially" in this context refers to an antigenic unit comprising an even number of T cell epitopes, the most hydrophobic T cell epitope being located as close to the center as possible.

For example, an antigenic unit comprises 5T cell epitopes, which are arranged as follows: 1-2-3 x-4-5; 1. 2, 3 x, 4 and 5 are each different T cell epitopes, -are T cell epitope linkers, which represent the most hydrophobic T cell epitope located in the center of the antigenic unit.

In another example, the antigenic unit comprises 6T cell epitopes arranged as 1-2-3 x-4-5-6 or alternatively as 1-2-4-3 x-5-6; 1. 2, 3 x, 4, 5 and 6 are each T cell epitopes, -are T cell epitope linkers, which represent the most hydrophobic T cell epitope located substantially centrally in the antigenic unit.

Alternatively, T cell epitopes may be alternately arranged between hydrophilic and hydrophobic T cell epitopes. Optionally, the GC-rich sequence encoding the T cell epitope is arranged in a manner that avoids 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.

T cell epitope linker

If the antigenic unit comprises a plurality of T cell epitopes, these epitopes are preferably separated by a T cell epitope linker. This ensures that each T cell epitope is presented to the immune system in an optimal manner. If the antigenic unit comprises n T cell epitopes, it preferably comprises n-1T cell epitope linkers, thereby separating each T cell epitope from one or two other T cell epitopes.

In one embodiment, the T cell epitope linker is designed to be non-immunogenic. The T cell epitope linker may be a rigid linker, meaning that the linker does not allow for substantial free movement of the two amino acid sequences to which it is attached relative to each other. Alternatively, it may be a flexible linker, i.e. a linker allowing the two amino acid sequences to which it is attached to move substantially freely relative to each other. Both types of joints are available. In one embodiment, the linker is a flexible linker allowing for optimal presentation of T cell epitopes to the immune system even though the antigenic unit contains a large number of T cell epitopes.

In one embodiment, the T cell epitope linker is a peptide consisting of 4 to 40 amino acids, e.g., 35, 30, 25 or 20 amino acids, such as 4 to 20 amino acids, e.g., 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.

In one embodiment, all T cell epitope linkers contained in the antigenic unit are identical. However, if one or more of these T cell epitopes comprise a sequence similar to a linker, it may be advantageous to replace adjacent T cell epitope linkers with linkers of a different sequence. In addition, if it is predicted that the T cell epitope/linker linkage itself constitutes an epitope, it is preferred to use T cell epitope linkers of different sequences.

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. The small size of these amino acids provides flexibility and allows for movement of the attached amino acid sequence. The incorporation of serine or threonine can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules and thus reduce adverse interactions between the linker and the antigen. In one embodiment, the flexible linker is a serine (S) and/or glycine (G) rich linker, i.e. a linker comprising several serine residues 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 variants thereof, such as GGGGSGGGGS (SEQ ID NO: 79), (GGGGS) m (SEQ ID NO: 80), (GGGSS) m (SEQ ID NO: 81), (GGSGG) m (SEQ ID NO: 82), (GGGSG) m (SEQ ID NO: 83) or (SGSSGS) m (SEQ ID NO: 84), wherein m is an integer from 1 to 5, such as 1, 2, 3, 4 or 5. 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 1 or at least 2 or at least 3 leucine residues, e.g. 1, 2, 3 or 4 leucine residues.

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).

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).

In another embodiment, the T cell epitope linker is a serine-glycine linker having a length of 10 amino acids and comprising 1 or 2 leucine residues.

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).

In yet another 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).

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 (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) or consists thereof.

In another embodiment, the T cell epitope linker is a rigid linker. Such rigid linkers can be used to isolate (larger) T cell epitopes efficiently 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 SACGELS (SEQ ID NO: 161).

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

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).

In yet another embodiment, the T cell epitope linker is a cleavable linker, e.g., a linker comprising one or more recognition sites for endopeptidases (e.g., endopeptidases such as furin, caspase, cathepsin, etc.).

Examples of T cell epitope linkers are disclosed in WO 2020/176797A1, paragraphs [0098] to [0099] and their cited sequences, in US 2019/0022202A1, paragraphs [0135] to [0139], WO 2017/118695A1 and WO 2021/219897A1, all of which are incorporated herein by reference.

Allergen

Vectors encoding constructs as described herein may be used to induce tolerance against a range of different protein allergens, such as T cell epitopes of allergens that may be encoded by a nucleic acid sequence comprised in a first nucleic acid sequence of the vector of the invention, including protein allergens that undergo post translational modification.

The one or more T cell epitopes comprised in the 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 shellfish allergen. In some embodiments, the allergen is tropomyosin. In other embodiments the allergen is arginine kinase, myosin light chain, troponin C or triose phosphate isomerase or actin. In some embodiments, the allergen is Pan b 1. In some embodiments, the antigenic unit comprises a Pan b 1T 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 1T cell epitopes (241-260), (210-230), (136-155), (76-95), (46-65) and (16-35). In some embodiments, the antigenic units comprise the totality of Met e 1T cell epitopes (241-260), (210-230), (136-155), (76-95), (46-65) and (16-35).

In some embodiments, the allergen is a cow's milk allergen. In 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.

In some embodiments, the allergen is an egg (egg) allergen. In some embodiments, the egg allergen is ovomucoid (ovicloid). In other embodiments, the egg allergen is ovalbumin (ovalbumin), ovotransferrin (ovotransferrin), conalbumin (conalbumin), gal d 3, egg lysozyme or ovomucin.

One T cell epitope known in the art and studied in the context of egg allergy is OVA (257-264) with the amino acid sequence SIINFEKL (SEQ ID NO: 120).

In one embodiment, the antigenic unit of the construct encoded in the vector of the invention comprises the T cell epitope OVA (257-264). Such vectors, or pharmaceutical compositions comprising such vectors, may be used to treat egg allergy.

In some embodiments, the allergen is a fish allergen. In some embodiments, the fish allergen is parvalbumin (parvalbumin). In other embodiments, the fish allergen is enolase, aldolase, or vitellogenin (vitellogenin).

In some embodiments, the allergen is a fruit allergen. In some embodiments, the fruit allergen is a pathogenic related protein 10, a suppressor protein (profilin), a nsLTP, a sweet protein-like protein (thaumatin-like protein), a gibberellin regulator protein, an isoflavone reductase-related protein, a class 1 chitinase, a beta 1,3 glucanase, a germinatin-like protein (germin like protein), an alkaline serine protease, a pathogenic related protein 1, a kiwi protease (actinin), a plant cystatin, kiwellin, a primary latex protein, cupin, or a 2S albumin. In some embodiments, the allergen is a plant allergen. In some embodiments, the plant allergen is a disease process-related protein 10, an inhibitor protein, a type 1 nsLTP, a nsLTP type 2 protein, an osmorexin-like protein, an isoflavone reductase-like protein, a β -fructofuranosidase, a PR protein TSI-1, a cyclophilin, or a FAD-containing oxidase.

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 soybean allergen is a Gly m lectin, a Gly m Bd28K, a Gly m 30kD, a Gly m CPI, or a Gly m TI.

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 woody nut or seed allergen. In some embodiments, the allergen is 11S globulin, 7S globulin, 2S globulin, PR10, PR-14nsLTP, oleosin (oleosin), or an inhibitor protein.

In other embodiments, the food allergy is an allergen selected from the group consisting of buckwheat, celery, pigment additives, eggs, fish, fruits, garlic, gluten, oats, beans, corn, milk, mustard, nuts, peanuts, poultry, rice, sesame, shellfish, soy, woody nuts, and wheat.

In some embodiments, the allergen is a bee venom allergen. In some embodiments, the bee venom allergen is phospholipase A2, hyaluronidase, acid phosphatase, melittin (melittin), allergen C/DPP, CRP/icarapin, or vitellogenin. In some embodiments, the allergen is a wasp allergen (vespid allergy). In some embodiments, the wasp allergen is phospholipase A1, hyaluronidase, protease, antigen 5, DPP IV, or vitellogenin.

In some embodiments, the allergen is a latex allergen (latex allergen). In some embodiments, the latex allergen is a 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.

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 stored dust allergen (storage dust allergen). In some embodiments, the house 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 1T cell epitope (111-139). In some embodiments, the house 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 house dust mite allergen is Blot t 1, blot 2, blot t 3, blot 4, blot 5, blot 8, blot 10, blot 12, or Blot 21.

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 g8, 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.

In some embodiments, the allergen is a mold allergen. In some embodiments, the mold allergen is an aspergillus fumigatus (Aspergillus fumigatus) allergen. In some embodiments, the A.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.

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.

In some embodiments, the allergen is a furred animal allergen. In some embodiments, the allergen is a canine allergen. In some embodiments, the canine allergen is Can f 1, 2, 3, 4, 5, or 6. In some embodiments, the allergen is a equine allergen. In some embodiments, the horse allergen is ecl 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, prealbumin, secretoglobin, or serum albumin.

In some embodiments, the allergen is a pollen allergen. In some embodiments, the allergen is a grass pollen allergen. In some embodiments, the grass pollen allergen is a moxiella (timothy grass), fescue (orchard grass), kentucky grass (Kentucky bluegrass), perennial rye (perennial rye), fescue (sweet vernal grass), bahia grass (bahia grass), johnson grass (johnson grass), or bermuda grass (Bermuda grass allergen) 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.

In some embodiments, the allergen is a tree pollen allergen. In some embodiments, the tree pollen allergen is an alder (alder), birch (birch), hornbeam (hornbeam), hazel (hazel), smut (European hophornbeam), chestnut (chestnut), beech (European beech), white oak (white oak), ash (ash), privet (private), olive (olive), clove (lilac), cypress (cypress), or cedar (cedar) pollen allergen. In some embodiments of the present invention, 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 2, cor a 6, cor a 8, cor a 9, cor a 10, cor a 11, cor a 12, 1Cor a 3, cor a 14, ost c 1, cas 5, cas 8 or Cas 9, fag s 1, que a 1, fra 1, cor Lig v 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 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 1, pla a 2 or Pla a 3. In some embodiments, the antigenic unit comprises a Bet v 1T cell epitope (139-152).

In some embodiments, the allergen is a weed pollen allergen (weed pollen allergen). In some embodiments, the weed allergen is a ragweed (ragweed), mugwort (mugwort), sunflower (sunflower), feverfew (feverfew), wallgrass (pellitory), plantain (engish plantain), arnica (annual starch), quinoa (goose fot), russian thistle (Russian thistle) or amaranth (amaranth polen) 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 mugwort 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 hela 1 or hela 2. In some embodiments, the hay pollen allergen is Par j 1, par j 2, par j 3 or Par j 4. In some embodiments, the plantain pollen allergen is plal 1. In some embodiments, the arnebia pollen allergen is Mer a 1. In some embodiments, the quinoa 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.

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

In some embodiments, the vectors of the present invention may be used to treat an allergic disorder selected from allergic rhinitis, asthma, atopic dermatitis, allergic gastroenteropathy, contact dermatitis, and drug allergy, or a combination thereof.

Allergic reactions to drugs affect more than 7% of the entire population. The vector-encoded constructs of the invention can be used to induce tolerance against immunogenic T cell epitopes present in such drugs and thus allow affected patients to continue and benefit from treatment with the drug.

Thus, in some embodiments, the allergen is comprised in a medicament having an adverse 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 for use in therapy.

Autoantigens

In another embodiment, the vectors of the invention encode constructs comprising one or more T cell epitopes contained in an autoantigen involved in an autoimmune disease. This allows antigen specificity to down-regulate the portion of the immune system responsible for autoimmune disease without inhibiting the immune system as a whole.

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, CNP enzyme, s100deg.P, 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 constructs encoded by the vectors of the invention comprise one or more T cell epitopes derived from one or more of the foregoing autoantigens.

The MS-associated T cell epitope is a T cell epitope from Myelin Oligodendrocyte Glycoprotein (MOG). MOGs are members of the immunoglobulin superfamily and are expressed only in the central nervous system. MOG (35-55) is capable of inducing autoantibody production and relapse remitting neurological disease, resulting in extensive plaque-like demyelination. Autoantibody responses to MOG (35-55) have been observed in MS patients and MOG (35-55) -induced C57/BL6 mice and Lewis rats in Experimental Autoimmune Encephalomyelitis (EAE). Another MOG T cell epitope is MOG (27-63).

Other MS-associated T cell epitopes known in the art and studied include those listed in table 3 below:

TABLE 3 Table 3

* T cell epitope-induced EAE was observed

In a preferred embodiment, the antigenic units of the constructs encoded by the vectors of the invention comprise one or more T cell epitopes selected from 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 to treat MS.

In some embodiments, the autoimmune disease is type 1 diabetes. In some embodiments, the autoantigen is the 65 kilodaltons isoform of glutamate decarboxylase (GAD 65), which is an autoantigen involved in type 1 diabetes. In some other embodiments, the autoantigen is insulin, IA-2, or ZnT8. In some other embodiments, the autoantigen is IGRP, chgA, IAPP, peripherin, tetraspanin-7, GRP78, uremic acid-3, or insulin gene enhancer protein isl-1. In some embodiments, the vector-encoded constructs of the invention comprise one or more T cell epitopes derived from one or more of the foregoing 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 (avenin b). In some embodiments, the antigenic unit comprises the T cell epitope alpha-gliadin (76-95). In some embodiments, the constructs encoded by the vectors of the invention comprise one or more T cell epitopes derived from one or more of the foregoing autoantigens.

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 (HSP 60). In some embodiments, the autoantigen is Band 3. In some embodiments, the autoantigen is nuclear ribonucleoprotein D1 (SmD 1). In some embodiments, the autoantigen is an acetylcholine receptor (AChR). In some embodiments, the autoantigen is myelin protein zero (P0). In some embodiments, the constructs encoded by the vectors of the invention comprise one or more T cell epitopes derived from one or more of the foregoing autoantigens

In one embodiment, the autoimmune disease is chronic inflammatory demyelinating polyneuropathy (chronic inflammatory demyelinating polyradiculoneuropathy, CIDP) and the autoantigen is clusterin 155. In another embodiment, the autoimmune disease is Hashimoto Thyroiditis (HT) and the autoantigen is thyroperoxidase and/or thyroglobulin. In another embodiment, the autoimmune disease is pemphigus larum and the autoantigen is desmosome-associated glycoprotein. In another embodiment, the autoimmune disease is pemphigus vulgaris and the autoantigen is desmoglein 3 (desmoglein 3). In another embodiment, the autoimmune disease is Thyropathy (TED) and the autoantigen is a calbindin (calpain). In another embodiment, the autoimmune disease is Grave's disease and the autoantigen is a thyroid stimulating hormone receptor. In another embodiment, the autoimmune disease is Primary Biliary Cirrhosis (PBC) and the autoantigens are anti-mitochondrial antibodies (AMA), anti-nuclear antibodies (ANA), rim-like/membrane (RL/M) and/or polynuclear dots (MND). In another embodiment, the autoimmune disease is myasthenia gravis and the autoantigen is an acetylcholine receptor. In another embodiment, the autoimmune disease is insulin resistant diabetes and the autoantigen is an 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 depolymerized protein-like metalloprotease domain containing thrombin-sensitive protein type 1 motif-5 (disintegrin-like and metalloprotease domain containing thrombospondin type motif-like 5, ADAMTS L5), phospholipase A2 group IVD (PLA 2G 4D), nuclear heterogeneous ribonucleoprotein A1 (hnRNP-A1), and keratin 17. In another embodiment, the autoimmune disease is rheumatoid arthritis and the autoantigen is selected from citrullinated protein, homocysteine protein and Fc portion of IgG. In some embodiments, the constructs encoded by the vectors of the invention comprise one or more T cell epitopes derived from one or more of the foregoing autoantigens.

Unit joint

The antigenic units are linked to the multimerising units, preferably by means of a unit linker. Thus, in one embodiment, the first nucleic acid sequence comprised in the vector of the invention encodes a unit linker linking the antigenic unit and the multimerization unit.

The unit linker may comprise a restriction site intended to facilitate 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).

Signal peptides

In one embodiment of the disclosure, at least one of the first nucleic acid sequence and the one or more other nucleic acid sequences encoding one or more immunosuppressive compounds further encodes a signal peptide. The signal peptide is located 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. In addition, the signal peptide is located at the N-terminus of the immunosuppressive compound. The signal peptide is designed to allow secretion of the first polypeptide and/or immunosuppressive compound from a cell comprising the vector of the invention. Preferably, each of the first nucleic acid sequence and the other nucleic acid sequences encoding one or more immunosuppressive compounds also encodes a signal peptide.

Any suitable signal peptide may be used. For the first polypeptide, preferably if the targeting unit is an antibody or a part thereof, such as an scFv, an example of a suitable signal peptide is a human Ig VH signal peptide. In one embodiment, the signal peptide is the native leader sequence of the protein as the targeting unit, i.e., the signal peptide naturally occurring at the N-terminus of the protein encoded as the targeting unit in the vector of the invention. Examples of such signal peptides are the signal peptide of human IL-10 (targeting unit is human IL-10) or the signal peptide of human TGF- β1 (targeting unit is human TGF- β1).

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

Thus, in one embodiment, the vector of the invention comprises a first nucleic acid sequence encoding a human IL-10 signal peptide (e.g., the human IL-10 signal peptide of SEQ ID NO: 69). In a preferred embodiment, such vectors comprise a first nucleic acid sequence encoding a human IL-10 targeting unit and also encoding a human IL-10 signal peptide. In another embodiment, the vector of the invention comprises a first nucleic acid sequence encoding a human Ig VH signal peptide (e.g., a human Ig VH signal peptide of SEQ ID NO: 2). In a preferred embodiment, such vectors comprise a first nucleic acid sequence encoding an scFv targeting unit (e.g., an anti-human CD205 targeting unit) and further encoding a human Ig VH signal peptide.

In one embodiment, the vector of the invention comprises a first nucleic acid sequence encoding a signal peptide selected from the group consisting of: human Ig VH signal peptide, hTGF- β1 signal peptide, hTGF- β2 signal peptide, hTGF- β3 signal peptide, hIL-10 signal peptide, hIL-2 signal peptide, hIL-4 signal peptide, hIL-6 signal peptide, hIL-11 signal peptide, hIL-13 signal peptide, hIL-27 signal peptide, hIL-35 signal peptide, hIL-37 signal peptide, hGM-CSF signal peptide, hFLT3L signal peptide, hCL 19 signal peptide, hCL 21 signal peptide, hICAM-1 signal peptide, human keratin signal peptide, hVSIG-3 signal peptide, hSCGB3A2 signal peptide, hCA-4 signal peptide, hPD-1 signal peptide, and hBTLA signal peptide, as set forth in any of the "sequence overview" sections herein.

In another embodiment, the vector of the invention comprises one or more additional nucleic acid sequences encoding one or more immunosuppressive compounds and further encoding a signal peptide selected from the group consisting of: hCLTA-4 signal peptide, hPD-1 signal peptide, hBTLA signal peptide, hLAG3 signal peptide, hNOX2 signal peptide, hSIGLEC7 signal peptide, hSIGLEC9 signal peptide, hTIM-3 signal peptide, hIL-10 signal peptide, hTGF- β1 signal peptide, hTGF- β2 signal peptide, hTGF- β3 signal peptide, hIL-27 signal peptide, hIL-2 signal peptide, hGM-CSF signal peptide, hFLT3L signal peptide, hIFN- γ signal peptide, and hIL-37 signal peptide, and hIL-35 signal peptide, as set forth in any of the foregoing signal peptides in the "sequence overview" section herein.

Sequence identity

Sequence identity can be determined as follows: a high level of sequence identity suggests the possibility that the second sequence is derived from the first sequence. Amino acid sequence identity requires the same amino acid sequence between two aligned sequences. Thus, a candidate sequence sharing 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 may be determined by computer analysis, such as, but not limited to, clustalW computer alignment program (Higgins D., thompson J., gibson T., thompson J.D., higgins D.G., gibson T.J.,1994.CLUSTAL W:improving the sensitivity of progressive multiple sequence alignment through sequence weighting,position-specific gap penalties and weight matrix choicel nucleic Acids Res.22:4673-4680), and default parameters suggested therein. Using this procedure, along with its default settings, the mature (biologically active) portion of the query subject is aligned with the reference polypeptide. The number of fully conserved residues is counted and divided by the length of the reference polypeptide. In this process, any tag sequence or fusion protein sequence that forms part of the query sequence is disregarded in the alignment and subsequent sequence identity determination.

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

Another preferred mathematical algorithm for comparing sequences is the Myers and Miller algorithm, CABIOS (1989). This algorithm was incorporated into the ALIGN program (version 2.0) which was part of the FASTA sequence alignment software package (Pearson WR, methods Mol Biol,2000, 132:185-219). Align calculates sequence identity based on global alignment. Align0 does not penalize gaps in the sequence ends. When amino acid sequences are compared using the ALIGN and ALIGN0 programs, the BLOSUM50 substitution matrix with a gap opening penalty/extension penalty of-12/-2 is preferably used.

Amino acid sequence variants may be prepared by introducing appropriate alterations to the nucleotide sequence encoding the first polypeptide and/or one or more immunostimulatory compounds, or by peptide synthesis. Such modifications include, for example, deletion of residues from within the amino acid sequence and/or insertion of residues into the amino acid sequence and/or substitution of residues in the amino acid sequence. The terms substitution/substitution, deletion/deletion and insertion/insertion as used herein in reference to amino acid sequence and sequence identity are well known and understood to those of skill in the art. Any combination of deletions, insertions and substitutions may be made to obtain the final first polypeptide and/or one or more immunostimulatory compounds, provided that the final protein possesses the desired characteristics. For example, deletion, insertion, or substitution of amino acid residues may result in silent changes and result in functionally equivalent polypeptides/immunostimulatory compounds.

Artificial amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the desired properties/attributes 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; amino acids having uncharged polar head groups with similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

Conservative substitutions, i.e., equivalent substitutions (like-to-like substitution), are contemplated herein, such as basic versus basic, acidic versus acidic, polar versus polar, and the like; and non-conservative substitutions, i.e., substitutions from one type of residue to another or alternatively include the incorporation of unnatural amino acids, such as ornithine, diaminobutyrate ornithine, norleucine, ornithine, pyridylalanine, thienylalanine, naphthylalanine, and phenylglycine. Conservative substitutions that may be made are, for example, within the following groups: 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), hydroxy amino acids (serine, threonine), large amino acids (phenylalanine, tryptophan) and small amino acids (glycine, alanine).

Substitutions may also be made with unnatural amino acids and the substituted residues include α and α -disubstituted amino acids, N-alkylamino acids, lactic acid, halogenated derivatives of natural amino acids such as trifluorotyrosine, p-CI-phenylalanine, p-Br-phenylalanine, p-I-phenylalanine, L-allyl-glycine, β -alanine, L-a-aminobutyric acid, L-y-aminobutyric acid, L-a-aminoisobutyric acid, L-e-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) such as 4-methyl-Phe, pentamethyl-Phe, L-Phe (4-amino) [ L-methyl-Phe ] L-phenyl ] N-phenyl (L-phenyl) amino acid, L-N-phenylamino acid, L-phenyl (L-phenyl) N-phenyl) and L-amino acid N-phenyl (L-phenyl) N-phenyl (L, L-phenyl) amino acid N-phenyl) N.

In the above paragraphs, # represents the hydrophobic nature of the substituent residue and # represents the hydrophilic nature of the substituent residue and # represents the amphipathic nature of the substituent residue. The variant amino acid sequence may comprise suitable spacer groups which may be inserted between any two amino acid residues of the sequence, including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacer groups such as glycine or β -alanine residues. Another form of variation includes the presence of one or more amino acid residues in a peptidomimetic form.

Polypeptides and multimeric/dimeric proteins

The vector of the invention encodes a first polypeptide as described above. As a result of administering the vector to the subject, the polypeptide (and the one or more immunosuppressive compounds) are expressed in vivo.

Due to the presence of multimerization units, such as dimerization units, multimeric proteins are formed upon expression of the polypeptide.

Multimeric proteins may be homomultimers or heteromultimers, for example, if the protein is a dimeric protein, the dimeric protein may be homodimeric, i.e., a dimeric protein in which the two polypeptide chains are identical and thus comprise the same units and thus comprise the same T cell epitope; or the dimeric protein may be a heterodimer comprising two polypeptide chains, wherein polypeptide chain 1 comprises a different T cell epitope in its antigenic unit than polypeptide 2. The latter may be of interest if the number of T cell epitopes incorporated into the antigenic unit exceeds the upper size limit of the antigenic unit. Preferably, the multimeric protein is a homomultimeric protein.

Host cell and vector preparation

The vectors of the invention are generally those suitable for transfecting a host cell and for a) expressing a first polypeptide and forming a multimeric protein consisting of a plurality of such first polypeptides encoded by a first nucleic acid sequence and b) expressing one or more immunosuppressive compounds encoded by other nucleic acid sequences, respectively.

In one embodiment, the host cell comprising the vector of the invention is a cell of a cell culture, such as 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 said subject, i.e. in vivo, as a result of administration of the vector to the subject.

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.

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

Methods of engineering and production of vectors of the invention (e.g., expression vectors, such as DNA and RNA plasmids or viral vectors) are well known and one of ordinary skill in the art can use such known methods to engineer/produce vectors of the invention. In addition, a variety of commercial manufacturers offer carrier design and production services.

In one aspect, the present disclosure relates to a method of producing a vector comprising:

wherein the vector allows co-expression of the first polypeptide and the one or more immunosuppressive compounds as separate molecules, the method comprising:

a) Transfecting cells with the vector in vitro;

b) Culturing the cells;

c) Optionally, lysing the cells to release the carrier from the cells; and

d) The vector or DNA plasmid is collected and optionally purified.

Pharmaceutical composition

In one embodiment of the present disclosure, a vector, such as a DNA plasmid, is used as a drug.

Thus, in one embodiment of the present disclosure, the carrier is provided in a pharmaceutical composition comprising the carrier and a pharmaceutically acceptable carrier or diluent.

Accordingly, in one aspect, the present disclosure relates to a pharmaceutical composition comprising (i) a pharmaceutically acceptable carrier or diluent and (ii) a carrier comprising:

Suitable pharmaceutically acceptable carriers or diluents include, but are not limited to, saline, buffered saline such as PBS, dextrose, water, glycerol, ethanol, isotonic aqueous buffers, and combinations thereof.

In one embodiment, the pharmaceutically acceptable carrier or diluent is an aqueous buffer. In another embodiment, the aqueous buffer is a Tyrode buffer, e.g., comprising 140mM NaCl, 6mM KCl, 3mM CaCl ₂ 、2mM MgCl ₂ Tyrode buffer at 10mM 4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid (Hepes) pH 7.4 and 10mM glucose.

The pharmaceutical composition may comprise a molecule that facilitates transfection of the host cell, i.e. a transfection agent.

The pharmaceutical composition may comprise an adjuvant. In one embodiment, the adjunct is selected from dexamethasone, enterotoxin cholera toxin B subunit (CTB), TLR2 ligand, worm-derived excretion/secretion (ES) product, rapamycin or vitamin D3 analogue and aryl hydrocarbon receptor ligand.

In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable amphiphilic block copolymer comprising blocks of poly (ethylene oxide) and poly (propylene oxide).

An "amphiphilic block copolymer" as used herein is a linear or branched copolymer comprising or consisting of poly (ethylene oxide) ("PEO") blocks and poly (propylene oxide) ("PPO") blocks. Common examples of useful PEO-PPO amphiphilic block copolymers have the general structure PEO-PPO-PEO (poloxamer), PPO PEO PPO, (PEO PPO-) 4ED (poloxamine)) and (PPO PEO-) 4ED (reverse poloxamine), where "ED" is ethylenediamido.

"poloxamer" is a linear amphiphilic block copolymer consisting of one poly (ethylene oxide) block coupled to one poly (propylene oxide) block coupled to one PEO block, i.e., the structure of formula EOa-POb-EOa, wherein EO is ethylene oxide, PO is propylene oxide, a is an integer from 2 to 130, and b is an integer from 15 to 67. Poloxamers are typically named by using a 3-digit numerical identifier, wherein the first 2 digits multiplied by 100 provide an approximate molecular mass of the PPO content, wherein the last digit multiplied by 10 represents an approximate percentage of PEO content. For example, "poloxamer 188" refers to a polymer comprising a PPO block of molecular weight about 1800 (corresponding to b of about 31 PPO) and PEO of about 80% (w/w) (corresponding to a of about 82). However, these values are known to vary to some extent and are commercially available, e.g., at the research level of poloxamer 188 according to the manufacturer's data sheet F68 and clinical grade->P188 showed a large variation in molecular weight (between 7,680 and 9,510) and the a and b values provided for these specific products were approximately 79 and 28, respectively. This reflects the heterogeneous nature of the block copolymer, indicating that the a and b values are the average numbers present in the final formulation.

"Poloxamine" or "sequential Poloxamine" (under the trademark "PolosoxamineCommercially available) are X-block copolymers carrying four PEO-PPO arms attached to a central ethylenediamine moiety through bonds between free OH groups contained in the PEO-PPO-arms and primary amine groups in the ethylenediamine moiety. Reverse poloxamine is also an X-block copolymer that carries four PPO-PEO arms attached to a central ethylenediamine moiety through bonds between free OH groups contained in the PPO-PEO arms and primary amine groups in ethylenediamine.

Preferred amphiphilic block copolymers are poloxamers or poloxamines. Preferred are poloxamers 407 and 188, especially poloxamer 188. The preferred poloxamine is the sequential poloxamine of formula (PEO-PPO) 4-ED. Particularly preferred is that the poloxamer is under the registered trademark respectively904. 704 and 304. These poloxamines are characterized as follows: 904 has a total average molecular weight of 6700, a total average molecular weight of PPO units of 4020, and a PEO percentage of about 40%; />704 has a total average molecular weight of 5500, a PPO unit total average molecular weight of 3300, and a PEO percentage of about 40%; and->304 has a total average molecular weight of 1650, a total average molecular weight of PPO units of 990, and a PEO percentage of about 40%.

In one embodiment, the pharmaceutical composition comprises the amphiphilic block copolymer in an amount of 0.2% w/v to 20% w/v, such as 0.2% w/v to 18% w/v, 0.2% w/v to 16% w/v, 0.2% w/v to 14% w/v, 0.2% w/v to 12% w/v, 0.2% w/v to 10% w/v, 0.2% w/v to 8% w/v, 0.2% w/v to 6% w/v, 0.2% w/v to 4% w/v, 0.4% w/v to 18% w/v, 0.6% w/v to 18% w/v, 0.8% w/v to 18% w/v, 1% w/v to 18% w/v, 2% w/v to 18% w/v, 1% w/v to 5% w/v, or 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, such as about 3% w/v.

The pharmaceutical composition may be formulated in any manner suitable for administration to a subject, for example, as a liquid formulation for injection, for example, for intradermal or intramuscular injection.

The pharmaceutical composition may be administered to the subject in any manner suitable for administration to the subject, such as by intradermal, intramuscular or subcutaneous injection, or by mucosal or epithelial administration, such as intranasal or oral administration.

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

The amount of vector (e.g., DNA plasmid) in the pharmaceutical composition may vary depending on whether the administration of the pharmaceutical composition is prophylactic or therapeutic treatment.

The pharmaceutical compositions of the invention generally comprise 0.1 to 10mg, for example about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1mg or e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10mg of a vector, such as a DNA plasmid.

In a preferred embodiment, the pharmaceutical composition is a sterile pharmaceutical composition.

Treatment of

In some aspects of the disclosure, vectors (e.g., DNA plasmids) are used for prophylactic or therapeutic treatment of conditions involving undesired immune responses, i.e., immune diseases, such as for prophylactic or therapeutic treatment of autoimmune diseases, allergic diseases, and transplant rejection.

Thus, in a further aspect, the present invention provides a method of treating a subject suffering from or in need of prophylaxis of an immune disorder selected from the group consisting of autoimmune disorders, allergic disorders and graft rejection, comprising administering to the subject a vector comprising:

In one embodiment, the invention provides a method of treating a subject suffering from or in need of prevention of an autoimmune disease, the method comprising administering to the subject a vector comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerization unit (e.g., a dimerization unit), and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen; and

In one embodiment, the invention provides a method of treating a subject having an allergic disease or in need of prevention of the allergic disease, the method comprising administering to the subject a vector comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerization unit (e.g., a dimerization unit), and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of an allergen; and

In one embodiment, the invention provides a method of treating a subject suffering from or in need of prevention of graft rejection, the method comprising administering to the subject a carrier comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerizing unit (e.g., a dimerization unit), and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of an alloantigen or a xenogeneic antigen; and

In the method of treatment, the carrier is preferably administered in a therapeutically or prophylactically effective amount. Such amounts of carrier may be administered in one administration, i.e. one dose, or in several administrations, i.e. repeated doses, i.e. in a series of doses, e.g. over a period of days, weeks or months or years.

The actual dosage administered can vary and depends on whether the treatment is prophylactic or therapeutic, the severity of the immune disorder being treated, parameters such as age, weight, sex, medical history, preexisting condition and general condition of the subject, and the judgment of the health care professional.

In the methods of treatment, the carrier may be administered in the form of a pharmaceutical composition and in the modes of administration described herein.

The treatment method of the present invention may continue as long as the clinician supervising patient care recognizes that the method is effective and requires treatment.

Indicators of therapeutic success are known in the art and include elevated antigen-specific regulatory T cell levels, reduced antigen-specific effector T cell levels (and elevated regulatory T cell levels), reduced effector T cell levels, reduced T cell activation levels (in ELISPOT, upon stimulation with an antigenic unit/T cell epitope in an antigenic unit), reduced basophil activation levels (in bas).

Likewise, a radioallergen adsorption assay (radioallergosorbent test, RAST) can be used to compare the level of allergen-specific IgE antibodies in blood samples from subjects before and after administration of the immunotherapeutic constructs, wherein lower allergen-specific IgE antibody levels are indicative of successful induction of tolerance.

Also disclosed herein is a vector comprising:

wherein the vector allows co-expression of the first polypeptide and the one or more immunosuppressive compounds as separate molecules for use in treating a subject suffering from or in need of prevention of an immune disorder selected from the group consisting of autoimmune disorders, allergic disorders and graft rejection, wherein the vector is administered to the subject.

Also disclosed herein is the use of a vector for the preparation of a medicament, the vector comprising:

wherein the vector allows co-expression of the first polypeptide and the one or more immunosuppressive compounds as separate molecules, the medicament for treating a subject suffering from or in need of prevention of an immune disorder selected from the group consisting of autoimmune disorders, allergic disorders, and graft rejection, wherein the medicament is administered to the subject.

Also disclosed herein is the use of a vector comprising:

wherein the vector allows co-expression of the first polypeptide and the one or more immunosuppressive compounds as separate molecules for use in treating a subject suffering from or in need of prophylaxis of an immune disorder selected from the group consisting of autoimmune disorders, allergic disorders and graft rejection.

Also disclosed herein are vectors comprising:

wherein the vector allows co-expression of the first polypeptide and the one or more immunosuppressive compounds as separate molecules when used in the therapeutic or prophylactic treatment of an immune disorder selected from autoimmune disorders, allergic disorders and graft rejection.

Also disclosed herein is the use of a vector comprising:

wherein the vector allows co-expression of the first polypeptide and the one or more immunosuppressive compounds as separate molecules for therapeutic or prophylactic treatment of an immune disorder selected from autoimmune disorders, allergic disorders and graft rejection.

Also disclosed herein is a medicament for treating or preventing an immune disease selected from autoimmune disease, allergic disease, and graft rejection in a subject suffering from or in need of prevention of such an immune disease by administering to the subject a vector comprising:

Examples

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

Example 1：

A DNA vector was designed and generated comprising a nucleotide sequence encoding murine Myelin Oligodendrocyte Glycoprotein (MOG) 27-63 and further encoding the following elements/units:

TABLE 4 Table 4

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* The MOG (27-63) sequence was obtained from Krienke et al, science 371,145-153,2021.

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 described above comprises the 35-55T cell epitope of the immunodominant (immunodomino) of MOG, MOG (35-55), which is the primary target of 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.

Hereinafter, "m", "mouse" and "mouse" are used interchangeably, and "h" and human are used interchangeably.

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

The DNA vector VB5052 (SEQ ID NO: 4) encodes a first polypeptide comprising a human macrophage inflammatory protein alpha variant targeting unit (hCCL 3L1, also known as LD78 beta or hMIP1 alpha) which is known to target APCs in an immunogenic manner, i.e. a first polypeptide/dimer protein comprising such targeting unit will induce a pro-inflammatory immune response in the subject to which they are administered (see e.g. WO 2011/161244 A1). VB5052 is the "pro-inflammatory form" of VB5049 and is used as a comparison of the construct of the invention.

The DNA vector VB5051 (SEQ ID NO: 5) encodes only the antigenic unit, namely MOG (27-63); single protein/peptide. VB5051 was used as a comparison for the construct of the invention.

Production of DNA vectors

All DNA vectors in this example section were generated by: sequences as described in the tables of this example section were ordered from netherlands Genscript Biotech b.v. and cloned into the expression vector pALD-CV77 (DNA plasmid).

Example 2：

The purpose of this study was to characterize the protein expression and secretion of the proteins encoded by DNA vectors VB5049 and VB5052 after transient transfection of mammalian cells.

HEK293 cells were obtained from ATCC and transiently transfected with DNA vector VB5049 or VB 5052. Briefly, 2x10 ⁵ Individual cells/well were plated in 24-well tissue culture plates containing 10% FBS growth medium and used2000 reagent (Invitrogen, thermo Fischer Scientific) was transfected with 1. Mu.g of the corresponding DNA vector. The transfected cells were then incubated with 5% CO ₂ Cell supernatants were then collected for 5 days at 37 ℃ to characterize secreted proteins encoded by VB5049 x or VB5052 x by sandwich ELISA on supernatants using anti-hIgG CH3 domain antibodies (detection antibody, 100 μl/well, 0.1 μg/ml mouse anti-human IgG Fc secondary antibody, biotin (05-4240, invitrogen)) and anti-MOG antibodies (capture antibody, 100 μl/well, 0.25 μg/ml mouse anti-MOG antibody (NYRMOG, sc-73330,Santa Cruz Biotechnology)). The results are shown in fig. 6A.

Secretion of IL-10 encoded by VB5049 was measured by sandwich ELISA using anti-mouse IL-10 antibodies (capture antibody, 100. Mu.l/well, 0.4. Mu.g/ml rat anti-mouse IL-10 antibodies (MAB 417, R & D Systems), detection antibody, 100. Mu.l/well, 0.2. Mu.g/ml goat anti-mouse IL-10 biotinylated antibodies (BAF 417, R & D Systems)). The results are shown in fig. 6B.

The secretion of the first polypeptide and IL-10 as two separate proteins from VB5049 was shown by sandwich ELISA using anti-murine MOG antibody (capture antibody, 100 μl/well, 0.25 μg/ml mouse anti-MOG antibody (NYRMOG, sc-73330,Santa Cruz Biotechnology)) and anti-murine IL-10 antibody (detection antibody, 100 μl/well, 0.2 μg/ml goat anti-mouse IL-10 biotinylated antibody (BAF 417, R & DSystems)). The results are shown in fig. 6C.

Secretion of the first polypeptide encoded by VB5052 was further shown by sandwich ELISA using anti-IgG CH3 domain antibodies (capture antibody, 100 μl/well, 1 μg/ml, mouse anti-human IgG (CH 3 domain), 153272, biorad) and anti-hCCL 3L1 antibodies (detection antibody, 100 μl/well, 0.2 μg/ml, goat anti-human CCL3L1 biotin antibody). The results are shown in fig. 6D.

As demonstrated by 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 CCL3L 1), both first polypeptides encoded by VB5049 and VB5052 respectively were secreted at high levels.

FIG. 6B shows IL-10 secretion levels in undiluted supernatants. The secretion level in the supernatant was estimated to be 2.25. Mu.g/ml by comparing the dilution curve of the supernatant with that of recombinant IL-10.

FIG. 6C shows ELISA results obtained with anti-MOG antibodies as capture antibodies and anti-murine IL-10 antibodies as detection antibodies. As expected, VB5049 produced a very weak signal, confirming that the first polypeptide and IL-10 were expressed as two separate molecules due to the presence of the T2A peptide.

The results depicted in fig. 6A-D show that: the first polypeptide/dimer protein (comprising targeting unit, dimerization unit and antigenic unit) encoded by DNA vectors VB5049 and VB5052 is secreted from transfected cells and for VB5049 IL-10 is also expressed and secreted as a separate protein.

To further characterize the expressed proteins, western Blot (WB) analysis was performed. Briefly, expi293F cells (3 x10 ⁶ Individual cells/ml, 1.6 ml) were inoculated into 6-well plates. Cells were transfected with 1 μg/ml plasmid DNA using the ExpiFectamine 293 reagent (Thermo Fisher sci.) and the plates were plated in humidified CO ₂ Cell incubator (8% CO) ₂ Incubation on a rotary shaker (19 mm diameter, 125 rpm) at 37 ℃. After 18 hours of incubation, an expictamine 293 transfection enhancer (Thermo Fisher sci.) was added to each well. Plates were incubated for an additional 28 hours, after which time supernatants were harvested.

Samples were prepared by mixing 105 μl of supernatant from transfected Expi293F cells with 37.5 μl of 4 xloemmli sample buffer (Bio-Rad) containing 7.5 μl of DTT (Thermo Fisher Sci.) or 7.5 μl of ultrapure water (for reducing and non-reducing conditions, respectively). The sample (reduced or non-reduced) was heated at 70℃for 10 minutes, after which 10. Mu.l to 4% -20% Criterion TGX dye free pre-gel (Bio-Rad) was added. SDS-PAGE was performed using Precision Plus Protein All Blue pre-stained protein standard (Bio-Rad) in 1 XTris/glycine/SDS running buffer (Bio-Rad). Proteins were transferred from the gel onto EtOH-activated Low Fluorescence (LF) 0.45 μm PVDF membrane (Bio-Rad) using a Tran-Blot Turbo semi-dry transfer system (Bio-Rad). PVDF membrane was blocked in EveryBlot buffer (Bio-Rad) for 5 min and used with mouse anti-MOG antibody (sc-73330,Santa Cruz Biotechnology) and rat anti-mouse IL-10 antibody (MAB 417, R)&D Systems) to detect the first polypeptide/dimer protein and IL-10, respectively. The membrane was incubated with the fluorochrome conjugated secondary antibody for 1 hour at room temperature, followed by washing and drying. By using Chemidoc ^TM MP imaging system (set Dyight 488 and 800, auto-optimal) acquires images.

Figures 7A and 7B show successful expression and secretion of the first polypeptide/dimer protein encoded by VB5049 and VB5052 and IL-10 encoded by VB 5049.

Fig. 7A shows that proteins expressed by VB5052 and VB5049, secreted from transfected cells, and electrophoresed on SDS-PAGE under reducing and non-reducing conditions can be detected. Membranes were probed with mouse anti-mouse MOG. Under reducing conditions, the first polypeptide encoded by VB5052 is detected as a monomer (31 kDa), whereas under non-reducing conditions it is detected as both a monomer (31 kDa, first polypeptide) and a dimer (61 kDa, dimer protein). Under reducing conditions, the first polypeptide encoded by VB5049 is detected as a monomer (51 kDa), whereas under non-reducing conditions it is detected as both a monomer (51 kDa, first polypeptide) and a dimer (101 kDa, dimer protein).

FIG. 7B shows the detection of IL-10 (18 kDa). No 71kDa protein was detected, indicating that IL-10 was expressed as a separate protein from VB 5049.

In summary, ELISA data and western blot data show that by using T2A peptide as a co-expression element, the complete first polypeptide/dimer protein can be co-expressed with mll 10 from DNA vectors.

Example 3：

The tolerance induction capacity of VB5049 was determined by calculating the ratio of IL-10/IFN- γ from measurements of IL-10 (anti-inflammatory cytokine associated with immune tolerance) and IFN- γ (marker of induction of inflammatory immune response). This ratio describes to what extent the immune response induced by VB5049 is biased towards tolerogenic characteristics.

The following study design was applied:

female 6 week old C57BL/6 mice were obtained from Janvier Labs (France). All animals were kept in the animal facility of radio Hospital (Norway Olympic). All animal protocols were approved by the norwegian food safety authorities (norwegian oslo). 5 mice/group were used for testing VB5049, VB5052 and VB5051, while 2 mice/group were used for negative control (PBS only). VB5052 is a pro-inflammatory form of VB5049 and is included as a comparison of VB 5049. The pro-inflammatory form of VB5049 x, VB5052 x, is expected to induce IFN- γ production. VB5051 is included as a comparison of VB 5049.

A dose of 50. Mu.g of the corresponding DNA vector dissolved in sterile PBS was administered to the interior of each tibialis muscle (2X 25. Mu.l, 1000. Mu.g/ml) by intramuscular needle injection followed by electroporation using the AgileP in vivo electroporation system (BTX, USA).

Spleens were collected 7 days after administration and triturated in a cell strainer to obtain a single cell suspension. Erythrocytes were lysed using potassium Ammonium Chloride (ACK) lysis buffer. After washing, spleen cells were counted using a NucleoCoulter NC-202 (ChemoMetec, denmark),re-suspended to a final concentration of 6x10 ⁶ Individual cells/ml, and at 6x10 ⁵ Individual cells/well were seeded in 96-well IFN- γ/IL-10 bicolor FluoroSpot plates. Spleen cells were then re-stimulated with 16.67 μg/ml MOG (35-55) peptide for 44 hours before testing for IFN- γ and IL-10 cytokine production in two-color FluoroSpot according to the manufacturer's protocol (Mabtech AB, sweden). Spot forming cells were measured in IRIS FluoroSpot and ELISpot reader (Mabtech AB) and analyzed using Apex software (Mabtech AB). The results were shown to be IL-10+ or IFN-gamma+ spots/10 ⁶ Average number of triplicate of individual splenocytes.

As can be seen from fig. 8, the IL-10/IFN- γ ratio obtained with VB5049 was high, indicating that VB5049 induces significantly higher levels of immunosuppressive cytokine IL-10 than inflammatory cytokine IFN- γ. This is not the case for VB5052, which shows an IL-10/IFN-gamma ratio of about 1, indicating that both cytokines are produced at equivalent levels after restimulation with MOG (35-55) peptide.

Total spleen cells obtained from mice administered with DNA vectors were also analyzed by polychromatic flow cytometry. Briefly, cells were re-stimulated with MOG (35-55) peptide for 16 hours, then harvested and counted. 2x10 ⁶ Individual cells/samples were used for flow cytometry analysis. Cells were first stained with an fixable vital dye (eFluor 780) for 10 minutes at room temperature in the dark. Thereafter, the cells were washed in 1 XPBS (centrifugation (400 g/10 min/4 ℃) and incubated with surface staining antibody (Ab) mixtures (anti-CD 3 BUV395, anti-CD 4BV785 and anti-CD 8) at 4℃for 30 min in the dark. After incubation, cells were washed and cell pellet resuspended in flow buffer (PBS, 10% FBS and 2mM EDTA). The cells were then fixed and permeabilized in the dark at 4 ℃ (eBioscience ^TM Foxp 3/transcription factor staining buffer set) for 45-60 minutes. Subsequently, the cells were washed in 1x permeabilization buffer, centrifuged as described previously, and the cell pellet was resuspended in an intracellular staining Ab mixture (anti-IFN-gamma APC, anti-IL-17Alexa fluor 488, BV421, anti-Foxp 3 PE). The cells were incubated with the intracellular staining Ab mixture for 30 minutes at 4 ℃ in the dark. The cells were then washed and resuspended in streaming buffer until streaming fine at BD Symphony A5Flow cytometry was performed on the cytometer. Compensation was set using single stain Ultra comp eBead for fluorescent dye conjugated abs and ArC reactive beads for fixable vital dyes. Flow cytometry files were analyzed using FlowJo software.

IFN-gamma and IL-17 are both pro-inflammatory cytokines contributing to the pathogenesis of chronic inflammatory and autoimmune diseases, including Experimental Autoimmune Encephalomyelitis (EAE) and multiple sclerosis. Thus, the tolerance-inducing construct must reliably induce tolerance without inadvertently sensitizing (sendize) the autoimmune response of the autoantigen (e.g., by inducing pro-inflammatory cytokines that may exacerbate autoimmunity).

Figures 9A and 9B show the lack of IFN- γ production upon administration of VB5049 as observed in the FluoroSpot assay by flow cytometry validation. In addition, flow cytometry also demonstrated that VB5049 did not induce IL-17 production. Conversely, as might be expected, administration of pro-inflammatory VB5052 would induce IFN- γ and IL-17 production. VB5051 administration induced some level of IFN-gamma production in spleen cells, but induced very low levels of IL-17.

The generation of MOG-specific regulatory T cells (tregs), i.e. T cells that act to suppress and control MOG-specific inflammatory immune responses and thus maintain self-tolerance, was determined by MOG-specific tetramer staining and flow cytometry (cd4+mog (35-55) -tet+foxp3+ cells).

Briefly, 1 to 2x10 will be pooled from each group ⁶ Individual spleen cells were transferred to 96-well V-bottom plates. Tetramers and antibodies were diluted in PBS containing 5% FBS and protected from light prior to use. All steps requiring cell washing were performed using PBS containing 5% FBS, unless otherwise specified. First, cells were treated with a T-Select MHC class II tetramer (10. Mu.l/well, H-2IAb MOG (35-55) tetramer-PE, TS-M704-1,MBL International Corporation) specific for MOG (35-55) or MOG (38-49) according to the manufacturer's instructionsMHC class II tetramer (1. Mu.g/ml, H-2IAb-GWYRSPFSRVVH->Tetrameric PE,2958, promimune). Without washing the cells, the FC receptor was blocked on ice for 5 min to prevent flow cytometry antibodies (Ab) from binding to FC receptor (0.25 μg/ml, truStain FcX ^TM Non-specific binding of PLUS (anti-mouse CD 16/32) antibodies, 156604, bioLegend). Cells were stained on ice for 30 min with a surface Ab mixture containing anti-mouse CD8 PE-Cy7 (0.25. Mu.g/ml, clone: 53-6.7, 100721,BD Biosciences), anti-mouse CD4 eFluor450 (0.25. Mu.g/ml, clone: GK1.5, 48-0041-82,Thermo Fischer/eBioscience) and anti-mouse CD25 PerCP-Cy5.5 (0.25. Mu.g/ml, clone: PC61, 102030, bioLegend). Cells were washed twice with PBS. Next, cells were stained with a fixable vital dye (150 μl/well, 1:8000 diluted in PBS, fixable vital dye 780, 565388,BD biosciences) for 10 minutes on ice. Cells were washed twice with PBS containing 5% FBS and fixed and permeabilized using Foxp 3/transcription factor staining buffer set (200. Mu.l/well, 00-5523-00,Thermo Fischer/eBioscience) according to the manufacturer's instructions. Cells were washed and stained with an intracellular Ab mixture containing anti-mouse FOXP3eFluor 660 (0.25. Mu.g/ml, clone: FJK-16s,50-5773-82,Thermo Fischer/eBioscience) and anti-mouse Ki-67 Alexa Fluor 488 (0.25. Mu.g/ml, clone: 11F6, 151204, biolegend) for 30 min on ice. Cells were washed twice, resuspended in 200. Mu.l of 5% FBS-containing PBS, and used for BD FACSymphosy ^TM A3 cell analyzer was used for analysis. The following controls were used as the controls using FlowJo ^TM Guidance of target cell populations gating by v10.8 software (BD Life Sciences): unstained controls (cells not treated with Ab) and fluorescence minus one (Fluorescence Minus One, FMO) controls (samples stained with all fluorophore-labeled Ab used herein, minus one fluorophore to be gated).

As shown in fig. 10, a higher percentage of MOG (35-55) -specific foxp3+ cells were detected after a single administration of VB5049 x compared to VB5052 x and VB5051 x. For VB5052, the MOG-specific Treg observed was the result of a natural feedback loop of VB 5052-stimulated inflammatory immune response (as discussed in more detail in example 5).

Example 3 thus demonstrates that VB5049 induces a higher anti-inflammatory/inflammatory cytokine ratio (IL-10/IFN- γ) than the pro-inflammatory control construct VB5052 and shows a lack of inflammatory IFN- γ production. Example 3 further shows that VB5049 induces higher numbers of MOG (35-55) -specific foxp3+ cells than either VB5052 or VB 5051. Taken together, these results demonstrate that administration of VB5049 elicits a greater antigen-specific tolerogenic response than administration of VB5051 and VB 5052.

Example 4

Designing and generating a DNA vector comprising a nucleotide sequence encoding MOG (27-63) and further encoding the following elements/units:

TABLE 5

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In addition, DNA vectors VB5049 (SEQ ID NO: 32), VB5052 (SEQ ID NO: 33) and VB501 (SEQ ID NO: 34) were designed and produced. These vectors are identical to VB5049, VB5052 and VB501 (table 4) but comprise the nucleotide sequence encoding MOG (27-63) of SEQ ID No. 18, but not the nucleotide sequence encoding MOG (27-63) of SEQ ID No. 16.

The DNA vectors VB5049, VB5052 and VB5062-VB5065 encode the same first polypeptide comprising an antigenic unit with MOG (27-63):

VB5049 additionally codes for mIL-10, the latter expressed as a separate molecule

VB5062 additionally codes for mTGF-. Beta.1, the latter expressed as a separate molecule

VB5063 additionally encodes the extracellular domain of mCTLA-4, which is expressed as a separate molecule

VB5064 additionally codes for mIL-2, the latter expressed as a separate molecule

VB5065 additionally encodes mIFN-gamma expressed as a separate molecule

VB5048 encodes only the first polypeptide but not the immunosuppressive compound. It serves as a comparison with the vector of the invention

VB5052 is the "pro-inflammatory" form of the DNA vector listed above and serves as a comparison with the vector of the invention

VB5051 encodes only MOG (27-63) and serves as a comparison with the vector of the invention

Characterization of expression and secretion of proteins encoded by DNA vectors

Expression and secretion of proteins encoded by VB5049, VB5052, VB5062, VB5063, VB5064 and VB5065 were characterized as follows:

briefly, expi293F cells (1.7x10 ⁶ Individual cells/ml, 1 ml) were inoculated into 96-well culture plates. Cells were transfected with 0.64 μg/ml plasmid DNA using the ExpiFectamine 293 reagent (Thermo Fisher Sci.) and the plates were incubated in humidified CO ₂ Cell incubator (8% CO) ₂ Incubation on a rotary shaker (3 mm diameter, 900 rpm) at 37 ℃. Supernatants were harvested 72 hours post transfection.

Sandwich ELISA was performed in the supernatant to characterize secreted first polypeptide/dimer protein using anti-MOG antibodies (capture antibody, mouse anti-MOG antibody, 0.25 μg/ml,100 μl/well, sc-73330,Santa Cruz Biotechnology) and anti-IgG CH3 domain antibodies (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 polypeptide/dimer proteins were highly expressed and secreted.

The expression and secretion of the encoded immunosuppressive compounds mIL-10, mTGF- β1, mCTLA-4, mIL-2 and mIFN- γ were characterized by sandwich ELISA using antibodies against mIL-10, anti-hTGF- β1 (also binding mTGF- β1), anti-mCTLA-4, anti-mIL-2 and anti-mIFN- γ, respectively. The results are shown in FIGS. 12A-12E.

The following antibodies were used:

(A) Capture antibody: 1. Mu.g/ml rat anti-murine IL-10 antibody, 100. Mu.l/well, MAB417, R & D Systems. Detection of antibodies: 0.2. Mu.g/ml, goat anti-murine IL-10 biotinylated antibody, 100. Mu.l/well, BAF417, R & D Systems.

(B) Capture antibody: 2. Mu.g/ml TGF-. Beta.1 antibody, 100. Mu.l/well, MAB2402, RD Systems. Detection of antibodies: 0.8 μg/ml chicken anti-human TGF-beta 1 biotinylated antibody, 100 μl/well, BAF240, RD Systems.

(C) Capture antibody: goat anti-murine CTLA-4 antibody at 0.8. Mu.g/ml, 100. Mu.l/well, AF476, RD Systems. Detection of antibodies: goat anti-murine CTLA-4 biotinylated antibody at 0.8. Mu.g/ml, 100. Mu.l/well, BAF476, RD Systems.

(D) Capture antibody: 2. Mu.g/ml rat anti-murine IL-2 antibody, 100. Mu.l/well, 503701, bioLegend. Detection of antibodies: 2. Mu.g/ml rat anti-murine IL-2 biotinylated antibody, 100. Mu.l/well, 503803, biolegend.

(E) Capture antibody: 2. Mu.g/ml rat anti-murine IFN-. Gamma.antibody, 100. Mu.l/well, 505502, bioLegend. Detection of antibodies: 2. Mu.g/ml rat anti-murine IFN-. Gamma.biotinylated antibody, 100. Mu.l/well, 505704, bioLegend.

As shown in fig. 12A-E, all five immunosuppressive compounds were expressed and secreted from their respective vectors.

The expression and secretion of the first polypeptide/dimer protein and the immunosuppressive compound as separate proteins were verified by sandwich ELISA using anti-MOG antibodies and antibodies to the immunosuppressive compounds mIL-10, mTGF- β1, mCTLA-4 and mIL-2, respectively. The results are shown in FIGS. 13A-13D.

The following antibodies were used:

capture antibody: mouse anti-MOG antibody, 0.25. Mu.g/ml, 100. Mu.l/well, sc-73330,Santa Cruz Biotechnology. Detection of antibodies:

(A) Goat anti-mouse anti-IL-10 biotinylated antibody at 0.2. Mu.g/ml, 100. Mu.l/well, BAF417, R & D Systems.

(B) 0.8 μg/ml chicken anti-human TGF-beta 1 biotinylated antibody, 100 μl/well, BAF240, RD Systems.

(C) Goat anti-mouse CTLA-4 biotinylated antibody at 0.8. Mu.g/ml, 100. Mu.l/well, BAF476, RD Systems.

(D) 2. Mu.g/ml rat anti-mouse IFN-. Gamma.biotinylated antibody, 100. Mu.l/well, 505704, bioLegend.

As shown in fig. 13A-D, the first polypeptide and the immunosuppressive compound are not expressed as fusion proteins, but are expressed and secreted as separate molecules.

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 encoding the same first polypeptide but encoding different immunosuppressive compounds. VB5048 encoding the same first polypeptide as the aforementioned DNA vector but not encoding an immunosuppressive compound was included as a comparison.

Samples (expanding total specimen volume at a given ratio) were prepared by mixing 14. Mu.l of supernatant from transfected Expi293F cells with 5. Mu.l of 4 XLaemmli sample buffer (Bio-Rad) containing 1. Mu.l of DTT (Cayman Chemical) or 1. Mu.l of ultrapure water (for reducing and non-reducing conditions, respectively). The sample (reduced or non-reduced) was heated at 70 ℃ for 10 minutes before being added to 4% -20% Criterion TGX dye-free pre-gel (Bio-Rad). SDS-PAGE was performed using Precision Plus Protein All Blue pre-stained protein standard (Bio-Rad) in 1 XTris/glycine/SDS running buffer (Bio-Rad). Proteins were transferred from the gel onto EtOH-activated Low Fluorescence (LF) 0.45 μm PVDF membrane (Bio-Rad) using a Trans-Blot Turbo semi-dry transfer system (Bio-Rad). PVDF membrane was blocked in EveryBlot buffer (Bio-Rad) for 5 min and used with mouse anti-MOG antibody (sc-73330,Santa Cruz Biotechnology), rat anti-mouse IL-10 antibody (MAB 417, R)&DSsystems), goat anti-murine CTLA-4 (AF 467, R&D Systems) or rat anti-murine IL-2 (503702) to detect MOG, mIL-10, mCTLA-4 or mIL-2, respectively. The membrane was incubated with a fluorescent dye conjugated species-specific secondary antibody for 1 hour at room temperature, followed by washing and drying. To detect mIL10 in Dyight channel 488, the membrane was re-probed with Dyight-488 secondary antibody. The membrane was re-activated in ethanol and TBST. The membrane was blocked and the secondary antibody conjugated with Dyight 488-was incubated for 1 hour at room temperature, followed by washing and drying. By using Chemidoc ^TM The MP imaging system acquires images.The results are shown in fig. 14.

Western blot analysis validated the ELISA results, indicating that VB5049, VB5062, VB5063 and VB5064 express two proteins: the first polypeptide/dimer protein (FIGS. 14A and B, reducing and non-reducing conditions) and an immunosuppressive compound as the second protein, namely mIL-10 (FIG. 14C), mCTLA-4 (FIG. 14D) or mIL-2 (FIG. 14E). Importantly, no additional bands were observed for the anti-IL-10, anti-CTLA-4 and anti-IL-2 probed membranes, indicating that the ribosome was successfully hopped at the T2A sequence, allowing the first polypeptide and immunosuppressive compound to be expressed as separate proteins from a single DNA plasmid.

In summary, ELISA data and western blot data show that by using T2A peptide as co-expression element, the complete second multimeric protein comprising targeting unit, dimerization unit and antigenic unit can be co-expressed together with immunosuppressive compound from DNA plasmid.

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 previously described in this example 4.

Secretion of MOG (27-63) peptides was characterized by direct ELISA by coating with supernatant and detection using anti-MOG antibodies (capture antibody, 100 μl/well, 3.3 μg/ml mouse anti-MOG antibody, sc-73330,Santa Cruz Biotechnology). FIG. 15 shows that MOG (27-63) peptide is expressed from VB5051 and secreted from mammalian cells transfected with the vector.

Example 5：

Tolerance induction capacity of VB5049 was assessed in the spleen from mice administered with VB5049 (see example 4) and was determined from IL-10/IFN- γ ratio calculated from IL-10 (anti-inflammatory cytokine associated with immune tolerance) signal and IFN- γ (marker inducing inflammatory immune response) signal generated in a two-color FluoroSpot assay after restimulation with MOG (35-55) peptide and from detection of cd4+foxp3+ T cells. Further, the absence of IFN-gamma and IL-17 production (two pro-inflammatory cytokines known to be involved in MS lesions) was demonstrated. The results obtained were compared with the response elicited by administration of VB5052 or VB5051 (see example 4 for a description of units/elements and these vectors).

The study design and method employed was similar to those described for VB5049 in example 3, except that for VB5049, different administration schedules were tested to verify the results obtained in example 3 and to further extend the data beyond single administration regimen.

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. Spleen was triturated in a cell strainer to obtain a single cell suspension, and splenocytes were restimulated with MOG (35-55) peptide for 44 hours or no restimulation, after which IFN- γ and IL-10 production were detected in a two-color FluoroSpot assay performed as described in example 3.

As shown in fig. 16A, in unstimulated spleen cells harvested from mice administered VB5049, VB5051 or VB5052, a degree of IL-10 production was detected, while only low background levels of IFN- γ were observed. When splenocytes were re-stimulated with MOG (35-55), no significant production of IFN- γ was detected for the group of mice administered with VB5049 or VB5051, while in contrast significantly elevated levels of IFN- γ were detected in splenocytes from mice administered with VB5052 (fig. 16B). In order to avoid excessive inflammation and to finally subside the inflammation, it is important that the production of pro-inflammatory cytokines such as IFN-gamma is regulated by a negative feedback mechanism, including the production of anti-inflammatory cytokines such as IL-10 (see e.g. Sugimoto et al, front immunol.2016, volume 7, 160). Thus, the increased IL-10 levels observed in response to VB5052 can be explained by the feedback mechanism that controls the inflammatory response induced. As shown in figure 17, significantly higher IL-10/IFN- γ ratios were detected for VB5049 compared to VB5052, indicating a higher immunosuppressive potential for VB5049 compared to VB 5052.

Foxp3, IFN- γ and IL-17 were detected by flow cytometry:

the presence of Foxp3 expressing cd4+ T cells was identified in the spleen cell population by flow cytometry. Cd4+foxp3+ T cells can suppress effector T cells and inflammatory immune responses, thereby maintaining self-tolerance. Flow cytometry was performed as described in example 3.

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 a higher level of inhibitory T cells induced by VB5049 compared to VB 5051. The pro-inflammatory cytokines IFN-gamma and IL-17 administered in response to VB5049 were not detected by flow cytometry as shown in FIGS. 19A and 19B, respectively. In contrast, both pro-inflammatory cytokines were detected in response to administration of the pro-inflammatory comparison vector VB 5052.

Example 5 thus shows that administration of VB5049 encoding a first polypeptide comprising an anti-DEC 205 targeting unit, a dimerization unit and a dimerization unit comprising MOG (27-63) and further encoding IL-10 results in a higher anti-inflammatory/inflammatory cytokine ratio (IL-10/IFN- γ) than administration of VB5051 encoding MOG (27-63) alone. In addition, administration of VB5049 resulted in a lack of inflammatory IFN-gamma and IL-17 cytokine production, as well as a higher percentage of CD4+Foxp3+ T cells compared to VB 5051. Taken together, these results indicate that VB5049 can elicit a greater antigen-specific tolerogenic response than antigen alone (VB 5051) in an anti-inflammatory manner as opposed to its pro-inflammatory form VB 5052. These results also confirm the results of example 3 and show that tolerogenic properties of VB5049 remain after repeated administration to mice.

Example 6：

Tolerance induction capacity of VB5062 was assessed in spleens from mice administered VB5062 (see table 5/example 4) and was determined according to IL-10/IFN- γ ratio calculated from IL-10 (anti-inflammatory cytokine associated with immune tolerance) signal and IFN- γ (marker inducing inflammatory immune response) signal generated in a two-color FluoroSpot assay after restimulation with MOG (35-55) peptide and foxp3+ MOG (38-49) specific regulatory T cell (Treg) assay by using MOG (38-49) specific tetramer ex vivo detection. The results obtained were compared with the immunogenicity elicitation elicited by VB5052 and the tolerance-inducing capacity of VB5051 (for a description of units/elements and these vectors, see example 4).

Briefly, 50 μg of DNA vector VB5062, VB5051 or VB5052 was administered intramuscularly to mice (5/group) followed by electroporation. Spleens were harvested 7 days after administration and triturated in a cell strainer to obtain a single cell suspension. Splenocytes were restimulated with MOG (35-55) peptide for 44 hours or no restimulation, after which production of IFN- γ and IL-10 was detected in a two-color FluoroSpot assay performed as described in example 3.

As shown in fig. 20A, IL-10 production was detected in unstimulated spleen cells harvested from mice administered VB5062, VB5051 or VB5052, while only low background levels of IFN- γ were observed. When splenocytes were re-stimulated with MOG (35-55), no significant production of IFN- γ was detected for the group of mice administered with VB5062 or VB5051, in contrast to significantly elevated levels of IFN- γ detected in splenocytes from mice administered with VB5052 (fig. 20B). As described in example 5, the elevated IL-10 levels observed in response to VB5052 can be explained by a potential feedback mechanism controlling the induced inflammatory response. As shown in figure 21, a significantly higher IL-10/IFN- γ ratio was detected for VB5062 compared to VB5052, indicating a higher immunosuppressive potential of VB5062 compared to VB 5052.

MOG (38-49) tetramer staining and flow cytometry were performed as described in example 3. Although similar IL-10 levels were detected for VB5062 and VB5051 in the two-color FluoSpot assay (FIGS. 20A-B), further single cell analysis showed an increase in the percentage of CD4+ MOG (38-49) -specific Foxp3+ cells in VB 5062-administered mice compared to VB 5051-administered mice, as shown in FIG. 22.

Example 6 thus shows that administration of VB5062 encoding a first polypeptide comprising an anti-DEC 205 targeting unit, a dimerization unit and an antigenic unit comprising MOG (27-63) and further encoding an immunosuppressive compound tgfβ1 results in a higher anti-inflammatory cytokine/inflammatory cytokine ratio (IL-10/IFN- γ). In addition, splenocytes from mice administered VB5062 showed a lack of inflammatory IFN- γ cytokine production compared to those obtained from mice administered with the pro-inflammatory form of VB 5052. Administration of VB5062 induced a higher percentage of MOG (38-49) -specific Foxp3+ cells than VB 5051. Taken together, these results indicate that VB5062 can elicit a greater antigen-specific tolerogenic response than antigen alone (VB 5051) in an anti-inflammatory manner as opposed to its pro-inflammatory form VB 5052.

Example 7：

The tolerance-inducing capacity of VB5063 was determined and compared to the immunogenicity of VB5052 and the tolerance-inducing capacity of VB5051 as described in example 6 (see table 5/example 4 for a description of units/elements and these vectors).

As shown in fig. 23A, IL-10 production was detected in unstimulated spleen cells harvested from mice administered VB5062, VB5051 or VB5052, while only low background levels of IFN- γ were observed. When splenocytes were re-stimulated with MOG (35-55), no significant production of IFN- γ was detected for the group of mice administered with VB5063 or VB5051, in contrast to significantly elevated levels of IFN- γ detected in splenocytes from mice administered with VB5052 (fig. 23B). Significantly higher IL-10 levels were detected in both unstimulated and restimulated splenocytes harvested from mice administered VB5063 with MOG (35-55) compared to the levels detected in mice administered VB 5051. As described in example 5, the elevated IL-10 levels observed in response to VB5052 can be explained by a potential feedback mechanism controlling the inflammatory response. As shown in figure 24, significantly higher IL-10/IFN- γ ratios were detected for VB5063 compared to VB5052, indicating a higher immunosuppressive potential of VB5063 compared to VB 5052.

As shown in figure 25, a higher percentage of MOG (38-49) -specific foxp3+ cells was detected in response to VB5063 compared to VB5051 administration, indicating an increased immunosuppressive Treg production in response to VB5063 administration.

Further, to determine the percentage of actively proliferating Treg cells induced in response to administration of VB5063, ki67 expression (nuclear marker strictly associated with dividing cells) was analyzed in spleen cells harvested from mice administered VB5063 or VB 5051. As shown in fig. 26, a higher percentage of ki67+ cells in the Treg (cd4+cd25+foxp3+) cell population was detected ex vivo in mice administered VB5063 compared to VB 5051.

Example 7 thus shows that administration of VB5063 encoding a first polypeptide comprising an anti-DEC 205 targeting unit, a dimerization unit and an antigenic unit comprising MOG (27-63) and further encoding an immunosuppressive compound CTLA-4 results in a higher anti-inflammatory cytokine/inflammatory cytokine ratio (IL-10/IFN- γ). In addition, splenocytes from mice administered VB5063 showed a lack of inflammatory IFN- γ cytokine production compared to the pro-inflammatory form of VB 5052. Further, VB5063 induced a higher percentage of MOG (38-49) -specific Foxp3+ expanded Treg cells and CD4+CD5+Foxp3+Ki67+ expanded Treg cells than VB 5051. Taken together, these results indicate that VB5063 can elicit a greater antigen-specific tolerogenic response than antigen alone (VB 5051) in an anti-inflammatory manner as opposed to its pro-inflammatory form VB 5052.

Example 8：

The tolerance-inducing capacity of VB5064 was determined and compared to the immunogenicity of VB5052 and the tolerance-inducing capacity of VB5051 as described in example 6 (see table 5/example 4 for a description of units/elements and these vectors).

As shown in fig. 27A, IL-10 production was detected in unstimulated spleen cells harvested from mice administered VB5064, VB5051 or VB5052, while only low background levels of IFN- γ were observed. When splenocytes were re-stimulated with MOG (35-55), no significant production of IFN- γ was detected for the group of mice administered with VB5064 or VB5051, in contrast to significantly elevated levels of IFN- γ detected in splenocytes from mice administered with VB5052 (fig. 27B). As described in example 5, the elevated IL-10 levels observed in response to VB5052 can be explained by a potential feedback mechanism controlling the inflammatory response. As shown in figure 28, significantly higher IL-10/IFN- γ ratios were detected for VB5064 compared to VB5052, indicating a higher immunosuppressive potential for VB5064 compared to VB 5052.

Although similar IL-10 levels were detected for VB5064 and VB5051 in the two-color FluoSpot assay (FIGS. 27A-B), a higher percentage of MOG (38-49) -specific Foxp3+ cells were detected in response to VB5064 than VB5051 (FIG. 29).

Further, to determine the percentage of actively proliferating Treg cells induced in response to administration of VB5064, expression of Ki67 (nuclear marker strictly associated with dividing cells) was analyzed in spleen cells harvested from mice administered with VB5064 or VB 5051. As shown in fig. 30, a higher percentage of ki67+ cells in the Treg cd4+cd25+foxp3+) cell population was detected ex vivo in spleen cells of mice administered VB5064 compared to VB 5051.

Example 8 thus shows that administration of VB5064 encoding a first polypeptide comprising an anti-DEC 205 targeting unit, a dimerization unit and an antigenic unit comprising MOG (27-63) and further encoding an immunosuppressive compound IL-2 results in a higher anti-inflammatory cytokine/inflammatory cytokine ratio (IL-10/IFN- γ). In addition, splenocytes from mice administered VB5064 showed a lack of inflammatory IFN- γ cytokine production compared to those obtained from mice administered with the pro-inflammatory form of VB 5052. Further, VB5064 induced a higher percentage of MOG (38-49) -specific Foxp3+ proliferative Treg cells and CD4+CD5+Foxp3+Ki67+ proliferative Treg cells than VB 5051. Taken together, these results indicate that VB5064 can elicit a greater antigen-specific tolerogenic response than antigen alone (VB 5051) in an anti-inflammatory manner as opposed to its pro-inflammatory form VB 5052.

Example 9

TABLE 6

The DNA vectors VB5049, VB5044 and VB5054 encode the same first polypeptide comprising an antigenic unit with MOG (27-63) and further encode the following immunosuppressive compounds expressed as separate molecules due to the presence of the coexpression elements listed in table 6:

VB5049: mIL-10 (used as control in Western blot analysis)

VB 5044: mIL-10 and mTGF-beta 1

VB 5054: mIL-10 and mTGF-. Beta.1 and mGM-CSF

Characterization of expression and secretion of proteins encoded by DNA vectors

Expression and secretion of the proteins encoded by VB5044 and VB5054 were characterized as described in example 4. As shown in fig. 31, the first polypeptide/dimer protein from both vectors was highly expressed and secreted.

The expression and secretion of the encoded immunosuppressive compounds mIL-10, mTGF- β1 and mGM-CSF were characterized by sandwich ELISA using antibodies to mIL-10, hTGF- β1 and mGM-CSF, respectively. The results are shown in figures 32A-C, where all immunosuppressive compounds encoded by VB5044 or VB5054 are shown to be expressed and secreted. Even the third immunosuppressive compound mGM-CSF encoded by vector VB5054 was highly expressed and secreted (fig. 32C).

The expression and secretion of the first polypeptide/dimer protein and the immunosuppressive compound as separate proteins was verified by sandwich ELISA using antibodies against MOG and the anti-immunosuppressive compounds mll-10, mTGF- β1 and mGM-CSF. The results show that the ribosome-jump peptides T2A and P2A are highly potent and that all the first polypeptide/dimer protein and immunosuppressive compound are expressed and secreted as separate proteins (fig. 33A-C).

Characterization of intact proteins expressed from VB5044 and VB5054 by Western blotting

Supernatants from transfected Expi293F cells were subjected to Western Blot (WB) analysis to further characterize the proteins encoded by VB5044 and VB 5054. VB5049 encoding the same first polypeptide and immunosuppressive compound IL-10 was included as a control.

WB was performed as described in example 4. PVDF membranes were probed with mouse anti-MOG (sc-73330,Santa Cruz Biotechnology), rat anti-murine IL-10 (MAB 417, R & D Systems), rabbit anti-tgfβ1 (USB 1042777-biotin, united States Biological) or goat anti-murine GM-CSF (BAF 415, R & D Systems) to detect the first polypeptide/dimer protein, mll-10, mTGF- β1 and mGM-CSF, respectively. The results are shown in FIGS. 34A-E.

WB analysis validated ELISA results, indicating that VB5044 and VB5054 expressed three and four proteins, respectively: the first polypeptide/dimer protein (FIGS. 34A and B, reducing and non-reducing conditions), and two or three immunosuppressive compounds, namely mIL-10 (FIG. 34C), mTGF- β1 (FIG. 34D), and mGM-CSF (FIG. 34E). Importantly, no additional bands were observed for membranes probed with anti-IL-10, anti-TGF- β1 and anti-GM-CSF, indicating that the ribosome was successfully hopped at the 2A sequence, resulting in expression of the first polypeptide and immunosuppressive compound as separate proteins from a single DNA plasmid.

In summary, ELISA data and WB data show that by using different 2A peptides as co-expression elements, complete dimeric proteins comprising targeting units, dimerization units and antigenic units can be co-expressed from DNA vectors together with several immunosuppressive compounds.

Example 10

TABLE 7

The DNA vectors VB5068, VB5069 and VB5070 encode a first polypeptide comprising an antigenic unit with MOG (27-63) and further encode the immunosuppressive compound IL-10, said first polypeptide and immunosuppressive compound being expressed as separate molecules due to the presence of the T2A peptide co-expression element. The first polypeptides encoded by these vectors comprise 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

Expression and secretion of the proteins encoded by VB5068, VB5069 and VB5070 were characterized as described in example 4. As shown in fig. 35, the first polypeptide/dimer protein from all three vectors was highly expressed and secreted.

The expression and secretion of the full-length first polypeptide/dimer protein encoded by VB5068 and VB5070 was verified by sandwich ELISA using antibodies against MOG and the corresponding targeting units mSCGB3A2 and mPD-1. FIGS. 36A and 36B show that the full-length protein is highly expressed and secreted from both vectors.

Taken together, these results indicate that cells transfected with DNA vectors VB5068 and VB5070 express and secrete proteins comprising the targeting unit (mSCGB 3A2 or mPD-1), the CH 3-containing dimerization unit and the mouse MOG (27-63) antigen.

Secretion of the immunosuppressive compound mIL-10 encoded by VB5068 and VB5069 was characterized by sandwich ELISA using antibodies against mouse IL-10. FIG. 37 shows that mIL-10 is highly expressed and secreted from both vectors.

The expression and secretion of the first polypeptide/dimer protein and the immunosuppressive compound IL-10 as separate proteins was verified by sandwich ELISA using antibodies against MOG and murine IL-10. The results show that the ribosome-jump peptide T2A is highly potent and that the first polypeptide/dimer protein and the mll-10 are expressed and secreted as separate proteins (fig. 38).

Characterization of intact proteins expressed from VB5069 and VB5070

Supernatants from transfected Expi293F cells were subjected to western blot analysis to further characterize the proteins encoded by DNA vectors VB5069 and VB 5070.

Western blot was 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-murine IL-10 (MAB 417, R & D Systems) to detect the immunosuppressive compound mIL-10. The results are shown in fig. 39A and 39B.

Western blot analysis validated the ELISA results, indicating that VB5069 and VB5070 express two proteins: a first polypeptide (FIG. 39A, reduced supernatant sample) and mIL-10 (FIG. 39B). Importantly, no additional bands were observed for the membranes probed with anti-IL-10, indicating that ribosomes successfully jump at the T2A sequence, resulting in the expression of two separate proteins from a single DNA vector.

In summary and in view of the previous examples disclosed herein, ELISA data and WB data show that by using a T2A peptide as a co-expression element, an intact first polypeptide comprising a targeting unit other than scFv specific for murine CD205 can be co-expressed from a DNA vector together with an immunosuppressive compound (mll-10).

Example 11

The tolerance induction capacity of VB5068 (see table 7) was determined and compared to the immunogenicity of VB5052 (pro-inflammatory form of VB 5068) and the tolerance induction capacity of VB5051 as described in example 6 (see example 4 for a description of units/elements and these vectors).

As shown in fig. 40A, IL-10 production was detected in unstimulated spleen cells harvested from mice administered VB5068, VB5051 or VB5052, while only low background levels of IFN- γ were observed. When splenocytes were re-stimulated with MOG (35-55), no significant IFN- γ production was detected for the group of mice administered with VB5068 or VB5051, in contrast to elevated levels of IFN- γ detected in splenocytes from mice administered with VB5052 (fig. 40B). As described in example 5, the elevated IL-10 levels observed in response to VB5052 can be explained by a potential feedback mechanism controlling the inflammatory response.

Although similar IL-10 levels were detected in spleen cells from mice administered VB5068 or VB5051 in the two-color FluoSpot assay (FIG. 40A/B), a higher percentage of MOG (38-49) -specific Foxp3+ cells were detected by flow cytometry in response to VB5068 compared to VB5051 (FIG. 41).

By incorporating an additional phenotypic marker CD25 for Treg identification, a higher observed percentage of MOG (38-49) -specific foxp3+ cells (suggesting Treg) in response to VB5068 detection was validated and confirmed (fig. 41). Spleen cells harvested from mice administered VB5068 and analyzed by flow cytometry showed higher levels of cd4+cd25+foxp3+mog (38-49) -tet+ Treg than detected in the spleen from mice administered either VB5051 or VB5052 (fig. 42).

Further, to determine the percentage of actively proliferating Treg cells induced in response to administration of VB5064, expression of Ki67 (nuclear marker strictly associated with dividing cells) was analyzed in spleen cells harvested from mice administered VB5068 or VB 5051. As shown in fig. 43, a higher percentage of ki67+ cells was detected ex vivo in the Treg (cd4+cd25+foxp3+) cell population in mice administered VB5068 compared to VB5051 and VB 5052.

To assess and verify the percentage of tregs induced and detected ex vivo following administration of VB5068 (fig. 41 and 42), treg induction was also assessed following re-stimulation of splenocytes with MOG (35-55). In another experiment, splenocytes from mice administered VB5068, VB5051 or VB5052 were restimulated with MOG (35-55) peptide for 16 hours and analyzed by flow cytometry. As shown in fig. 44, a higher percentage of cd4+cd25+foxp3+ tregs was detected in spleen cells harvested from mice administered VB5068 than in spleens from mice administered VB5051 or VB 5052.

Example 11 shows that administration of VB5068 to mice, which VB5068 encodes a first polypeptide comprising an anti-SCGB 3A2 targeting unit, a dimerization unit and an antigenic unit comprising MOG (27-63), and further encodes the immunosuppressive compound IL-10, results in a higher anti-inflammatory cytokine/inflammatory cytokine ratio (IL-10/IFN-gamma). In addition, splenocytes from mice administered VB5068 showed a lack of inflammatory IFN- γ cytokine production compared to splenocytes from mice administered with the pro-inflammatory form of VB 5052. In addition, VB5068 induced a higher percentage of MOG (38-49) -specific Foxp3+ Treg and CD4+CD5+Foxp3+Ki67+ expanded Treg cells than both VB5051 and VB 5052. Taken together, these results indicate that VB5064 can elicit a greater antigen-specific tolerogenic response than antigen alone (VB 5051) in an anti-inflammatory manner as opposed to its pro-inflammatory form VB 5052. Further, the data presented in example 11 shows the versatility of the vector of the invention, indicating that various targeting units (anti-CD 205 and SCGB3A2 targeting units) allow targeting of antigens comprised in antigenic units to APCs in a tolerogenic manner.

Example 12

Designing and generating a DNA vector comprising a nucleotide sequence encoding a T cell epitope of shrimp allergen tropomyosin from the species penaeus vannamei (Metapenaeus ensis) and further encoding the following elements/units:

TABLE 8

Tropomyosin is the major allergen in shellfish. Six major T cell epitopes from tropomyosin (Met e 1 allergen) of the shrimp species penaeus vannamei are identified in the Met e 1 hypersensitive Balb/c mouse model. Oral immunotherapy with peptides of these six T cell epitopes effectively reduced allergic reactions to shrimp tropomyosin (Wai et al, int J Mol Sci 20 (18), 4656,2015).

DNA vector VB5076 encodes a first polypeptide comprising an antigenic unit that comprises a T cell epitope 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 the immunosuppressive compound mIL10.

Characterization of expression and secretion of the protein encoded by the DNA vector VB5076

Expression and secretion of the protein encoded by VB5076 was characterized as described in example 4. As shown in fig. 45, the first polypeptide/dimer protein from VB5076 is expressed and secreted at high levels.

Secretion of the immunosuppressive compound mIL-10 encoded by VB5076 was characterized by sandwich ELISA using antibodies against mouse IL-10. FIG. 46 shows that mIL-10 is highly expressed and secreted from VB 5076.

Characterization of intact proteins expressed from VB5076

Supernatants from transfected Expi293F cells were subjected to western blot analysis to further characterize the protein encoded by VB 5076. Commercial antibodies directed against the first polypeptide/dimer protein expressed by VB5076 that are compatible with western blotting cannot be determined; thus, only the immunosuppressive compound mIL-10 can be detected.

Western blot was performed as described in example 4. PVDF membranes were probed with rat anti-murine IL-10 (MAB 417, R & D Systems) to detect the immunosuppressive compound mIL-10. The results are shown in FIG. 47.

For membranes probed with anti-IL-10, a major band was observed at the expected molecular weight of IL-10, indicating that the ribosome was successfully hopped at the T2A sequence, resulting in the expression of mIL10 from VB5076 as a separate molecule.

In summary, ELISA data and western blot data show that by using T2A peptide as co-expression element, a first polypeptide/dimer protein comprising an antigenic unit with several T cell epitopes can be co-expressed from a DNA plasmid together with another protein (immunosuppressive compound).

Sequence overview

SEQ ID NO:1

The amino acid sequence of one embodiment of the dimerization unit consists of hinge region 1 from human IgG3 (amino acids 1-12), hinge region 4 from human IgG3 (amino acids 13-27), glycine-serine linker (amino acids 28-37), CH3 domain from human IgG3 (amino acids 38-144)

Signal peptide of mIg VH

VB5049 amino acid sequence

Amino acids 1-19: a mouse immunoglobulin heavy chain signal peptide; amino acids 20-265: a mouse single chain variable fragment (scFv) against DEC205; amino acids 266-409: dimerization unit (SEQ ID NO: 1): a hinge region 1 from human IgG3, a hinge region 4 from human IgG3, a glycine-serine linker, a CH3 domain from human IgG 3; amino acids 410-414: a unit joint; amino acids 415-451: murine MOG 27-63 (MOG 35-55 underlined); amino acids 452-454: a joint; amino acids 455-472: co-expression element T2A peptide; amino acids 473-490: signal peptide murine IL-10, natural leader sequence; amino acids 491-650: murine IL-10

VB5052 amino acid sequence

Amino acids 1-23: the signal peptide human CCL3L1; amino acids 24-93: human CCL3L1; amino acids 94-237: dimerization unit (SEQ ID NO: 1): a hinge region 1 from human IgG3, a hinge region 4 from human IgG3, a glycine-serine linker, a CH3 domain from human IgG 3; amino acids 238-242: a unit joint; amino acids 243-279: mouse MOG 27-63 (MOG 35-55 underlined)

VB5051 amino acid sequence

Amino acids 1-19: a mouse immunoglobulin heavy chain signal peptide; amino acids 20-56: mouse MOG 27-63 (MOG 35-55 underlined)

T2A

P2A

E2A

F2A

Nucleotide sequence encoding amino acids 1-27 of SEQ ID NO. 1

Nucleotide sequence encoding amino acids 38-144 of SEQ ID NO. 1

Nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 1

Signal peptide hCCL3L1

hCCL3L1

Murine CD205 specific scFv

MOG(27-63)*

Signal peptide mIL-10

mIL-10

MOG(27-63)

VB5062

VB5063

VB5064

VB5065

Signal peptide mTGF-beta 1

mTGF-β1

Signal peptide mCLTA-4

mCLTA-4

Signal peptide mIL-2

mIL-2

Signal peptide mIFN-gamma

mIFN-γ

VB5049

VB5052

VB5051

VB5048

VB5044

VB5054

Signal peptide mGM-CSF

mGM-CSF

VB5068

VB5069

VB5070

Signal peptide mSCGB3A2

mSCGB3A2

Signal peptide mVSIG3

mVSIG3 extracellular domain

Signal peptide mPD-1

mPD-1 extracellular domain

VB5076

Met e 1

hCTLA4 extracellular domain, signal peptide underlined

hPD-1 extracellular domain, signal peptide underlined

hIL-10, signal peptide underlined

hTGF beta-1, signal peptide underlined

hIL-2, signal peptide underlined

hGM-CSF, signal peptide underlined

hIFN-gamma, signal peptide underlined

hTGFβ-2

hTGFβ-3

hTGFβ-1

hTGFβ-2

hTGFβ-3

/>

hIL-10

hSCGB3A2, signal peptide underlined

hSCGB3A2

/>

hVSIG-3 extracellular domain, signal peptide underlined

hVSIG-3 extracellular domain

Description of the embodiments

1. A carrier, comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerizing unit (e.g., a dimerization unit), and an antigenic unit (antigenic unit), wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, allergen, alloantigen, or xenogeneic antigen; and

2. The vector according to embodiment 1, wherein the one or more immunosuppressive compounds induce immune tolerance.

3. The vector according to embodiment 1, wherein the one or more immunosuppressive compounds increase immune tolerance.

4. The vector according to embodiment 1, wherein the one or more immunosuppressive compounds maintain immune tolerance.

5. The vector according to embodiment 1, wherein the one or more immunosuppressive compounds induce and/or increase and/or maintain immune tolerance

6. The vector according to any one of embodiments 1 to 5, wherein the one or more immunosuppressive compounds facilitate presentation of the one or more T cell epitopes in the antigenic unit in a tolerance-inducible manner (tolerance inducing manner), such as facilitating and/or supporting presentation of the one or more T cell epitopes in the antigenic unit in a tolerance-inducible manner.

7. The vector according to any one of embodiments 1 to 6, wherein the one or more immunosuppressive compounds facilitate induction of tolerance-maintaining cells (tolerance maintaining cell), such as promoting and/or supporting induction of tolerance-maintaining cells.

8. The vector according to any one of embodiments 1 to 7, wherein the one or more immunosuppressive compounds help maintain tolerogenic cells.

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

10. The vector according to 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.

11. The vector according to any one of embodiments 9 to 10, wherein the inhibitory checkpoint molecule is a human inhibitory checkpoint molecule, preferably a human inhibitory checkpoint molecule selected from the group consisting of: hTLA-4 with hLTA-4 as SEQ ID NO:51, hTLA-4 with hPD-1 as SEQ ID NO:52, hBTLA, hLAG3, hNOX2, hSIGLEC7, hSIGLEC9 and hTIM-3.

12. The vector according to any one of embodiments 1 to 8, wherein the immunosuppressive 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.

13. The vector according to embodiment 12, having the cytokine is a human cytokine selected from the group consisting of: hIL-10 as shown in SEQ ID NO. 53, hTGF-beta 1 as shown in SEQ ID NO. 54, hTGF-beta 2, hTGF-beta 3, hIL-27, hIL-2 as shown in SEQ ID NO. 55, hGM-CSF as shown in SEQ ID NO. 56, hIFN-gamma as shown in SEQ ID NO. 57, hIL-37 and hIL-35.

14. The vector according to any one of claims 1 to 13, comprising a plurality of other nucleic acid sequences encoding a plurality of immunosuppressive compounds, such as 2, 3, 4, 5, 6, 7 or 8 different immunosuppressive compounds.

15. The vector according to embodiment 14, wherein the plurality of immunosuppressive compounds are different immunosuppressive compounds that are produced at different levels or promote tolerance-inducing environment (tolerance-inducing environment).

16. The vector according to any one of embodiments 14 to 15, wherein said different immunosuppressive compounds induce tolerance and increase tolerance and maintain tolerance or induce tolerance and increase tolerance or induce tolerance and maintain tolerance.

17. The vector according to any one of the preceding embodiments, wherein the vector comprises one or more co-expression elements.

18. The vector according to embodiment 17, wherein the one or more co-expression elements cause transcription of the first polypeptide and the one or more immunosuppressive compounds on a single transcript and independent translation into the first polypeptide alone and the one or more immunosuppressive compounds alone.

19. The vector according to any one of embodiments 17 to 18, wherein the one or more co-expression elements are IRES elements or nucleic acid sequences encoding a 2A self-cleaving peptide.

20. The vector according to any one of embodiments 17 to 18, wherein the vector comprises more than one co-expression element, which is an IRES element or a nucleic acid sequence encoding a 2A self-cleaving peptide or an IRES element and a nucleic acid sequence encoding a 2A self-cleaving peptide.

21. The vector according to any one of embodiments 17 to 20, wherein the 2A self-cleaving peptide is selected from the group consisting of: T2A peptide, P2A peptide, E2A peptide, and F2A peptide.

22. The vector according to any one of embodiments 17 to 21, wherein the 2A self-cleaving peptide is selected from the group consisting of: T2A peptide having an amino acid sequence having 80% to 100% sequence identity to the amino acid sequence of SEQ ID NO. 6, P2A peptide having an amino acid sequence having 80% to 100% sequence identity to the amino acid sequence of SEQ ID NO. 7, E2A peptide having an amino acid sequence having 80% to 100% sequence identity to the amino acid sequence of SEQ ID NO. 8, and F2A peptide having an amino acid sequence having 80% to 100% sequence identity to the amino acid sequence of SEQ ID NO. 9.

23. The vector according to any one of embodiments 17 to 22, wherein the 2A self-cleaving peptide is selected from the group consisting of: T2A peptide having the amino acid sequence of SEQ ID NO. 6, P2A peptide having the amino acid sequence of SEQ ID NO. 7, E2A peptide having the amino acid sequence of SEQ ID NO. 8 and F2A peptide having the amino acid sequence of SEQ ID NO. 9.

24. The vector according to embodiment 17, wherein the one or more co-expression elements cause transcription of the first polypeptide and the one or more immunosuppressive compounds as separate transcripts.

25. The vector according to embodiment 24, wherein the one or more co-expression elements is a bi-directional promoter.

26. The vector according to embodiment 24, wherein the one or more co-expression elements are promoters and wherein the vector comprises a separate promoter for each of the nucleic acid sequences encoding the first polypeptide and the one or more immunosuppressive compounds.

27. The vector according to embodiment 24, wherein the one or more co-expression elements are a bi-directional promoter and a promoter.

28. The vector according to any one of embodiments 17 to 27, wherein the vector comprises one or more co-expression elements selected from the group consisting of IRES elements, nucleic acid sequences encoding 2A self-cleaving peptides, bi-directional promoters and promoters.

29. The vector according to any one of embodiments 1 to 28, wherein the antigenic unit comprises one or more T cell epitopes of a self antigen.

30. The vector according to embodiment 29, wherein said antigenic unit comprises a plurality of T cell epitopes of self-antigens.

31. The vector according to embodiment 29, wherein said antigenic unit comprises a plurality of T cell epitopes of a plurality of different autoantigens.

32. The vector according to any one of embodiments 29 to 31, wherein the autoantigen is associated with multiple sclerosis.

33. The vector according to any one of embodiments 29 to 32, wherein the one or more T cell epitopes are selected from the group consisting of: MOGs such as MOG (35-55), MBPs such as MBP (84-104) and MBP (76-112), PLPs such as PLP (139-151), PLP (131-159) and PLP (178-191), MAG, MOBP, CNPase, S100deg.P and transaldolase.

34. The vector according to any one of embodiments 29 to 33, wherein the antigenic units comprise 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).

35. The vector according to any one of embodiments 29 to 31, wherein the autoantigen is associated with type 1 diabetes.

36. The vector according to embodiment 35, wherein said autoantigen is selected from the group consisting of: glutamate decarboxylase 65 kilodaltons isoform (GAD 65), insulin, IA-2, znT8, IGRP, chgA, IAPP, peripheral protein (periherein), tetraspanin-7, GRP78, uremic acid-3 and insulin gene enhancer protein isl-1.

37. The vector according to any one of embodiments 29 to 31, wherein the autoantigen is associated with celiac disease.

38. The vector according to embodiment 37, wherein the autoantigen is selected from the group consisting of alpha-gliadin, gamma-gliadin (e.g., alpha-gliadin (76-95)), omega-gliadin, low molecular weight glutenin, high molecular weight glutenin, hordein, secalin and avenin b.

39. The vector according to any one of embodiments 29 to 31, wherein the autoantigen is associated with rheumatoid arthritis.

40. The vector according to embodiment 39, wherein the self antigen is selected from the group consisting of collagen, heat shock protein 60 (HSP 60), band 3, ribonucleoprotein D1 (SmD 1), acetylcholine receptor (AChR), and myelin protein zero (P0).

41. The vector according to any one of embodiments 29 to 31, wherein 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 multiple radiculoneopathy, hashimoto's thyroiditis, largehead pemphigus (pemphigus foliaceus), pemphigus vulgaris (pemphigus vulgaris), thyroiditis (thyroid eye disease), grave's disease, primary biliary cirrhosis, myasthenia gravis, insulin resistant diabetes, hemolytic anemia, and psoriasis, and/or wherein the antigenic unit comprises one or more T cell epitopes selected from one or more autoantigens selected from the group consisting of neurin 155, thyroid peroxidase, thyroglobulin, desmosome-related glycoprotein, desmoglein 3 (calpain), thyroid stimulating hormone receptor, anti-mitochondrial antibodies (AMA), anti-nuclear antibodies (ananas), rim-like/membrane (RL/M), polynuclear dots (MND), acetylcholinergic receptors, insulin receptors, erythrocyte cathepsin (LL-37), thrombospondin-containing, granulin-1-type 5, granulin-like domain 5-type 5, and human granulin-1 (fca-5, 5-granulin-type 5-granulin-G domain, and human granulin-1-5-homogranulin-type 5-G domain (fca).

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

43. The vector according to embodiment 42, wherein the antigenic unit comprises a plurality of T cell epitopes of an allergen.

44. The vector according to embodiment 42, wherein the antigenic unit comprises a plurality of T cell epitopes of a plurality of different allergens.

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

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 shellfish allergens, cow's milk allergens, egg allergens, fish allergens, fruit allergens, wheat allergens, peanut allergens, woody nut allergens, soybean allergens, seed allergens, buckwheat allergens, celery allergens, garlic allergens, gluten allergens, oat allergens, legume allergens, corn allergens, milk allergens, mustard allergens, nut allergens, poultry allergens, meat allergens, rice allergens, and sesame allergens.

47. The vector according to any one of embodiments 42 to 45, wherein the antigenic units comprise one or more T cell epitopes of one or more mussel allergens selected from tropomyosin, arginine kinase, myosin light chain, troponin C, triose phosphate isomerase, and actin.

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

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, wasp allergens, latex allergens, dust mite allergens, house dust mite allergens, stored dust mite allergens, cockroach allergens, mould allergens, fungus allergens, trichogenous animal allergens such as canine allergens, feline allergens and equine allergens, pollen allergens such as pasture pollen allergens (grass pollen allergen), tree pollen allergens, weed pollen allergens (weed pollen allergen), insect allergens and drug allergens.

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

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

52. The vector according to embodiment 51, wherein the antigenic unit comprises a plurality of T cell epitopes of an alloantigen.

53. The vector according to embodiment 51, wherein the antigenic unit comprises a plurality of T cell epitopes of a plurality of different alloantigens.

54. The 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.

55. The vector according to embodiment 54, wherein said antigenic unit comprises a plurality of T cell epitopes of a heterologous antigen.

56. The vector according to embodiment 54, wherein said antigenic unit comprises a plurality of T cell epitopes of a plurality of different heterologous antigens.

57. The vector according to any one of embodiments 1 to 56, wherein the antigenic unit comprises a plurality of T cell epitopes, said plurality of T cell epitopes being discontinuous T cell epitopes.

58. The vector according to any one of embodiments 1 to 56, wherein the antigenic unit comprises a plurality of T cell epitopes, the plurality of T cell epitopes being the smallest T cell epitope comprised in one or more hotspots.

59. The vector according to any one of embodiments 1 to 56, wherein the antigenic unit comprises a plurality of T cell epitopes from the group consisting of discontinuous T cell epitopes and minimal T cell epitopes comprised in one or more hot spots.

60. The vector according to any one of the preceding embodiments, wherein the one or more T cell epitopes are 7 to about 200 amino acids long, such as 7 to 150 amino acids, preferably 7 to 100 amino acids, e.g. 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 or 15 to 30 or 20 to 75 amino acids or 25 to 50 amino acids.

61. The vector according to embodiment 60, wherein the one or more T cell epitopes have a length suitable for MHC (major histocompatibility complex) presentation, such as 7 to 11 amino acids in length for MHC class I presentation or about 15 amino acids in length for MHC class II presentation.

62. The vector according to any of the preceding embodiments, 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 amino acids, 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.

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

64. The vector according to any one of the preceding embodiments, wherein the antigenic unit comprises a plurality of discontinuous T cell epitopes separated by T cell epitope linkers.

65. The vector according to any one of the preceding embodiments, wherein the targeting unit is or comprises a moiety that interacts with a surface molecule on an antigen presenting cell without activating the cell.

66. The vector according to any one of the preceding embodiments, 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.

67. The vector according to any one of embodiments 65 to 66, wherein the surface molecule is selected from the group consisting of: TGF-beta receptors (e.g., TGF-beta R1, TGF-beta R2, and TGF-beta R3), IL-10R (e.g., IL-10RA and IL-10 RB), 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 (ASGR 1/ASGR 2), CD80, CD86, clec9A, clec12A, clec12B, DCIR2, langerin, MR, DC-Sign, treml4, dectin-1, PDL2, HVEM, CD163, and CD141.

68. The vector according to any one of embodiments 65 to 66, wherein the surface molecule is a surface molecule present on a human antigen presenting cell and wherein the surface molecule is selected from the group consisting of: hTGF beta receptors (e.g., hTGF beta R1, hTGF beta R2, and hTGF beta R3), hIL-10R (e.g., hIL-10RA, and hIL-10 RB), hIL-2R, hIL-4R, hIL-6R, hIL-11R, hIL-13R, hIL-27R, hIL-35R, hIL-37-R, hGM-CSFR, hFLT3, hCR 7, hCD11b, hCD11c, hCD103, hCD14, hCD36, hCD205, hCD109, hVISTA, hMARCO, hMHCII, hCD83, hSIGLEC, hClec A (hMGL), hASGR1/hASGR 2), hCD80, hCD86, hlec 9A, hClec A, hClec12B, hDCIR2, langerin, hMR, hDC-Sign, hTreml4, hDectin-1, hPDL2, hHVEM, hCD163, and D141.

69. The vector according to any one of embodiments 65 to 68, wherein the moiety is a natural ligand, an antibody or a portion thereof (e.g., scFv) or a synthetic ligand.

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: TGF-beta (e.g., TGF-beta 1, TGF-beta 2, and TGF-beta 3), 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 CD 54), 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).

71. The vector according to any one of embodiments 65 to 68, wherein said moiety is a human natural ligand selected from the group consisting of: hTGF beta, hIL-10, hIL-2 (hIL-2 as SEQ ID NO: 55), hIL-4, hIL-6, hIL-11, hIL-13, hIL-27, hIL-35, hIL-37, hGM-CSF (hGM-CSF as SEQ ID NO: 56), hFLT3L, hCCL, hCCL21, hICAM-1 (intercellular adhesion molecule 1, also referred to as CD 54), human keratin, hVSIG-3 (preferably the extracellular domain of hVSIG-3), hSCGB3A2, hCDLA-4 (preferably the extracellular domain of hCDLA-4, e.g., the extracellular domain of hCDLA 4 of SEQ ID NO: 51), hPD-1 (preferably the extracellular domain of PD-1, e.g., the extracellular domain of hPD-1 of SEQ ID NO: 52), and hBTLA (preferably the extracellular domain of hBTLA).

72. The vector according to any one of embodiments 65 to 70, wherein the targeting unit is or comprises IL1-10, tgfβ (such as tgfβ -1, tgfβ -2 and tgfβ -3), SCGB3A2 or VSIG-3 (preferably the extracellular domain of VSIG-3).

73. The vector according to any one of embodiments 65 to 70, wherein the targeting unit is or comprises hIL1-10, hTGF beta (e.g., hTGF beta-1, hTGF beta-2, and hTGF beta-3), hSCGB3A2, or hVSIG-3 (preferably the extracellular domain of hVSIG-3).

74. The vector according to any one of the preceding embodiments, 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 minor fibrous egg, and a tetramerization unit, such as a domain derived from p53, and wherein the multimerization unit optionally comprises hinge regions, such as hinge exon h1 and hinge exon h4, preferably hinge exon h1 and hinge exon h4 of IgG 3.

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

76. The vector according to any one of embodiments 74 to 75, wherein the hinge region is Ig-derived.

77. The vector according to any one of embodiments 74 to 76, wherein the multimerization unit is a dimerization unit and the dimerization unit further comprises another dimerization promoting domain.

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

79. The vector according to any one of embodiments 77 to 78, wherein the other domain is a carboxy-terminal C domain derived from IgG, preferably derived from IgG 3.

80. The 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.

81. The vector according to embodiment 80, wherein the dimerization unit linker connects the hinge region and another domain that promotes dimerization.

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 of human IgG 3.

83. The vector according to embodiment 82, wherein said dimerization unit comprises an amino acid sequence having at least 80% sequence identity to amino acid sequence SEQ ID NO. 1.

84. The vector according to embodiment 83, wherein said dimerization unit consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO. 1.

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

86. The vector according to any one of the preceding embodiments, wherein the first nucleic acid sequence encodes a first polypeptide further comprising a unit linker connecting the antigenic unit to the multimerization unit, and wherein the unit linker is a non-immunogenic linker and/or a flexible or rigid linker.

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.

88. The vector according to embodiment 87, wherein said signal peptide is a native leader sequence of said protein as a targeting unit.

89. The vector according to any one of embodiments 87 to 88, wherein the signal peptide is selected from the group consisting of: human Ig VH signal peptide, hTGF-beta 1 signal peptide, hTGF-beta 2 signal peptide, hTGF-beta 3 signal peptide, hIL-10 signal peptide, hIL-2 signal peptide, hIL-4 signal peptide, hIL-6 signal peptide, hIL-11 signal peptide, hIL-13 signal peptide, hIL-27 signal peptide, hIL-35 signal peptide, hIL-37 signal peptide, hGM-CSF signal peptide, hFLT3L signal peptide, hCL 19 signal peptide, hCL 21 signal peptide, hICAM-1 signal peptide, human keratin signal peptide, hVSIG-3 signal peptide, hSCGB3A2 signal peptide, hCA-4 signal peptide, hPD-1 signal peptide, and hBTLA signal peptide.

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

91. The vector according to embodiment 90, wherein the signal peptide is a natural leader sequence of an immunosuppressive compound.

92. The vector according to any one of embodiments 90 to 91, wherein the signal peptide is selected from the group consisting of: hCLTA-4 signal peptide, hPD-1 signal peptide, hBTLA signal peptide, hLAG3 signal peptide, hNOX2 signal peptide, hSIGLEC7 signal peptide, hSIGLEC9 signal peptide, hTIM-3 signal peptide, hIL-10 signal peptide, hTGF-beta 1 signal peptide, hTGF-beta 2 signal peptide, hTGF-beta 3 signal peptide, hIL-27 signal peptide, hIL-2 signal peptide, hGM-CSF signal peptide, hFLT3L signal peptide, hIFN-gamma signal peptide, and hIL-37 signal peptide.

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.

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.

95. A method of producing a vector as defined in any one of embodiments 1 to 94, the method comprising:

a) Transfecting cells with the vector in vitro;

b) Culturing the cells;

c) Optionally, lysing the cells to release the carrier from the cells; and is also provided with

d) The vector is collected and optionally purified.

96. A host cell comprising a vector as defined in any one of embodiments 1 to 94, such as a host cell selected from a prokaryotic cell, a yeast cell, an insect cell, a higher eukaryotic cell (e.g. a cell from an animal or a human).

97. A carrier as defined in any one of embodiments 1 to 94 for use as a medicament.

98. A pharmaceutical composition comprising a carrier as defined in any one of embodiments 1 to 94 and a pharmaceutically acceptable carrier or diluent.

99. The pharmaceutical composition according to embodiment 98, wherein the pharmaceutically 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 buffer, and combinations thereof.

100. The pharmaceutical composition according to any one of embodiments 98 to 99, wherein the composition further comprises a transfection agent.

101. The composition according to any one of embodiments 98 to 100, wherein the composition further comprises a pharmaceutically acceptable amphiphilic block copolymer comprising blocks of poly (ethylene oxide) and poly (propylene oxide), e.g., further comprises a pharmaceutically 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.

102. A composition according to any of embodiments 98 to 101, wherein the composition further comprises an adjuvant, such as an adjuvant selected from the group consisting of: dexamethasone, enterotoxin cholera toxin B subunit (CTB), TLR2 ligand, worm-derived excretion/secretion (ES) product, rapamycin vitamin D3 analogue and aryl hydrocarbon receptor ligand.

103. The pharmaceutical composition according to any one of embodiments 98 to 102, wherein the composition comprises 0.1 to 10mg of the vector, e.g., the DNA plasmid.

104. A method of treating a subject suffering from or in need of prophylaxis of an immune disorder selected from the group consisting of autoimmune disorders, allergic disorders and graft rejection, comprising administering to the subject a vector as defined in any one of embodiments 1 to 94 or a pharmaceutical composition as defined in any one of embodiments 98 to 103.

105. The method according to embodiment 104, wherein the carrier or pharmaceutical composition is administered in a therapeutically effective amount or a prophylactically effective amount.

106. The method according to any one of embodiments 104 to 105, wherein the carrier or pharmaceutical composition is administered by intradermal, intramuscular or subcutaneous injection or by mucosal or epithelial application, such as intranasal or oral.

107. A method of treating a subject suffering from or in need of prophylaxis of an autoimmune disease, the method comprising administering to the subject a vector comprising a vector as defined in any one of embodiments 1 to 41, or a pharmaceutical composition comprising such a vector as defined in any one of embodiments 98 to 103.

108. The method according to embodiment 107, wherein the carrier or pharmaceutical composition is administered in a therapeutically effective amount.

109. The method according to any one of embodiments 107 to 108, wherein the carrier or pharmaceutical composition is administered by intradermal, intramuscular or subcutaneous injection or by mucosal or epithelial application, such as intranasal or oral.

110. A method of treating a subject suffering from or in need of prevention of an allergic disease, the method comprising administering to the subject a vector comprising a vector as defined in any one of embodiments 1 to 28 or 42 to 50, or a pharmaceutical composition comprising such a vector as defined in any one of embodiments 98 to 103.

111. The method according to embodiment 110, wherein the carrier or pharmaceutical composition is administered in a therapeutically effective amount.

112. The method according to any one of embodiments 110 to 111, wherein the carrier or pharmaceutical composition is administered by intradermal, intramuscular or subcutaneous injection or by mucosal or epithelial application, such as intranasal or oral.

113. A method for treating a subject suffering from or in need of prevention of graft rejection, the method comprising administering to the subject a carrier comprising a carrier as defined in any one of embodiments 1 to 28 or 51 to 56, or a pharmaceutical composition comprising such a carrier as defined in any one of embodiments 98 to 103.

114. The method according to embodiment 113, wherein the carrier or pharmaceutical composition is administered in a therapeutically effective amount.

115. The method according to any one of embodiments 113 to 114, wherein the carrier or pharmaceutical composition is administered by intradermal, intramuscular or subcutaneous injection or by mucosal or epithelial application, such as intranasal or oral.

Sequence listing

<110> Kande treatment Co., ltd (Nykode Therapeutics AS)

Co-expression of <120> constructs and immunosuppressive compounds

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<170> PatentIn version 3.5

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Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Gly Gln

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Gly Lys Asn Ala Thr Gly Met Glu Val Gly Trp Tyr Arg Ser Pro Phe

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Cys Leu Leu Leu Leu Thr Gly Met Arg Ile Ser Arg Gly Gln Tyr Ser

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Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln

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Lys Thr Pro Leu Gly Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp

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Thr Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly

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Gly Ser Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

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Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

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Gln Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp

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Gln Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His

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Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu

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Gly Gly Leu Ser Pro Gly Lys Asn Ala Thr Gly Met Glu Val Gly Trp

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Tyr Arg Ser Pro Phe Ser Arg Val Val His Leu Tyr Arg Asn Gly Lys

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Asp Gln Asp Ala Glu Ala Gln Pro

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Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Thr Leu Lys Gly

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Val Gln Cys Ser Pro Gly Lys Asn Ala Thr Gly Met Glu Val Gly Trp

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Tyr Arg Ser Pro Phe Ser Arg Val Val His Leu Tyr Arg Asn Gly Lys

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Asp Gln Asp Ala Glu Ala Gln Pro

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<213> artificial sequence

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<223> nucleotide sequence encoding amino acids 38-144 of SEQ ID NO. 1

<400> 11

ggacagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 60

aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 120

tgggagagca gcgggcagcc ggagaacaac tacaacacca cgcctcccat gctggactcc 180

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 240

aacatcttct catgctccgt gatgcatgag gctctgcaca accgcttcac gcagaagagc 300

ctctccctgt ctccgggtaa a 321

<210> 12

<211> 432

<212> DNA

<213> artificial sequence

<220>

<223> nucleotide sequence encoding the amino acid sequence of SEQ ID NO. 1

<400> 12

gagctcaaaa ccccacttgg tgacacaact cacacagagc ccaaatcttg tgacacacct 60

cccccgtgcc caaggtgccc aggcggtgga agcagcggag gtggaagtgg aggacagccc 120

cgagaaccac aggtgtacac cctgccccca tcccgggagg agatgaccaa gaaccaggtc 180

agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc 240

agcgggcagc cggagaacaa ctacaacacc acgcctccca tgctggactc cgacggctcc 300

ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacatcttc 360

tcatgctccg tgatgcatga ggctctgcac aaccgcttca cgcagaagag cctctccctg 420

tctccgggta aa 432

<210> 13

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> Signal peptide hCCL3L1

<400> 13

Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala

1 5 10 15

Leu Cys Asn Gln Val Leu Ser

20

<210> 14

<211> 70

<212> PRT

<213> artificial sequence

<220>

<223> hCCL3L1

<400> 14

Ala Pro Leu Ala Ala Asp Thr Pro Thr Ala Cys Cys Phe Ser Tyr Thr

1 5 10 15

Ser Arg Gln Ile Pro Gln Asn Phe Ile Ala Asp Tyr Phe Glu Thr Ser

20 25 30

Ser Gln Cys Ser Lys Pro Ser Val Ile Phe Leu Thr Lys Arg Gly Arg

35 40 45

Gln Val Cys Ala Asp Pro Ser Glu Glu Trp Val Gln Lys Tyr Val Ser

50 55 60

Asp Leu Glu Leu Ser Ala

65 70

<210> 15

<211> 246

<212> PRT

<213> artificial sequence

<220>

<223> scFv specific for murine CD205

<400> 15

Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Thr Ser Leu Gly

1 5 10 15

Asn Ser Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile Lys Gly Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Ser Gly Asn Ala Pro Gln Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ile Phe Thr Ile Ser Asn Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln His Tyr Gln Ser Phe Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Leu Glu

115 120 125

Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys

130 135 140

Ala Ala Ser Gly Phe Thr Phe Asn Asp Phe Tyr Met Asn Trp Ile Arg

145 150 155 160

Gln Pro Pro Gly Gln Ala Pro Glu Trp Leu Gly Val Ile Arg Asn Lys

165 170 175

Gly Asn Gly Tyr Thr Thr Glu Val Asn Thr Ser Val Lys Gly Arg Phe

180 185 190

Thr Ile Ser Arg Asp Asn Thr Gln Asn Ile Leu Tyr Leu Gln Met Asn

195 200 205

Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Gly Gly

210 215 220

Pro Tyr Tyr Tyr Ser Gly Asp Asp Ala Pro Tyr Trp Gly Gln Gly Val

225 230 235 240

Met Val Thr Val Ser Ser

245

<210> 16

<211> 37

<212> PRT

<213> artificial sequence

<220>

<223> MOG (27-63)*

<400> 16

Ser Pro Gly Lys Asn Ala Thr Gly Met Glu Val Gly Trp Tyr Arg Ser

1 5 10 15

Pro Phe Ser Arg Val Val His Leu Tyr Arg Asn Gly Lys Asp Gln Asp

20 25 30

Ala Glu Ala Gln Pro

35

<210> 17

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Signal peptide mIL-10

<400> 17

Met Pro Gly Ser Ala Leu Leu Cys Cys Leu Leu Leu Leu Thr Gly Met

1 5 10 15

Arg Ile

<210> 18

<211> 160

<212> PRT

<213> artificial sequence

<220>

<223> mIL-10

<400> 18

Ser Arg Gly Gln Tyr Ser Arg Glu Asp Asn Asn Cys Thr His Phe Pro

1 5 10 15

Val Gly Gln Ser His Met Leu Leu Glu Leu Arg Thr Ala Phe Ser Gln

20 25 30

Val Lys Thr Phe Phe Gln Thr Lys Asp Gln Leu Asp Asn Ile Leu Leu

35 40 45

Thr Asp Ser Leu Met Gln Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

50 55 60

Leu Ser Glu Met Ile Gln Phe Tyr Leu Val Glu Val Met Pro Gln Ala

65 70 75 80

Glu Lys His Gly Pro Glu Ile Lys Glu His Leu Asn Ser Leu Gly Glu

85 90 95

Lys Leu Lys Thr Leu Arg Met Arg Leu Arg Arg Cys His Arg Phe Leu

100 105 110

Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Ser Asp Phe

115 120 125

Asn Lys Leu Gln Asp Gln Gly Val Tyr Lys Ala Met Asn Glu Phe Asp

130 135 140

Ile Phe Ile Asn Cys Ile Glu Ala Tyr Met Met Ile Lys Met Lys Ser

145 150 155 160

<210> 19

<211> 37

<212> PRT

<213> artificial sequence

<220>

<223> MOG (27-63)

<400> 19

Ser Pro Gly Lys Asn Ala Thr Gly Met Glu Val Gly Trp Tyr Arg Ser

1 5 10 15

Pro Phe Ser Arg Val Val His Leu Tyr Arg Asn Gly Lys Asp Gln Asp

20 25 30

Ala Glu Gln Ala Pro

35

<210> 20

<211> 862

<212> PRT

<213> artificial sequence

<220>

<223> VB5062

<400> 20

Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Thr Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Thr

20 25 30

Ser Leu Gly Asn Ser Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile

35 40 45

Lys Gly Trp Leu Ala Trp Tyr Gln Gln Lys Ser Gly Asn Ala Pro Gln

50 55 60

Leu Leu Ile Tyr Lys Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ile Phe Thr Ile Ser Asn

85 90 95

Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln His Tyr Gln Ser

100 105 110

Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys

130 135 140

Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

145 150 155 160

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Phe Tyr Met Asn

165 170 175

Trp Ile Arg Gln Pro Pro Gly Gln Ala Pro Glu Trp Leu Gly Val Ile

180 185 190

Arg Asn Lys Gly Asn Gly Tyr Thr Thr Glu Val Asn Thr Ser Val Lys

195 200 205

Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Gln Asn Ile Leu Tyr Leu

210 215 220

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala

225 230 235 240

Arg Gly Gly Pro Tyr Tyr Tyr Ser Gly Asp Asp Ala Pro Tyr Trp Gly

245 250 255

Gln Gly Val Met Val Thr Val Ser Ser Glu Leu Lys Thr Pro Leu Gly

260 265 270

Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys

275 280 285

Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Gly Gln

290 295 300

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

305 310 315 320

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

325 330 335

Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn

340 345 350

Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

355 360 365

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile

370 375 380

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln

385 390 395 400

Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly Gly Leu Ser Pro

405 410 415

Gly Lys Asn Ala Thr Gly Met Glu Val Gly Trp Tyr Arg Ser Pro Phe

420 425 430

Ser Arg Val Val His Leu Tyr Arg Asn Gly Lys Asp Gln Asp Ala Glu

435 440 445

Gln Ala Pro Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly

450 455 460

Asp Val Glu Glu Asn Pro Gly Pro Met Pro Pro Ser Gly Leu Arg Leu

465 470 475 480

Leu Pro Leu Leu Leu Pro Leu Pro Trp Leu Leu Val Leu Thr Pro Gly

485 490 495

Arg Pro Ala Ala Gly Leu Ser Thr Cys Lys Thr Ile Asp Met Glu Leu

500 505 510

Val Lys Arg Lys Arg Ile Glu Ala Ile Arg Gly Gln Ile Leu Ser Lys

515 520 525

Leu Arg Leu Ala Ser Pro Pro Ser Gln Gly Glu Val Pro Pro Gly Pro

530 535 540

Leu Pro Glu Ala Val Leu Ala Leu Tyr Asn Ser Thr Arg Asp Arg Val

545 550 555 560

Ala Gly Glu Ser Ala Asp Pro Glu Pro Glu Pro Glu Ala Asp Tyr Tyr

565 570 575

Ala Lys Glu Val Thr Arg Val Leu Met Val Asp Arg Asn Asn Ala Ile

580 585 590

Tyr Glu Lys Thr Lys Asp Ile Ser His Ser Ile Tyr Met Phe Phe Asn

595 600 605

Thr Ser Asp Ile Arg Glu Ala Val Pro Glu Pro Pro Leu Leu Ser Arg

610 615 620

Ala Glu Leu Arg Leu Gln Arg Leu Lys Ser Ser Val Glu Gln His Val

625 630 635 640

Glu Leu Tyr Gln Lys Tyr Ser Asn Asn Ser Trp Arg Tyr Leu Gly Asn

645 650 655

Arg Leu Leu Thr Pro Thr Asp Thr Pro Glu Trp Leu Ser Phe Asp Val

660 665 670

Thr Gly Val Val Arg Gln Trp Leu Asn Gln Gly Asp Gly Ile Gln Gly

675 680 685

Phe Arg Phe Ser Ala His Cys Ser Cys Asp Ser Lys Asp Asn Lys Leu

690 695 700

His Val Glu Ile Asn Gly Ile Ser Pro Lys Arg Arg Gly Asp Leu Gly

705 710 715 720

Thr Ile His Asp Met Asn Arg Pro Phe Leu Leu Leu Met Ala Thr Pro

725 730 735

Leu Glu Arg Ala Gln His Leu His Ser Ser Arg His Arg Arg Ala Leu

740 745 750

Asp Thr Asn Tyr Cys Phe Ser Ser Thr Glu Lys Asn Cys Cys Val Arg

755 760 765

Gln Leu Tyr Ile Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp Ile His

770 775 780

Glu Pro Lys Gly Tyr His Ala Asn Phe Cys Leu Gly Pro Cys Pro Tyr

785 790 795 800

Ile Trp Ser Leu Asp Thr Gln Tyr Ser Lys Val Leu Ala Leu Tyr Asn

805 810 815

Gln His Asn Pro Gly Ala Ser Ala Ser Pro Cys Cys Val Pro Gln Ala

820 825 830

Leu Glu Pro Leu Pro Ile Val Tyr Tyr Val Gly Arg Lys Pro Lys Val

835 840 845

Glu Gln Leu Ser Asn Met Ile Val Arg Ser Cys Lys Cys Ser

850 855 860

<210> 21

<211> 633

<212> PRT

<213> artificial sequence

<220>

<223> VB5063

<400> 21

Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Thr Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Thr

20 25 30

Ser Leu Gly Asn Ser Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile

35 40 45

Lys Gly Trp Leu Ala Trp Tyr Gln Gln Lys Ser Gly Asn Ala Pro Gln

50 55 60

Leu Leu Ile Tyr Lys Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ile Phe Thr Ile Ser Asn

85 90 95

Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln His Tyr Gln Ser

100 105 110

Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys

130 135 140

Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

145 150 155 160

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Phe Tyr Met Asn

165 170 175

Trp Ile Arg Gln Pro Pro Gly Gln Ala Pro Glu Trp Leu Gly Val Ile

180 185 190

Arg Asn Lys Gly Asn Gly Tyr Thr Thr Glu Val Asn Thr Ser Val Lys

195 200 205

Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Gln Asn Ile Leu Tyr Leu

210 215 220

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala

225 230 235 240

Arg Gly Gly Pro Tyr Tyr Tyr Ser Gly Asp Asp Ala Pro Tyr Trp Gly

245 250 255

Gln Gly Val Met Val Thr Val Ser Ser Glu Leu Lys Thr Pro Leu Gly

260 265 270

Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys

275 280 285

Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Gly Gln

290 295 300

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

305 310 315 320

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

325 330 335

Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn

340 345 350

Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

355 360 365

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile

370 375 380

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln

385 390 395 400

Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly Gly Leu Ser Pro

405 410 415

Gly Lys Asn Ala Thr Gly Met Glu Val Gly Trp Tyr Arg Ser Pro Phe

420 425 430

Ser Arg Val Val His Leu Tyr Arg Asn Gly Lys Asp Gln Asp Ala Glu

435 440 445

Gln Ala Pro Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly

450 455 460

Asp Val Glu Glu Asn Pro Gly Pro Met Ala Cys Leu Gly Leu Arg Arg

465 470 475 480

Tyr Lys Ala Gln Leu Gln Leu Pro Ser Arg Thr Trp Pro Phe Val Ala

485 490 495

Leu Leu Thr Leu Leu Phe Ile Pro Val Phe Ser Glu Ala Ile Gln Val

500 505 510

Thr Gln Pro Ser Val Val Leu Ala Ser Ser His Gly Val Ala Ser Phe

515 520 525

Pro Cys Glu Tyr Ser Pro Ser His Asn Thr Asp Glu Val Arg Val Thr

530 535 540

Val Leu Arg Gln Thr Asn Asp Gln Met Thr Glu Val Cys Ala Thr Thr

545 550 555 560

Phe Thr Glu Lys Asn Thr Val Gly Phe Leu Asp Tyr Pro Phe Cys Ser

565 570 575

Gly Thr Phe Asn Glu Ser Arg Val Asn Leu Thr Ile Gln Gly Leu Arg

580 585 590

Ala Val Asp Thr Gly Leu Tyr Leu Cys Lys Val Glu Leu Met Tyr Pro

595 600 605

Pro Pro Tyr Phe Val Gly Met Gly Asn Gly Thr Gln Ile Tyr Val Ile

610 615 620

Asp Pro Glu Pro Cys Pro Asp Ser Asp

625 630

<210> 22

<211> 583

<212> PRT

<213> artificial sequence

<220>

<223> VB5064

<400> 22

Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Thr Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Thr

20 25 30

Ser Leu Gly Asn Ser Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile

35 40 45

Lys Gly Trp Leu Ala Trp Tyr Gln Gln Lys Ser Gly Asn Ala Pro Gln

50 55 60

Leu Leu Ile Tyr Lys Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ile Phe Thr Ile Ser Asn

85 90 95

Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln His Tyr Gln Ser

100 105 110

Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys

130 135 140

Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

145 150 155 160

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Phe Tyr Met Asn

165 170 175

Trp Ile Arg Gln Pro Pro Gly Gln Ala Pro Glu Trp Leu Gly Val Ile

180 185 190

Arg Asn Lys Gly Asn Gly Tyr Thr Thr Glu Val Asn Thr Ser Val Lys

195 200 205

Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Gln Asn Ile Leu Tyr Leu

210 215 220

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala

225 230 235 240

Arg Gly Gly Pro Tyr Tyr Tyr Ser Gly Asp Asp Ala Pro Tyr Trp Gly

245 250 255

Gln Gly Val Met Val Thr Val Ser Ser Glu Leu Lys Thr Pro Leu Gly

260 265 270

Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys

275 280 285

Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Gly Gln

290 295 300

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

305 310 315 320

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

325 330 335

Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn

340 345 350

Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

355 360 365

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile

370 375 380

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln

385 390 395 400

Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly Gly Leu Met Tyr

405 410 415

Ser Met Gln Leu Ala Ser Cys Val Thr Leu Thr Leu Val Leu Leu Val

420 425 430

Asn Ser Ala Pro Thr Ser Ser Ser Thr Ser Ser Ser Thr Ala Glu Ala

435 440 445

Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln His Leu Glu Gln

450 455 460

Leu Leu Met Asp Leu Gln Glu Leu Leu Ser Arg Met Glu Asn Tyr Arg

465 470 475 480

Asn Leu Lys Leu Pro Arg Met Leu Thr Phe Lys Phe Tyr Leu Pro Lys

485 490 495

Gln Ala Thr Glu Leu Lys Asp Leu Gln Cys Leu Glu Asp Glu Leu Gly

500 505 510

Pro Leu Arg His Val Leu Asp Leu Thr Gln Ser Lys Ser Phe Gln Leu

515 520 525

Glu Asp Ala Glu Asn Phe Ile Ser Asn Ile Arg Val Thr Val Val Lys

530 535 540

Leu Lys Gly Ser Asp Asn Thr Phe Glu Cys Gln Phe Asp Asp Glu Ser

545 550 555 560

Ala Thr Val Val Asp Phe Leu Arg Arg Trp Ile Ala Phe Cys Gln Ser

565 570 575

Ile Ile Ser Thr Ser Pro Gln

580

<210> 23

<211> 569

<212> PRT

<213> artificial sequence

<220>

<223> VB5065

<400> 23

Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Thr Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Thr

20 25 30

Ser Leu Gly Asn Ser Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile

35 40 45

Lys Gly Trp Leu Ala Trp Tyr Gln Gln Lys Ser Gly Asn Ala Pro Gln

50 55 60

Leu Leu Ile Tyr Lys Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ile Phe Thr Ile Ser Asn

85 90 95

Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln His Tyr Gln Ser

100 105 110

Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys

130 135 140

Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

145 150 155 160

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Phe Tyr Met Asn

165 170 175

Trp Ile Arg Gln Pro Pro Gly Gln Ala Pro Glu Trp Leu Gly Val Ile

180 185 190

Arg Asn Lys Gly Asn Gly Tyr Thr Thr Glu Val Asn Thr Ser Val Lys

195 200 205

Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Gln Asn Ile Leu Tyr Leu

210 215 220

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala

225 230 235 240

Arg Gly Gly Pro Tyr Tyr Tyr Ser Gly Asp Asp Ala Pro Tyr Trp Gly

245 250 255

Gln Gly Val Met Val Thr Val Ser Ser Glu Leu Lys Thr Pro Leu Gly

260 265 270

Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys

275 280 285

Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Gly Gln

290 295 300

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

305 310 315 320

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

325 330 335

Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn

340 345 350

Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

355 360 365

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile

370 375 380

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln

385 390 395 400

Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly Gly Leu Met Asn

405 410 415

Ala Thr His Cys Ile Leu Ala Leu Gln Leu Phe Leu Met Ala Val Ser

420 425 430

Gly Cys Tyr Cys His Gly Thr Val Ile Glu Ser Leu Glu Ser Leu Asn

435 440 445

Asn Tyr Phe Asn Ser Ser Gly Ile Asp Val Glu Glu Lys Ser Leu Phe

450 455 460

Leu Asp Ile Trp Arg Asn Trp Gln Lys Asp Gly Asp Met Lys Ile Leu

465 470 475 480

Gln Ser Gln Ile Ile Ser Phe Tyr Leu Arg Leu Phe Glu Val Leu Lys

485 490 495

Asp Asn Gln Ala Ile Ser Asn Asn Ile Ser Val Ile Glu Ser His Leu

500 505 510

Ile Thr Thr Phe Phe Ser Asn Ser Lys Ala Lys Lys Asp Ala Phe Met

515 520 525

Ser Ile Ala Lys Phe Glu Val Asn Asn Pro Gln Val Gln Arg Gln Ala

530 535 540

Phe Asn Glu Leu Ile Arg Val Val His Gln Leu Leu Pro Glu Ser Ser

545 550 555 560

Leu Arg Lys Arg Lys Arg Ser Arg Cys

565

<210> 24

<211> 29

<212> PRT

<213> artificial sequence

<220>

<223> Signal peptide mTGF beta 1

<400> 24

Met Pro Pro Ser Gly Leu Arg Leu Leu Pro Leu Leu Leu Pro Leu Pro

1 5 10 15

Trp Leu Leu Val Leu Thr Pro Gly Arg Pro Ala Ala Gly

20 25

<210> 25

<211> 361

<212> PRT

<213> artificial sequence

<220>

<223> mTGF beta 1

<400> 25

Leu Ser Thr Cys Lys Thr Ile Asp Met Glu Leu Val Lys Arg Lys Arg

1 5 10 15

Ile Glu Ala Ile Arg Gly Gln Ile Leu Ser Lys Leu Arg Leu Ala Ser

20 25 30

Pro Pro Ser Gln Gly Glu Val Pro Pro Gly Pro Leu Pro Glu Ala Val

35 40 45

Leu Ala Leu Tyr Asn Ser Thr Arg Asp Arg Val Ala Gly Glu Ser Ala

50 55 60

Asp Pro Glu Pro Glu Pro Glu Ala Asp Tyr Tyr Ala Lys Glu Val Thr

65 70 75 80

Arg Val Leu Met Val Asp Arg Asn Asn Ala Ile Tyr Glu Lys Thr Lys

85 90 95

Asp Ile Ser His Ser Ile Tyr Met Phe Phe Asn Thr Ser Asp Ile Arg

100 105 110

Glu Ala Val Pro Glu Pro Pro Leu Leu Ser Arg Ala Glu Leu Arg Leu

115 120 125

Gln Arg Leu Lys Ser Ser Val Glu Gln His Val Glu Leu Tyr Gln Lys

130 135 140

Tyr Ser Asn Asn Ser Trp Arg Tyr Leu Gly Asn Arg Leu Leu Thr Pro

145 150 155 160

Thr Asp Thr Pro Glu Trp Leu Ser Phe Asp Val Thr Gly Val Val Arg

165 170 175

Gln Trp Leu Asn Gln Gly Asp Gly Ile Gln Gly Phe Arg Phe Ser Ala

180 185 190

His Cys Ser Cys Asp Ser Lys Asp Asn Lys Leu His Val Glu Ile Asn

195 200 205

Gly Ile Ser Pro Lys Arg Arg Gly Asp Leu Gly Thr Ile His Asp Met

210 215 220

Asn Arg Pro Phe Leu Leu Leu Met Ala Thr Pro Leu Glu Arg Ala Gln

225 230 235 240

His Leu His Ser Ser Arg His Arg Arg Ala Leu Asp Thr Asn Tyr Cys

245 250 255

Phe Ser Ser Thr Glu Lys Asn Cys Cys Val Arg Gln Leu Tyr Ile Asp

260 265 270

Phe Arg Lys Asp Leu Gly Trp Lys Trp Ile His Glu Pro Lys Gly Tyr

275 280 285

His Ala Asn Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Ser Leu Asp

290 295 300

Thr Gln Tyr Ser Lys Val Leu Ala Leu Tyr Asn Gln His Asn Pro Gly

305 310 315 320

Ala Ser Ala Ser Pro Cys Cys Val Pro Gln Ala Leu Glu Pro Leu Pro

325 330 335

Ile Val Tyr Tyr Val Gly Arg Lys Pro Lys Val Glu Gln Leu Ser Asn

340 345 350

Met Ile Val Arg Ser Cys Lys Cys Ser

355 360

<210> 26

<211> 35

<212> PRT

<213> artificial sequence

<220>

<223> Signal peptide mCLTA-4

<400> 26

Met Ala Cys Leu Gly Leu Arg Arg Tyr Lys Ala Gln Leu Gln Leu Pro

1 5 10 15

Ser Arg Thr Trp Pro Phe Val Ala Leu Leu Thr Leu Leu Phe Ile Pro

20 25 30

Val Phe Ser

35

<210> 27

<211> 126

<212> PRT

<213> artificial sequence

<220>

<223> mCLTA-4 extracellular Domain

<220>

<221> Domain

<222> (1)..(126)

<223> extracellular Domain

<400> 27

Glu Ala Ile Gln Val Thr Gln Pro Ser Val Val Leu Ala Ser Ser His

1 5 10 15

Gly Val Ala Ser Phe Pro Cys Glu Tyr Ser Pro Ser His Asn Thr Asp

20 25 30

Glu Val Arg Val Thr Val Leu Arg Gln Thr Asn Asp Gln Met Thr Glu

35 40 45

Val Cys Ala Thr Thr Phe Thr Glu Lys Asn Thr Val Gly Phe Leu Asp

50 55 60

Tyr Pro Phe Cys Ser Gly Thr Phe Asn Glu Ser Arg Val Asn Leu Thr

65 70 75 80

Ile Gln Gly Leu Arg Ala Val Asp Thr Gly Leu Tyr Leu Cys Lys Val

85 90 95

Glu Leu Met Tyr Pro Pro Pro Tyr Phe Val Gly Met Gly Asn Gly Thr

100 105 110

Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp

115 120 125

<210> 28

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Signal peptide mIL-2

<400> 28

Met Tyr Ser Met Gln Leu Ala Ser Cys Val Thr Leu Thr Leu Val Leu

1 5 10 15

Leu Val Asn Ser

20

<210> 29

<211> 149

<212> PRT

<213> artificial sequence

<220>

<223> mIL-2

<400> 29

Ala Pro Thr Ser Ser Ser Thr Ser Ser Ser Thr Ala Glu Ala Gln Gln

1 5 10 15

Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln His Leu Glu Gln Leu Leu

20 25 30

Met Asp Leu Gln Glu Leu Leu Ser Arg Met Glu Asn Tyr Arg Asn Leu

35 40 45

Lys Leu Pro Arg Met Leu Thr Phe Lys Phe Tyr Leu Pro Lys Gln Ala

50 55 60

Thr Glu Leu Lys Asp Leu Gln Cys Leu Glu Asp Glu Leu Gly Pro Leu

65 70 75 80

Arg His Val Leu Asp Leu Thr Gln Ser Lys Ser Phe Gln Leu Glu Asp

85 90 95

Ala Glu Asn Phe Ile Ser Asn Ile Arg Val Thr Val Val Lys Leu Lys

100 105 110

Gly Ser Asp Asn Thr Phe Glu Cys Gln Phe Asp Asp Glu Ser Ala Thr

115 120 125

Val Val Asp Phe Leu Arg Arg Trp Ile Ala Phe Cys Gln Ser Ile Ile

130 135 140

Ser Thr Ser Pro Gln

145

<210> 30

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> Signal peptide mIFN gamma

<400> 30

Met Asn Ala Thr His Cys Ile Leu Ala Leu Gln Leu Phe Leu Met Ala

1 5 10 15

Val Ser Gly Cys Tyr Cys

20

<210> 31

<211> 133

<212> PRT

<213> artificial sequence

<220>

<223> mIFN gamma

<400> 31

His Gly Thr Val Ile Glu Ser Leu Glu Ser Leu Asn Asn Tyr Phe Asn

1 5 10 15

Ser Ser Gly Ile Asp Val Glu Glu Lys Ser Leu Phe Leu Asp Ile Trp

20 25 30

Arg Asn Trp Gln Lys Asp Gly Asp Met Lys Ile Leu Gln Ser Gln Ile

35 40 45

Ile Ser Phe Tyr Leu Arg Leu Phe Glu Val Leu Lys Asp Asn Gln Ala

50 55 60

Ile Ser Asn Asn Ile Ser Val Ile Glu Ser His Leu Ile Thr Thr Phe

65 70 75 80

Phe Ser Asn Ser Lys Ala Lys Lys Asp Ala Phe Met Ser Ile Ala Lys

85 90 95

Phe Glu Val Asn Asn Pro Gln Val Gln Arg Gln Ala Phe Asn Glu Leu

100 105 110

Ile Arg Val Val His Gln Leu Leu Pro Glu Ser Ser Leu Arg Lys Arg

115 120 125

Lys Arg Ser Arg Cys

130

<210> 32

<211> 650

<212> PRT

<213> artificial sequence

<220>

<223> VB5049

<400> 32

Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Thr Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Thr

20 25 30

Ser Leu Gly Asn Ser Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile

35 40 45

Lys Gly Trp Leu Ala Trp Tyr Gln Gln Lys Ser Gly Asn Ala Pro Gln

50 55 60

Leu Leu Ile Tyr Lys Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ile Phe Thr Ile Ser Asn

85 90 95

Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln His Tyr Gln Ser

100 105 110

Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys

130 135 140

Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

145 150 155 160

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Phe Tyr Met Asn

165 170 175

Trp Ile Arg Gln Pro Pro Gly Gln Ala Pro Glu Trp Leu Gly Val Ile

180 185 190

Arg Asn Lys Gly Asn Gly Tyr Thr Thr Glu Val Asn Thr Ser Val Lys

195 200 205

Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Gln Asn Ile Leu Tyr Leu

210 215 220

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala

225 230 235 240

Arg Gly Gly Pro Tyr Tyr Tyr Ser Gly Asp Asp Ala Pro Tyr Trp Gly

245 250 255

Gln Gly Val Met Val Thr Val Ser Ser Glu Leu Lys Thr Pro Leu Gly

260 265 270

Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys

275 280 285

Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Gly Gln

290 295 300

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

305 310 315 320

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

325 330 335

Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn

340 345 350

Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

355 360 365

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile

370 375 380

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln

385 390 395 400

Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly Gly Leu Ser Pro

405 410 415

Gly Lys Asn Ala Thr Gly Met Glu Val Gly Trp Tyr Arg Ser Pro Phe

420 425 430

Ser Arg Val Val His Leu Tyr Arg Asn Gly Lys Asp Gln Asp Ala Glu

435 440 445

Gln Ala Pro Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly

450 455 460

Asp Val Glu Glu Asn Pro Gly Pro Met Pro Gly Ser Ala Leu Leu Cys

465 470 475 480

Cys Leu Leu Leu Leu Thr Gly Met Arg Ile Ser Arg Gly Gln Tyr Ser

485 490 495

Arg Glu Asp Asn Asn Cys Thr His Phe Pro Val Gly Gln Ser His Met

500 505 510

Leu Leu Glu Leu Arg Thr Ala Phe Ser Gln Val Lys Thr Phe Phe Gln

515 520 525

Thr Lys Asp Gln Leu Asp Asn Ile Leu Leu Thr Asp Ser Leu Met Gln

530 535 540

Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln

545 550 555 560

Phe Tyr Leu Val Glu Val Met Pro Gln Ala Glu Lys His Gly Pro Glu

565 570 575

Ile Lys Glu His Leu Asn Ser Leu Gly Glu Lys Leu Lys Thr Leu Arg

580 585 590

Met Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser

595 600 605

Lys Ala Val Glu Gln Val Lys Ser Asp Phe Asn Lys Leu Gln Asp Gln

610 615 620

Gly Val Tyr Lys Ala Met Asn Glu Phe Asp Ile Phe Ile Asn Cys Ile

625 630 635 640

Glu Ala Tyr Met Met Ile Lys Met Lys Ser

645 650

<210> 33

<211> 280

<212> PRT

<213> artificial sequence

<220>

<223> VB5052

<400> 33

Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala

1 5 10 15

Leu Cys Asn Gln Val Leu Ser Ala Pro Leu Ala Ala Asp Thr Pro Thr

20 25 30

Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile

35 40 45

Ala Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Ser Val Ile

50 55 60

Phe Leu Thr Lys Arg Gly Arg Gln Val Cys Ala Asp Pro Ser Glu Glu

65 70 75 80

Trp Ala Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu

85 90 95

Lys Thr Pro Leu Gly Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp

100 105 110

Thr Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly

115 120 125

Gly Ser Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

130 135 140

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

145 150 155 160

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly

165 170 175

Gln Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp

180 185 190

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

195 200 205

Gln Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His

210 215 220

Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu

225 230 235 240

Gly Gly Leu Ser Pro Gly Lys Asn Ala Thr Gly Met Glu Val Gly Trp

245 250 255

Tyr Arg Ser Pro Phe Ser Arg Val Val His Leu Tyr Arg Asn Gly Lys

260 265 270

Asp Gln Asp Ala Glu Gln Ala Pro

275 280

<210> 34

<211> 56

<212> PRT

<213> artificial sequence

<220>

<223> VB5051

<400> 34

Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Thr Leu Lys Gly

1 5 10 15

Val Gln Cys Ser Pro Gly Lys Asn Ala Thr Gly Met Glu Val Gly Trp

20 25 30

Tyr Arg Ser Pro Phe Ser Arg Val Val His Leu Tyr Arg Asn Gly Lys

35 40 45

Asp Gln Asp Ala Glu Gln Ala Pro

50 55

<210> 35

<211> 451

<212> PRT

<213> artificial sequence

<220>

<223> VB5048

<400> 35

Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Thr Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Thr

20 25 30

Ser Leu Gly Asn Ser Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile

35 40 45

Lys Gly Trp Leu Ala Trp Tyr Gln Gln Lys Ser Gly Asn Ala Pro Gln

50 55 60

Leu Leu Ile Tyr Lys Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ile Phe Thr Ile Ser Asn

85 90 95

Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln His Tyr Gln Ser

100 105 110

Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys

130 135 140

Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

145 150 155 160

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Phe Tyr Met Asn

165 170 175

Trp Ile Arg Gln Pro Pro Gly Gln Ala Pro Glu Trp Leu Gly Val Ile

180 185 190

Arg Asn Lys Gly Asn Gly Tyr Thr Thr Glu Val Asn Thr Ser Val Lys

195 200 205

Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Gln Asn Ile Leu Tyr Leu

210 215 220

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala

225 230 235 240

Arg Gly Gly Pro Tyr Tyr Tyr Ser Gly Asp Asp Ala Pro Tyr Trp Gly

245 250 255

Gln Gly Val Met Val Thr Val Ser Ser Glu Leu Lys Thr Pro Leu Gly

260 265 270

Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys

275 280 285

Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Gly Gln

290 295 300

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

305 310 315 320

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

325 330 335

Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn

340 345 350

Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

355 360 365

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile

370 375 380

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln

385 390 395 400

Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly Gly Leu Ser Pro

405 410 415

Gly Lys Asn Ala Thr Gly Met Glu Val Gly Trp Tyr Arg Ser Pro Phe

420 425 430

Ser Arg Val Val His Leu Tyr Arg Asn Gly Lys Asp Gln Asp Ala Glu

435 440 445

Gln Ala Pro

450

<210> 36

<211> 1062

<212> PRT

<213> artificial sequence

<220>

<223> VB5044

<400> 36

Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Thr Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Thr

20 25 30

Ser Leu Gly Asn Ser Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile

35 40 45

Lys Gly Trp Leu Ala Trp Tyr Gln Gln Lys Ser Gly Asn Ala Pro Gln

50 55 60

Leu Leu Ile Tyr Lys Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ile Phe Thr Ile Ser Asn

85 90 95

Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln His Tyr Gln Ser

100 105 110

Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys

130 135 140

Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

145 150 155 160

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Phe Tyr Met Asn

165 170 175

Trp Ile Arg Gln Pro Pro Gly Gln Ala Pro Glu Trp Leu Gly Val Ile

180 185 190

Arg Asn Lys Gly Asn Gly Tyr Thr Thr Glu Val Asn Thr Ser Val Lys

195 200 205

Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Gln Asn Ile Leu Tyr Leu

210 215 220

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala

225 230 235 240

Arg Gly Gly Pro Tyr Tyr Tyr Ser Gly Asp Asp Ala Pro Tyr Trp Gly

245 250 255

Gln Gly Val Met Val Thr Val Ser Ser Glu Leu Lys Thr Pro Leu Gly

260 265 270

Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys

275 280 285

Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Gly Gln

290 295 300

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

305 310 315 320

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

325 330 335

Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn

340 345 350

Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

355 360 365

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile

370 375 380

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln

385 390 395 400

Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly Gly Leu Ser Pro

405 410 415

Gly Lys Asn Ala Thr Gly Met Glu Val Gly Trp Tyr Arg Ser Pro Phe

420 425 430

Ser Arg Val Val His Leu Tyr Arg Asn Gly Lys Asp Gln Asp Ala Glu

435 440 445

Gln Ala Pro Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly

450 455 460

Asp Val Glu Glu Asn Pro Gly Pro Met Pro Gly Ser Ala Leu Leu Cys

465 470 475 480

Cys Leu Leu Leu Leu Thr Gly Met Arg Ile Ser Arg Gly Gln Tyr Ser

485 490 495

Arg Glu Asp Asn Asn Cys Thr His Phe Pro Val Gly Gln Ser His Met

500 505 510

Leu Leu Glu Leu Arg Thr Ala Phe Ser Gln Val Lys Thr Phe Phe Gln

515 520 525

Thr Lys Asp Gln Leu Asp Asn Ile Leu Leu Thr Asp Ser Leu Met Gln

530 535 540

Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln

545 550 555 560

Phe Tyr Leu Val Glu Val Met Pro Gln Ala Glu Lys His Gly Pro Glu

565 570 575

Ile Lys Glu His Leu Asn Ser Leu Gly Glu Lys Leu Lys Thr Leu Arg

580 585 590

Met Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser

595 600 605

Lys Ala Val Glu Gln Val Lys Ser Asp Phe Asn Lys Leu Gln Asp Gln

610 615 620

Gly Val Tyr Lys Ala Met Asn Glu Phe Asp Ile Phe Ile Asn Cys Ile

625 630 635 640

Glu Ala Tyr Met Met Ile Lys Met Lys Ser Gly Ser Gly Ala Thr Asn

645 650 655

Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro

660 665 670

Met Pro Pro Ser Gly Leu Arg Leu Leu Pro Leu Leu Leu Pro Leu Pro

675 680 685

Trp Leu Leu Val Leu Thr Pro Gly Arg Pro Ala Ala Gly Leu Ser Thr

690 695 700

Cys Lys Thr Ile Asp Met Glu Leu Val Lys Arg Lys Arg Ile Glu Ala

705 710 715 720

Ile Arg Gly Gln Ile Leu Ser Lys Leu Arg Leu Ala Ser Pro Pro Ser

725 730 735

Gln Gly Glu Val Pro Pro Gly Pro Leu Pro Glu Ala Val Leu Ala Leu

740 745 750

Tyr Asn Ser Thr Arg Asp Arg Val Ala Gly Glu Ser Ala Asp Pro Glu

755 760 765

Pro Glu Pro Glu Ala Asp Tyr Tyr Ala Lys Glu Val Thr Arg Val Leu

770 775 780

Met Val Asp Arg Asn Asn Ala Ile Tyr Glu Lys Thr Lys Asp Ile Ser

785 790 795 800

His Ser Ile Tyr Met Phe Phe Asn Thr Ser Asp Ile Arg Glu Ala Val

805 810 815

Pro Glu Pro Pro Leu Leu Ser Arg Ala Glu Leu Arg Leu Gln Arg Leu

820 825 830

Lys Ser Ser Val Glu Gln His Val Glu Leu Tyr Gln Lys Tyr Ser Asn

835 840 845

Asn Ser Trp Arg Tyr Leu Gly Asn Arg Leu Leu Thr Pro Thr Asp Thr

850 855 860

Pro Glu Trp Leu Ser Phe Asp Val Thr Gly Val Val Arg Gln Trp Leu

865 870 875 880

Asn Gln Gly Asp Gly Ile Gln Gly Phe Arg Phe Ser Ala His Cys Ser

885 890 895

Cys Asp Ser Lys Asp Asn Lys Leu His Val Glu Ile Asn Gly Ile Ser

900 905 910

Pro Lys Arg Arg Gly Asp Leu Gly Thr Ile His Asp Met Asn Arg Pro

915 920 925

Phe Leu Leu Leu Met Ala Thr Pro Leu Glu Arg Ala Gln His Leu His

930 935 940

Ser Ser Arg His Arg Arg Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser

945 950 955 960

Thr Glu Lys Asn Cys Cys Val Arg Gln Leu Tyr Ile Asp Phe Arg Lys

965 970 975

Asp Leu Gly Trp Lys Trp Ile His Glu Pro Lys Gly Tyr His Ala Asn

980 985 990

Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Ser Leu Asp Thr Gln Tyr

995 1000 1005

Ser Lys Val Leu Ala Leu Tyr Asn Gln His Asn Pro Gly Ala Ser

1010 1015 1020

Ala Ser Pro Cys Cys Val Pro Gln Ala Leu Glu Pro Leu Pro Ile

1025 1030 1035

Val Tyr Tyr Val Gly Arg Lys Pro Lys Val Glu Gln Leu Ser Asn

1040 1045 1050

Met Ile Val Arg Ser Cys Lys Cys Ser

1055 1060

<210> 37

<211> 1224

<212> PRT

<213> artificial sequence

<220>

<223> VB5054

<400> 37

Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Thr Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Thr

20 25 30

Ser Leu Gly Asn Ser Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile

35 40 45

Lys Gly Trp Leu Ala Trp Tyr Gln Gln Lys Ser Gly Asn Ala Pro Gln

50 55 60

Leu Leu Ile Tyr Lys Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ile Phe Thr Ile Ser Asn

85 90 95

Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln His Tyr Gln Ser

100 105 110

Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys

130 135 140

Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

145 150 155 160

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Phe Tyr Met Asn

165 170 175

Trp Ile Arg Gln Pro Pro Gly Gln Ala Pro Glu Trp Leu Gly Val Ile

180 185 190

Arg Asn Lys Gly Asn Gly Tyr Thr Thr Glu Val Asn Thr Ser Val Lys

195 200 205

Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Gln Asn Ile Leu Tyr Leu

210 215 220

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala

225 230 235 240

Arg Gly Gly Pro Tyr Tyr Tyr Ser Gly Asp Asp Ala Pro Tyr Trp Gly

245 250 255

Gln Gly Val Met Val Thr Val Ser Ser Glu Leu Lys Thr Pro Leu Gly

260 265 270

Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys

275 280 285

Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Gly Gln

290 295 300

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

305 310 315 320

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

325 330 335

Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn

340 345 350

Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

355 360 365

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile

370 375 380

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln

385 390 395 400

Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly Gly Leu Ser Pro

405 410 415

Gly Lys Asn Ala Thr Gly Met Glu Val Gly Trp Tyr Arg Ser Pro Phe

420 425 430

Ser Arg Val Val His Leu Tyr Arg Asn Gly Lys Asp Gln Asp Ala Glu

435 440 445

Gln Ala Pro Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly

450 455 460

Asp Val Glu Glu Asn Pro Gly Pro Met Pro Gly Ser Ala Leu Leu Cys

465 470 475 480

Cys Leu Leu Leu Leu Thr Gly Met Arg Ile Ser Arg Gly Gln Tyr Ser

485 490 495

Arg Glu Asp Asn Asn Cys Thr His Phe Pro Val Gly Gln Ser His Met

500 505 510

Leu Leu Glu Leu Arg Thr Ala Phe Ser Gln Val Lys Thr Phe Phe Gln

515 520 525

Thr Lys Asp Gln Leu Asp Asn Ile Leu Leu Thr Asp Ser Leu Met Gln

530 535 540

Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln

545 550 555 560

Phe Tyr Leu Val Glu Val Met Pro Gln Ala Glu Lys His Gly Pro Glu

565 570 575

Ile Lys Glu His Leu Asn Ser Leu Gly Glu Lys Leu Lys Thr Leu Arg

580 585 590

Met Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser

595 600 605

Lys Ala Val Glu Gln Val Lys Ser Asp Phe Asn Lys Leu Gln Asp Gln

610 615 620

Gly Val Tyr Lys Ala Met Asn Glu Phe Asp Ile Phe Ile Asn Cys Ile

625 630 635 640

Glu Ala Tyr Met Met Ile Lys Met Lys Ser Gly Ser Gly Ala Thr Asn

645 650 655

Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro

660 665 670

Met Pro Pro Ser Gly Leu Arg Leu Leu Pro Leu Leu Leu Pro Leu Pro

675 680 685

Trp Leu Leu Val Leu Thr Pro Gly Arg Pro Ala Ala Gly Leu Ser Thr

690 695 700

Cys Lys Thr Ile Asp Met Glu Leu Val Lys Arg Lys Arg Ile Glu Ala

705 710 715 720

Ile Arg Gly Gln Ile Leu Ser Lys Leu Arg Leu Ala Ser Pro Pro Ser

725 730 735

Gln Gly Glu Val Pro Pro Gly Pro Leu Pro Glu Ala Val Leu Ala Leu

740 745 750

Tyr Asn Ser Thr Arg Asp Arg Val Ala Gly Glu Ser Ala Asp Pro Glu

755 760 765

Pro Glu Pro Glu Ala Asp Tyr Tyr Ala Lys Glu Val Thr Arg Val Leu

770 775 780

Met Val Asp Arg Asn Asn Ala Ile Tyr Glu Lys Thr Lys Asp Ile Ser

785 790 795 800

His Ser Ile Tyr Met Phe Phe Asn Thr Ser Asp Ile Arg Glu Ala Val

805 810 815

Pro Glu Pro Pro Leu Leu Ser Arg Ala Glu Leu Arg Leu Gln Arg Leu

820 825 830

Lys Ser Ser Val Glu Gln His Val Glu Leu Tyr Gln Lys Tyr Ser Asn

835 840 845

Asn Ser Trp Arg Tyr Leu Gly Asn Arg Leu Leu Thr Pro Thr Asp Thr

850 855 860

Pro Glu Trp Leu Ser Phe Asp Val Thr Gly Val Val Arg Gln Trp Leu

865 870 875 880

Asn Gln Gly Asp Gly Ile Gln Gly Phe Arg Phe Ser Ala His Cys Ser

885 890 895

Cys Asp Ser Lys Asp Asn Lys Leu His Val Glu Ile Asn Gly Ile Ser

900 905 910

Pro Lys Arg Arg Gly Asp Leu Gly Thr Ile His Asp Met Asn Arg Pro

915 920 925

Phe Leu Leu Leu Met Ala Thr Pro Leu Glu Arg Ala Gln His Leu His

930 935 940

Ser Ser Arg His Arg Arg Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser

945 950 955 960

Thr Glu Lys Asn Cys Cys Val Arg Gln Leu Tyr Ile Asp Phe Arg Lys

965 970 975

Asp Leu Gly Trp Lys Trp Ile His Glu Pro Lys Gly Tyr His Ala Asn

980 985 990

Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Ser Leu Asp Thr Gln Tyr

995 1000 1005

Ser Lys Val Leu Ala Leu Tyr Asn Gln His Asn Pro Gly Ala Ser

1010 1015 1020

Ala Ser Pro Cys Cys Val Pro Gln Ala Leu Glu Pro Leu Pro Ile

1025 1030 1035

Val Tyr Tyr Val Gly Arg Lys Pro Lys Val Glu Gln Leu Ser Asn

1040 1045 1050

Met Ile Val Arg Ser Cys Lys Cys Ser Gly Ser Gly Glu Gly Arg

1055 1060 1065

Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro

1070 1075 1080

Met Trp Leu Gln Asn Leu Leu Phe Leu Gly Ile Val Val Tyr Ser

1085 1090 1095

Leu Ser Ala Pro Thr Arg Ser Pro Ile Thr Val Thr Arg Pro Trp

1100 1105 1110

Lys His Val Glu Ala Ile Lys Glu Ala Leu Asn Leu Leu Asp Asp

1115 1120 1125

Met Pro Val Thr Leu Asn Glu Glu Val Glu Val Val Ser Asn Glu

1130 1135 1140

Phe Ser Phe Lys Lys Leu Thr Cys Val Gln Thr Arg Leu Lys Ile

1145 1150 1155

Phe Glu Gln Gly Leu Arg Gly Asn Phe Thr Lys Leu Lys Gly Ala

1160 1165 1170

Leu Asn Met Thr Ala Ser Tyr Tyr Gln Thr Tyr Cys Pro Pro Thr

1175 1180 1185

Pro Glu Thr Asp Cys Glu Thr Gln Val Thr Thr Tyr Ala Asp Phe

1190 1195 1200

Ile Asp Ser Leu Lys Thr Phe Leu Thr Asp Ile Pro Phe Glu Cys

1205 1210 1215

Lys Lys Pro Val Gln Lys

1220

<210> 38

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> Signal peptide mGM-CSF

<400> 38

Met Trp Leu Gln Asn Leu Leu Phe Leu Gly Ile Val Val Tyr Ser Leu

1 5 10 15

Ser

<210> 39

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> mGM-CSF

<400> 39

Ala Pro Thr Arg Ser Pro Ile Thr Val Thr Arg Pro Trp Lys His Val

1 5 10 15

Glu Ala Ile Lys Glu Ala Leu Asn Leu Leu Asp Asp Met Pro Val Thr

20 25 30

Leu Asn Glu Glu Val Glu Val Val Ser Asn Glu Phe Ser Phe Lys Lys

35 40 45

Leu Thr Cys Val Gln Thr Arg Leu Lys Ile Phe Glu Gln Gly Leu Arg

50 55 60

Gly Asn Phe Thr Lys Leu Lys Gly Ala Leu Asn Met Thr Ala Ser Tyr

65 70 75 80

Tyr Gln Thr Tyr Cys Pro Pro Thr Pro Glu Thr Asp Cys Glu Thr Gln

85 90 95

Val Thr Thr Tyr Ala Asp Phe Ile Asp Ser Leu Lys Thr Phe Leu Thr

100 105 110

Asp Ile Pro Phe Glu Cys Lys Lys Pro Val Gln Lys

115 120

<210> 40

<211> 476

<212> PRT

<213> artificial sequence

<220>

<223> VB5068

<400> 40

Met Lys Leu Val Ser Ile Phe Leu Leu Val Thr Ile Gly Ile Cys Gly

1 5 10 15

Tyr Ser Ala Thr Ala Leu Leu Ile Asn Arg Leu Pro Val Val Asp Lys

20 25 30

Leu Pro Val Pro Leu Asp Asp Ile Ile Pro Ser Phe Asp Pro Leu Lys

35 40 45

Met Leu Leu Lys Thr Leu Gly Ile Ser Val Glu His Leu Val Thr Gly

50 55 60

Leu Lys Lys Cys Val Asp Glu Leu Gly Pro Glu Ala Ser Glu Ala Val

65 70 75 80

Lys Lys Leu Leu Glu Ala Leu Ser His Leu Val Glu Leu Lys Thr Pro

85 90 95

Leu Gly Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr Pro Pro

100 105 110

Pro Cys Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly

115 120 125

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

130 135 140

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

145 150 155 160

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu

165 170 175

Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe

180 185 190

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

195 200 205

Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe

210 215 220

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly Gly Leu

225 230 235 240

Ser Pro Gly Lys Asn Ala Thr Gly Met Glu Val Gly Trp Tyr Arg Ser

245 250 255

Pro Phe Ser Arg Val Val His Leu Tyr Arg Asn Gly Lys Asp Gln Asp

260 265 270

Ala Glu Gln Ala Pro Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr

275 280 285

Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Pro Gly Ser Ala Leu

290 295 300

Leu Cys Cys Leu Leu Leu Leu Thr Gly Met Arg Ile Ser Arg Gly Gln

305 310 315 320

Tyr Ser Arg Glu Asp Asn Asn Cys Thr His Phe Pro Val Gly Gln Ser

325 330 335

His Met Leu Leu Glu Leu Arg Thr Ala Phe Ser Gln Val Lys Thr Phe

340 345 350

Phe Gln Thr Lys Asp Gln Leu Asp Asn Ile Leu Leu Thr Asp Ser Leu

355 360 365

Met Gln Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met

370 375 380

Ile Gln Phe Tyr Leu Val Glu Val Met Pro Gln Ala Glu Lys His Gly

385 390 395 400

Pro Glu Ile Lys Glu His Leu Asn Ser Leu Gly Glu Lys Leu Lys Thr

405 410 415

Leu Arg Met Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn

420 425 430

Lys Ser Lys Ala Val Glu Gln Val Lys Ser Asp Phe Asn Lys Leu Gln

435 440 445

Asp Gln Gly Val Tyr Lys Ala Met Asn Glu Phe Asp Ile Phe Ile Asn

450 455 460

Cys Ile Glu Ala Tyr Met Met Ile Lys Met Lys Ser

465 470 475

<210> 41

<211> 625

<212> PRT

<213> artificial sequence

<220>

<223> VB5069

<400> 41

Met Thr Arg Arg Arg Ser Ala Pro Ala Ser Trp Leu Leu Val Ser Leu

1 5 10 15

Leu Gly Val Ala Thr Ser Leu Glu Val Ser Glu Ser Pro Gly Ser Val

20 25 30

Gln Val Ala Arg Gly Gln Thr Ala Val Leu Pro Cys Ala Phe Ser Thr

35 40 45

Ser Ala Ala Leu Leu Asn Leu Asn Val Ile Trp Met Val Ile Pro Leu

50 55 60

Ser Asn Ala Asn Gln Pro Glu Gln Val Ile Leu Tyr Gln Gly Gly Gln

65 70 75 80

Met Phe Asp Gly Ala Leu Arg Phe His Gly Arg Val Gly Phe Thr Gly

85 90 95

Thr Met Pro Ala Thr Asn Val Ser Ile Phe Ile Asn Asn Thr Gln Leu

100 105 110

Ser Asp Thr Gly Thr Tyr Gln Cys Leu Val Asn Asn Leu Pro Asp Arg

115 120 125

Gly Gly Arg Asn Ile Gly Val Thr Gly Leu Thr Val Leu Val Pro Pro

130 135 140

Ser Ala Pro Gln Cys Gln Ile Gln Gly Ser Gln Asp Leu Gly Ser Asp

145 150 155 160

Val Ile Leu Leu Cys Ser Ser Glu Glu Gly Ile Pro Arg Pro Thr Tyr

165 170 175

Leu Trp Glu Lys Leu Asp Asn Thr Leu Lys Leu Pro Pro Thr Ala Thr

180 185 190

Gln Asp Gln Val Gln Gly Thr Val Thr Ile Arg Asn Ile Ser Ala Leu

195 200 205

Ser Ser Gly Leu Tyr Gln Cys Val Ala Ser Asn Ala Ile Gly Thr Ser

210 215 220

Thr Cys Leu Leu Asp Leu Gln Val Ile Ser Pro Gln Pro Arg Ser Val

225 230 235 240

Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Glu Pro Lys Ser

245 250 255

Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly Gly Ser Ser

260 265 270

Gly Gly Gly Ser Gly Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

275 280 285

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

290 295 300

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

305 310 315 320

Ser Gly Gln Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met Leu Asp

325 330 335

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

340 345 350

Arg Trp Gln Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu Ala

355 360 365

Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

370 375 380

Gly Leu Gly Gly Leu Ser Pro Gly Lys Asn Ala Thr Gly Met Glu Val

385 390 395 400

Gly Trp Tyr Arg Ser Pro Phe Ser Arg Val Val His Leu Tyr Arg Asn

405 410 415

Gly Lys Asp Gln Asp Ala Glu Gln Ala Pro Gly Ser Gly Glu Gly Arg

420 425 430

Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met

435 440 445

Pro Gly Ser Ala Leu Leu Cys Cys Leu Leu Leu Leu Thr Gly Met Arg

450 455 460

Ile Ser Arg Gly Gln Tyr Ser Arg Glu Asp Asn Asn Cys Thr His Phe

465 470 475 480

Pro Val Gly Gln Ser His Met Leu Leu Glu Leu Arg Thr Ala Phe Ser

485 490 495

Gln Val Lys Thr Phe Phe Gln Thr Lys Asp Gln Leu Asp Asn Ile Leu

500 505 510

Leu Thr Asp Ser Leu Met Gln Asp Phe Lys Gly Tyr Leu Gly Cys Gln

515 520 525

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Val Glu Val Met Pro Gln

530 535 540

Ala Glu Lys His Gly Pro Glu Ile Lys Glu His Leu Asn Ser Leu Gly

545 550 555 560

Glu Lys Leu Lys Thr Leu Arg Met Arg Leu Arg Arg Cys His Arg Phe

565 570 575

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Ser Asp

580 585 590

Phe Asn Lys Leu Gln Asp Gln Gly Val Tyr Lys Ala Met Asn Glu Phe

595 600 605

Asp Ile Phe Ile Asn Cys Ile Glu Ala Tyr Met Met Ile Lys Met Lys

610 615 620

Ser

625

<210> 42

<211> 554

<212> PRT

<213> artificial sequence

<220>

<223> VB5070

<400> 42

Met Trp Val Arg Gln Val Pro Trp Ser Phe Thr Trp Ala Val Leu Gln

1 5 10 15

Leu Ser Trp Gln Ser Gly Trp Leu Leu Glu Val Pro Asn Gly Pro Trp

20 25 30

Arg Ser Leu Thr Phe Tyr Pro Ala Trp Leu Thr Val Ser Glu Gly Ala

35 40 45

Asn Ala Thr Phe Thr Cys Ser Leu Ser Asn Trp Ser Glu Asp Leu Met

50 55 60

Leu Asn Trp Asn Arg Leu Ser Pro Ser Asn Gln Thr Glu Lys Gln Ala

65 70 75 80

Ala Phe Cys Asn Gly Leu Ser Gln Pro Val Gln Asp Ala Arg Phe Gln

85 90 95

Ile Ile Gln Leu Pro Asn Arg His Asp Phe His Met Asn Ile Leu Asp

100 105 110

Thr Arg Arg Asn Asp Ser Gly Ile Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

His Pro Lys Ala Lys Ile Glu Glu Ser Pro Gly Ala Glu Leu Val Val

130 135 140

Thr Glu Arg Ile Leu Glu Thr Ser Thr Arg Tyr Pro Ser Pro Ser Pro

145 150 155 160

Lys Pro Glu Gly Arg Phe Gln Gly Met Glu Leu Lys Thr Pro Leu Gly

165 170 175

Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys

180 185 190

Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Gly Gln

195 200 205

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

210 215 220

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

225 230 235 240

Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn

245 250 255

Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

260 265 270

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile

275 280 285

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln

290 295 300

Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly Gly Leu Ser Pro

305 310 315 320

Gly Lys Asn Ala Thr Gly Met Glu Val Gly Trp Tyr Arg Ser Pro Phe

325 330 335

Ser Arg Val Val His Leu Tyr Arg Asn Gly Lys Asp Gln Asp Ala Glu

340 345 350

Gln Ala Pro Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly

355 360 365

Asp Val Glu Glu Asn Pro Gly Pro Met Pro Gly Ser Ala Leu Leu Cys

370 375 380

Cys Leu Leu Leu Leu Thr Gly Met Arg Ile Ser Arg Gly Gln Tyr Ser

385 390 395 400

Arg Glu Asp Asn Asn Cys Thr His Phe Pro Val Gly Gln Ser His Met

405 410 415

Leu Leu Glu Leu Arg Thr Ala Phe Ser Gln Val Lys Thr Phe Phe Gln

420 425 430

Thr Lys Asp Gln Leu Asp Asn Ile Leu Leu Thr Asp Ser Leu Met Gln

435 440 445

Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln

450 455 460

Phe Tyr Leu Val Glu Val Met Pro Gln Ala Glu Lys His Gly Pro Glu

465 470 475 480

Ile Lys Glu His Leu Asn Ser Leu Gly Glu Lys Leu Lys Thr Leu Arg

485 490 495

Met Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser

500 505 510

Lys Ala Val Glu Gln Val Lys Ser Asp Phe Asn Lys Leu Gln Asp Gln

515 520 525

Gly Val Tyr Lys Ala Met Asn Glu Phe Asp Ile Phe Ile Asn Cys Ile

530 535 540

Glu Ala Tyr Met Met Ile Lys Met Lys Ser

545 550

<210> 43

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> Signal peptide mSCGB3A2

<400> 43

Met Lys Leu Val Ser Ile Phe Leu Leu Val Thr Ile Gly Ile Cys Gly

1 5 10 15

Tyr Ser Ala Thr Ala

20

<210> 44

<211> 70

<212> PRT

<213> artificial sequence

<220>

<223> mSCGB3A2

<400> 44

Leu Leu Ile Asn Arg Leu Pro Val Val Asp Lys Leu Pro Val Pro Leu

1 5 10 15

Asp Asp Ile Ile Pro Ser Phe Asp Pro Leu Lys Met Leu Leu Lys Thr

20 25 30

Leu Gly Ile Ser Val Glu His Leu Val Thr Gly Leu Lys Lys Cys Val

35 40 45

Asp Glu Leu Gly Pro Glu Ala Ser Glu Ala Val Lys Lys Leu Leu Glu

50 55 60

Ala Leu Ser His Leu Val

65 70

<210> 45

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> Signal peptide mVSIG3

<400> 45

Met Thr Arg Arg Arg Ser Ala Pro Ala Ser Trp Leu Leu Val Ser Leu

1 5 10 15

Leu Gly Val Ala Thr Ser

20

<210> 46

<211> 218

<212> PRT

<213> artificial sequence

<220>

<223> mVSIG3 extracellular Domain

<220>

<221> Domain

<222> (1)..(218)

<223> extracellular Domain

<400> 46

Leu Glu Val Ser Glu Ser Pro Gly Ser Val Gln Val Ala Arg Gly Gln

1 5 10 15

Thr Ala Val Leu Pro Cys Ala Phe Ser Thr Ser Ala Ala Leu Leu Asn

20 25 30

Leu Asn Val Ile Trp Met Val Ile Pro Leu Ser Asn Ala Asn Gln Pro

35 40 45

Glu Gln Val Ile Leu Tyr Gln Gly Gly Gln Met Phe Asp Gly Ala Leu

50 55 60

Arg Phe His Gly Arg Val Gly Phe Thr Gly Thr Met Pro Ala Thr Asn

65 70 75 80

Val Ser Ile Phe Ile Asn Asn Thr Gln Leu Ser Asp Thr Gly Thr Tyr

85 90 95

Gln Cys Leu Val Asn Asn Leu Pro Asp Arg Gly Gly Arg Asn Ile Gly

100 105 110

Val Thr Gly Leu Thr Val Leu Val Pro Pro Ser Ala Pro Gln Cys Gln

115 120 125

Ile Gln Gly Ser Gln Asp Leu Gly Ser Asp Val Ile Leu Leu Cys Ser

130 135 140

Ser Glu Glu Gly Ile Pro Arg Pro Thr Tyr Leu Trp Glu Lys Leu Asp

145 150 155 160

Asn Thr Leu Lys Leu Pro Pro Thr Ala Thr Gln Asp Gln Val Gln Gly

165 170 175

Thr Val Thr Ile Arg Asn Ile Ser Ala Leu Ser Ser Gly Leu Tyr Gln

180 185 190

Cys Val Ala Ser Asn Ala Ile Gly Thr Ser Thr Cys Leu Leu Asp Leu

195 200 205

Gln Val Ile Ser Pro Gln Pro Arg Ser Val

210 215

<210> 47

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> Signal peptide mPD-1

<400> 47

Met Trp Val Arg Gln Val Pro Trp Ser Phe Thr Trp Ala Val Leu Gln

1 5 10 15

Leu Ser Trp Gln Ser Gly Trp Leu

20

<210> 48

<211> 145

<212> PRT

<213> artificial sequence

<220>

<223> mPD-1 extracellular Domain

<220>

<221> Domain

<222> (1)..(145)

<223> extracellular Domain

<400> 48

Leu Glu Val Pro Asn Gly Pro Trp Arg Ser Leu Thr Phe Tyr Pro Ala

1 5 10 15

Trp Leu Thr Val Ser Glu Gly Ala Asn Ala Thr Phe Thr Cys Ser Leu

20 25 30

Ser Asn Trp Ser Glu Asp Leu Met Leu Asn Trp Asn Arg Leu Ser Pro

35 40 45

Ser Asn Gln Thr Glu Lys Gln Ala Ala Phe Cys Asn Gly Leu Ser Gln

50 55 60

Pro Val Gln Asp Ala Arg Phe Gln Ile Ile Gln Leu Pro Asn Arg His

65 70 75 80

Asp Phe His Met Asn Ile Leu Asp Thr Arg Arg Asn Asp Ser Gly Ile

85 90 95

Tyr Leu Cys Gly Ala Ile Ser Leu His Pro Lys Ala Lys Ile Glu Glu

100 105 110

Ser Pro Gly Ala Glu Leu Val Val Thr Glu Arg Ile Leu Glu Thr Ser

115 120 125

Thr Arg Tyr Pro Ser Pro Ser Pro Lys Pro Glu Gly Arg Phe Gln Gly

130 135 140

Met

145

<210> 49

<211> 784

<212> PRT

<213> artificial sequence

<220>

<223> VB5076

<400> 49

Met Asn Phe Gly Leu Arg Leu Ile Phe Leu Val Leu Thr Leu Lys Gly

1 5 10 15

Val Gln Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Thr

20 25 30

Ser Leu Gly Asn Ser Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile

35 40 45

Lys Gly Trp Leu Ala Trp Tyr Gln Gln Lys Ser Gly Asn Ala Pro Gln

50 55 60

Leu Leu Ile Tyr Lys Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg

65 70 75 80

Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ile Phe Thr Ile Ser Asn

85 90 95

Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln His Tyr Gln Ser

100 105 110

Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys

130 135 140

Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

145 150 155 160

Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Phe Tyr Met Asn

165 170 175

Trp Ile Arg Gln Pro Pro Gly Gln Ala Pro Glu Trp Leu Gly Val Ile

180 185 190

Arg Asn Lys Gly Asn Gly Tyr Thr Thr Glu Val Asn Thr Ser Val Lys

195 200 205

Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Gln Asn Ile Leu Tyr Leu

210 215 220

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala

225 230 235 240

Arg Gly Gly Pro Tyr Tyr Tyr Ser Gly Asp Asp Ala Pro Tyr Trp Gly

245 250 255

Gln Gly Val Met Val Thr Val Ser Ser Glu Leu Lys Thr Pro Leu Gly

260 265 270

Asp Thr Thr His Thr Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys

275 280 285

Pro Arg Cys Pro Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Gly Gln

290 295 300

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

305 310 315 320

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

325 330 335

Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn

340 345 350

Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

355 360 365

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile

370 375 380

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln

385 390 395 400

Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly Gly Leu Lys Glu

405 410 415

Val Asp Arg Leu Glu Asp Glu Leu Val Asn Glu Lys Glu Lys Tyr Lys

420 425 430

Ser Ile Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Tyr Lys Glu

435 440 445

Gln Ile Lys Thr Leu Thr Asn Lys Leu Lys Ala Ala Glu Ala Arg Ala

450 455 460

Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Gln Leu Lys Glu

465 470 475 480

Ala Arg Phe Leu Ala Glu Glu Ala Asp Arg Lys Tyr Asp Glu Val Gly

485 490 495

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ala Leu Asn Arg Arg Ile

500 505 510

Gln Leu Leu Glu Glu Asp Leu Glu Arg Ser Glu Glu Arg Gly Gly Gly

515 520 525

Gly Ser Gly Gly Gly Gly Ser Asp Leu Asp Gln Val Gln Glu Ser Leu

530 535 540

Leu Lys Ala Asn Asn Gln Leu Val Glu Lys Asp Gly Gly Gly Gly Ser

545 550 555 560

Gly Gly Gly Gly Ser Glu Gln Gln Asn Lys Glu Ala Asn Asn Arg Ala

565 570 575

Glu Lys Ser Glu Glu Glu Val His Asn Gly Ser Gly Glu Gly Arg Gly

580 585 590

Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met Pro

595 600 605

Gly Ser Ala Leu Leu Cys Cys Leu Leu Leu Leu Thr Gly Met Arg Ile

610 615 620

Ser Arg Gly Gln Tyr Ser Arg Glu Asp Asn Asn Cys Thr His Phe Pro

625 630 635 640

Val Gly Gln Ser His Met Leu Leu Glu Leu Arg Thr Ala Phe Ser Gln

645 650 655

Val Lys Thr Phe Phe Gln Thr Lys Asp Gln Leu Asp Asn Ile Leu Leu

660 665 670

Thr Asp Ser Leu Met Gln Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

675 680 685

Leu Ser Glu Met Ile Gln Phe Tyr Leu Val Glu Val Met Pro Gln Ala

690 695 700

Glu Lys His Gly Pro Glu Ile Lys Glu His Leu Asn Ser Leu Gly Glu

705 710 715 720

Lys Leu Lys Thr Leu Arg Met Arg Leu Arg Arg Cys His Arg Phe Leu

725 730 735

Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Ser Asp Phe

740 745 750

Asn Lys Leu Gln Asp Gln Gly Val Tyr Lys Ala Met Asn Glu Phe Asp

755 760 765

Ile Phe Ile Asn Cys Ile Glu Ala Tyr Met Met Ile Lys Met Lys Ser

770 775 780

<210> 50

<211> 171

<212> PRT

<213> artificial sequence

<220>

<223> Met e 1

<400> 50

Lys Glu Val Asp Arg Leu Glu Asp Glu Leu Val Asn Glu Lys Glu Lys

1 5 10 15

Tyr Lys Ser Ile Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Tyr

20 25 30

Lys Glu Gln Ile Lys Thr Leu Thr Asn Lys Leu Lys Ala Ala Glu Ala

35 40 45

Arg Ala Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Gln Leu

50 55 60

Lys Glu Ala Arg Phe Leu Ala Glu Glu Ala Asp Arg Lys Tyr Asp Glu

65 70 75 80

Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ala Leu Asn Arg

85 90 95

Arg Ile Gln Leu Leu Glu Glu Asp Leu Glu Arg Ser Glu Glu Arg Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Leu Asp Gln Val Gln Glu

115 120 125

Ser Leu Leu Lys Ala Asn Asn Gln Leu Val Glu Lys Asp Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Glu Gln Gln Asn Lys Glu Ala Asn Asn

145 150 155 160

Arg Ala Glu Lys Ser Glu Glu Glu Val His Asn

165 170

<210> 51

<211> 161

<212> PRT

<213> artificial sequence

<220>

<223> hCTLA4 extracellular Domain

<220>

<221> signal

<222> (1)..(35)

<223> Signal peptide

<220>

<221> Domain

<222> (36)..(161)

<223> extracellular Domain

<400> 51

Met Ala Cys Leu Gly Phe Gln Arg His Lys Ala Gln Leu Asn Leu Ala

1 5 10 15

Thr Arg Thr Trp Pro Cys Thr Leu Leu Phe Phe Leu Leu Phe Ile Pro

20 25 30

Val Phe Cys Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala

35 40 45

Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly

50 55 60

Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln

65 70 75 80

Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr

85 90 95

Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val

100 105 110

Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile

115 120 125

Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly

130 135 140

Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser

145 150 155 160

Asp

<210> 52

<211> 170

<212> PRT

<213> artificial sequence

<220>

<223> hPD-1 extracellular Domain

<220>

<221> signal

<222> (1)..(23)

<223> Signal peptide

<220>

<221> Domain

<222> (24)..(170)

<223> excelella domain

<400> 52

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val

165 170

<210> 53

<211> 178

<212> PRT

<213> artificial sequence

<220>

<223> hIL-10

<220>

<221> signal

<222> (1)..(18)

<223> Signal peptide

<400> 53

Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr Gly Val

1 5 10 15

Arg Ala Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His

20 25 30

Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe

35 40 45

Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu

50 55 60

Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys

65 70 75 80

Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro

85 90 95

Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu

100 105 110

Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg

115 120 125

Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn

130 135 140

Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu

145 150 155 160

Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile

165 170 175

Arg Asn

<210> 54

<211> 390

<212> PRT

<213> artificial sequence

<220>

<223> hTGF beta - 1

<220>

<221> signal

<222> (1)..(29)

<223> Signal peptide

<400> 54

Met Pro Pro Ser Gly Leu Arg Leu Leu Leu Leu Leu Leu Pro Leu Leu

1 5 10 15

Trp Leu Leu Val Leu Thr Pro Gly Arg Pro Ala Ala Gly Leu Ser Thr

20 25 30

Cys Lys Thr Ile Asp Met Glu Leu Val Lys Arg Lys Arg Ile Glu Ala

35 40 45

Ile Arg Gly Gln Ile Leu Ser Lys Leu Arg Leu Ala Ser Pro Pro Ser

50 55 60

Gln Gly Glu Val Pro Pro Gly Pro Leu Pro Glu Ala Val Leu Ala Leu

65 70 75 80

Tyr Asn Ser Thr Arg Asp Arg Val Ala Gly Glu Ser Ala Glu Pro Glu

85 90 95

Pro Glu Pro Glu Ala Asp Tyr Tyr Ala Lys Glu Val Thr Arg Val Leu

100 105 110

Met Val Glu Thr His Asn Glu Ile Tyr Asp Lys Phe Lys Gln Ser Thr

115 120 125

His Ser Ile Tyr Met Phe Phe Asn Thr Ser Glu Leu Arg Glu Ala Val

130 135 140

Pro Glu Pro Val Leu Leu Ser Arg Ala Glu Leu Arg Leu Leu Arg Leu

145 150 155 160

Lys Leu Lys Val Glu Gln His Val Glu Leu Tyr Gln Lys Tyr Ser Asn

165 170 175

Asn Ser Trp Arg Tyr Leu Ser Asn Arg Leu Leu Ala Pro Ser Asp Ser

180 185 190

Pro Glu Trp Leu Ser Phe Asp Val Thr Gly Val Val Arg Gln Trp Leu

195 200 205

Ser Arg Gly Gly Glu Ile Glu Gly Phe Arg Leu Ser Ala His Cys Ser

210 215 220

Cys Asp Ser Arg Asp Asn Thr Leu Gln Val Asp Ile Asn Gly Phe Thr

225 230 235 240

Thr Gly Arg Arg Gly Asp Leu Ala Thr Ile His Gly Met Asn Arg Pro

245 250 255

Phe Leu Leu Leu Met Ala Thr Pro Leu Glu Arg Ala Gln His Leu Gln

260 265 270

Ser Ser Arg His Arg Arg Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser

275 280 285

Thr Glu Lys Asn Cys Cys Val Arg Gln Leu Tyr Ile Asp Phe Arg Lys

290 295 300

Asp Leu Gly Trp Lys Trp Ile His Glu Pro Lys Gly Tyr His Ala Asn

305 310 315 320

Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Ser Leu Asp Thr Gln Tyr

325 330 335

Ser Lys Val Leu Ala Leu Tyr Asn Gln His Asn Pro Gly Ala Ser Ala

340 345 350

Ala Pro Cys Cys Val Pro Gln Ala Leu Glu Pro Leu Pro Ile Val Tyr

355 360 365

Tyr Val Gly Arg Lys Pro Lys Val Glu Gln Leu Ser Asn Met Ile Val

370 375 380

Arg Ser Cys Lys Cys Ser

385 390

<210> 55

<211> 153

<212> PRT

<213> artificial sequence

<220>

<223> hIL-2

<220>

<221> signal

<222> (1)..(20)

<223> Signal peptide

<400> 55

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu

20 25 30

Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile

35 40 45

Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe

50 55 60

Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu

65 70 75 80

Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

85 90 95

Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile

100 105 110

Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala

115 120 125

Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe

130 135 140

Cys Gln Ser Ile Ile Ser Thr Leu Thr

145 150

<210> 56

<211> 144

<212> PRT

<213> artificial sequence

<220>

<223> hGM-CSF

<220>

<221> signal

<222> (1)..(17)

<223> Signal peptide

<400> 56

Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile

1 5 10 15

Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His

20 25 30

Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp

35 40 45

Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe

50 55 60

Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys

65 70 75 80

Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met

85 90 95

Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser

100 105 110

Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys

115 120 125

Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu

130 135 140

<210> 57

<211> 166

<212> PRT

<213> artificial sequence

<220>

<223> hIFN- gamma

<220>

<221> signal

<222> (1)..(23)

<223> Signal peptide

<400> 57

Met Lys Tyr Thr Ser Tyr Ile Leu Ala Phe Gln Leu Cys Ile Val Leu

1 5 10 15

Gly Ser Leu Gly Cys Tyr Cys Gln Asp Pro Tyr Val Lys Glu Ala Glu

20 25 30

Asn Leu Lys Lys Tyr Phe Asn Ala Gly His Ser Asp Val Ala Asp Asn

35 40 45

Gly Thr Leu Phe Leu Gly Ile Leu Lys Asn Trp Lys Glu Glu Ser Asp

50 55 60

Arg Lys Ile Met Gln Ser Gln Ile Val Ser Phe Tyr Phe Lys Leu Phe

65 70 75 80

Lys Asn Phe Lys Asp Asp Gln Ser Ile Gln Lys Ser Val Glu Thr Ile

85 90 95

Lys Glu Asp Met Asn Val Lys Phe Phe Asn Ser Asn Lys Lys Lys Arg

100 105 110

Asp Asp Phe Glu Lys Leu Thr Asn Tyr Ser Val Thr Asp Leu Asn Val

115 120 125

Gln Arg Lys Ala Ile His Glu Leu Ile Gln Val Met Ala Glu Leu Ser

130 135 140

Pro Ala Ala Lys Thr Gly Lys Arg Lys Arg Ser Gln Met Leu Phe Arg

145 150 155 160

Gly Arg Arg Ala Ser Gln

165

<210> 58

<211> 442

<212> PRT

<213> artificial sequence

<220>

<223> hTGF beta 2

<400> 58

Met His Tyr Cys Val Leu Ser Ala Phe Leu Ile Leu His Leu Val Thr

1 5 10 15

Val Ala Leu Ser Leu Ser Thr Cys Ser Thr Leu Asp Met Asp Gln Phe

20 25 30

Met Arg Lys Arg Ile Glu Ala Ile Arg Gly Gln Ile Leu Ser Lys Leu

35 40 45

Lys Leu Thr Ser Pro Pro Glu Asp Tyr Pro Glu Pro Glu Glu Val Pro

50 55 60

Pro Glu Val Ile Ser Ile Tyr Asn Ser Thr Arg Asp Leu Leu Gln Glu

65 70 75 80

Lys Ala Ser Arg Arg Ala Ala Ala Cys Glu Arg Glu Arg Ser Asp Glu

85 90 95

Glu Tyr Tyr Ala Lys Glu Val Tyr Lys Ile Asp Met Pro Pro Phe Phe

100 105 110

Pro Ser Glu Thr Val Cys Pro Val Val Thr Thr Pro Ser Gly Ser Val

115 120 125

Gly Ser Leu Cys Ser Arg Gln Ser Gln Val Leu Cys Gly Tyr Leu Asp

130 135 140

Ala Ile Pro Pro Thr Phe Tyr Arg Pro Tyr Phe Arg Ile Val Arg Phe

145 150 155 160

Asp Val Ser Ala Met Glu Lys Asn Ala Ser Asn Leu Val Lys Ala Glu

165 170 175

Phe Arg Val Phe Arg Leu Gln Asn Pro Lys Ala Arg Val Pro Glu Gln

180 185 190

Arg Ile Glu Leu Tyr Gln Ile Leu Lys Ser Lys Asp Leu Thr Ser Pro

195 200 205

Thr Gln Arg Tyr Ile Asp Ser Lys Val Val Lys Thr Arg Ala Glu Gly

210 215 220

Glu Trp Leu Ser Phe Asp Val Thr Asp Ala Val His Glu Trp Leu His

225 230 235 240

His Lys Asp Arg Asn Leu Gly Phe Lys Ile Ser Leu His Cys Pro Cys

245 250 255

Cys Thr Phe Val Pro Ser Asn Asn Tyr Ile Ile Pro Asn Lys Ser Glu

260 265 270

Glu Leu Glu Ala Arg Phe Ala Gly Ile Asp Gly Thr Ser Thr Tyr Thr

275 280 285

Ser Gly Asp Gln Lys Thr Ile Lys Ser Thr Arg Lys Lys Asn Ser Gly

290 295 300

Lys Thr Pro His Leu Leu Leu Met Leu Leu Pro Ser Tyr Arg Leu Glu

305 310 315 320

Ser Gln Gln Thr Asn Arg Arg Lys Lys Arg Ala Leu Asp Ala Ala Tyr

325 330 335

Cys Phe Arg Asn Val Gln Asp Asn Cys Cys Leu Arg Pro Leu Tyr Ile

340 345 350

Asp Phe Lys Arg Asp Leu Gly Trp Lys Trp Ile His Glu Pro Lys Gly

355 360 365

Tyr Asn Ala Asn Phe Cys Ala Gly Ala Cys Pro Tyr Leu Trp Ser Ser

370 375 380

Asp Thr Gln His Ser Arg Val Leu Ser Leu Tyr Asn Thr Ile Asn Pro

385 390 395 400

Glu Ala Ser Ala Ser Pro Cys Cys Val Ser Gln Asp Leu Glu Pro Leu

405 410 415

Thr Ile Leu Tyr Tyr Ile Gly Lys Thr Pro Lys Ile Glu Gln Leu Ser

420 425 430

Asn Met Ile Val Lys Ser Cys Lys Cys Ser

435 440

<210> 59

<211> 412

<212> PRT

<213> artificial sequence

<220>

<223> hTGF beta 3

<400> 59

Met Lys Met His Leu Gln Arg Ala Leu Val Val Leu Ala Leu Leu Asn

1 5 10 15

Phe Ala Thr Val Ser Leu Ser Leu Ser Thr Cys Thr Thr Leu Asp Phe

20 25 30

Gly His Ile Lys Lys Lys Arg Val Glu Ala Ile Arg Gly Gln Ile Leu

35 40 45

Ser Lys Leu Arg Leu Thr Ser Pro Pro Glu Pro Thr Val Met Thr His

50 55 60

Val Pro Tyr Gln Val Leu Ala Leu Tyr Asn Ser Thr Arg Glu Leu Leu

65 70 75 80

Glu Glu Met His Gly Glu Arg Glu Glu Gly Cys Thr Gln Glu Asn Thr

85 90 95

Glu Ser Glu Tyr Tyr Ala Lys Glu Ile His Lys Phe Asp Met Ile Gln

100 105 110

Gly Leu Ala Glu His Asn Glu Leu Ala Val Cys Pro Lys Gly Ile Thr

115 120 125

Ser Lys Val Phe Arg Phe Asn Val Ser Ser Val Glu Lys Asn Arg Thr

130 135 140

Asn Leu Phe Arg Ala Glu Phe Arg Val Leu Arg Val Pro Asn Pro Ser

145 150 155 160

Ser Lys Arg Asn Glu Gln Arg Ile Glu Leu Phe Gln Ile Leu Arg Pro

165 170 175

Asp Glu His Ile Ala Lys Gln Arg Tyr Ile Gly Gly Lys Asn Leu Pro

180 185 190

Thr Arg Gly Thr Ala Glu Trp Leu Ser Phe Asp Val Thr Asp Thr Val

195 200 205

Arg Glu Trp Leu Leu Arg Arg Glu Ser Asn Leu Gly Leu Glu Ile Ser

210 215 220

Ile His Cys Pro Cys His Thr Phe Gln Pro Asn Gly Asp Ile Leu Glu

225 230 235 240

Asn Ile His Glu Val Met Glu Ile Lys Phe Lys Gly Val Asp Asn Glu

245 250 255

Asp Asp His Gly Arg Gly Asp Leu Gly Arg Leu Lys Lys Gln Lys Asp

260 265 270

His His Asn Pro His Leu Ile Leu Met Met Ile Pro Pro His Arg Leu

275 280 285

Asp Asn Pro Gly Gln Gly Gly Gln Arg Lys Lys Arg Ala Leu Asp Thr

290 295 300

Asn Tyr Cys Phe Arg Asn Leu Glu Glu Asn Cys Cys Val Arg Pro Leu

305 310 315 320

Tyr Ile Asp Phe Arg Gln Asp Leu Gly Trp Lys Trp Val His Glu Pro

325 330 335

Lys Gly Tyr Tyr Ala Asn Phe Cys Ser Gly Pro Cys Pro Tyr Leu Arg

340 345 350

Ser Ala Asp Thr Thr His Ser Thr Val Leu Gly Leu Tyr Asn Thr Leu

355 360 365

Asn Pro Glu Ala Ser Ala Ser Pro Cys Cys Val Pro Gln Asp Leu Glu

370 375 380

Pro Leu Thr Ile Leu Tyr Tyr Val Gly Arg Thr Pro Lys Val Glu Gln

385 390 395 400

Leu Ser Asn Met Val Val Lys Ser Cys Lys Cys Ser

405 410

<210> 60

<211> 1173

<212> DNA

<213> artificial sequence

<220>

<223> hTGF beta 1

<400> 60

atgccgccct ccgggctgcg gctgctgccg ctgctgctac cgctgctgtg gctactggtg 60

ctgacgcctg gccggccggc cgcgggacta tccacctgca agactatcga catggagctg 120

gtgaagcgga agcgcatcga ggccatccgc ggccagatcc tgtccaagct gcggctcgcc 180

agccccccga gccaggggga ggtgccgccc ggcccgctgc ccgaggccgt gctcgccctg 240

tacaacagca cccgcgaccg ggtggccggg gagagtgcag aaccggagcc cgagcctgag 300

gccgactact acgccaagga ggtcacccgc gtgctaatgg tggaaaccca caacgaaatc 360

tatgacaagt tcaagcagag tacacacagc atatatatgt tcttcaacac atcagagctc 420

cgagaagcgg tacctgaacc cgtgttgctc tcccgggcag agctgcgtct gctgaggctc 480

aagttaaaag tggagcagca cgtggagctg taccagaaat acagcaacaa ttcctggcga 540

tacctcagca accggctgct ggcacccagc gactcgccag agtggttatc ttttgatgtc 600

accggagttg tgcggcagtg gttgagccgt ggaggggaaa ttgagggctt tcgccttagc 660

gcccactgct cctgtgacag cagggataac acactgcaag tggacatcaa cgggttcact 720

accggccgcc gaggtgacct ggccaccatt catggcatga accggccttt cctgcttctc 780

atggccaccc cgctggagag ggcccagcat ctgcaaagct cccggcaccg ccgagccctg 840

gacaccaact attgcttcag ctccacggag aagaactgct gcgtgcggca gctgtacatt 900

gacttccgca aggacctcgg ctggaagtgg atccacgagc ccaagggcta ccatgccaac 960

ttctgcctcg ggccctgccc ctacatttgg agcctggaca cgcagtacag caaggtcctg 1020

gccctgtaca accagcataa cccgggcgcc tcggcggcgc cgtgctgcgt gccgcaggcg 1080

ctggagccgc tgcccatcgt gtactacgtg ggccgcaagc ccaaggtgga gcagctgtcc 1140

aacatgatcg tgcgctcctg caagtgcagc tga 1173

<210> 61

<211> 1329

<212> DNA

<213> artificial sequence

<220>

<223> hTGF beta 2

<400> 61

atgcactact gtgtgctgag cgcttttctg atcctgcatc tggtcacggt cgcgctcagc 60

ctgtctacct gcagcacact cgatatggac cagttcatgc gcaagaggat cgaggcgatc 120

cgcgggcaga tcctgagcaa gctgaagctc accagtcccc cagaagacta tcctgagccc 180

gaggaagtcc ccccggaggt gatttccatc tacaacagca ccagggactt gctccaggag 240

aaggcgagcc ggagggcggc cgcctgcgag cgcgagagga gcgacgaaga gtactacgcc 300

aaggaggttt acaaaataga catgccgccc ttcttcccct ccgaaactgt ctgcccagtt 360

gttacaacac cctctggctc agtgggcagc ttgtgctcca gacagtccca ggtgctctgt 420

gggtaccttg atgccatccc gcccactttc tacagaccct acttcagaat tgttcgattt 480

gacgtctcag caatggagaa gaatgcttcc aatttggtga aagcagagtt cagagtcttt 540

cgtttgcaga acccaaaagc cagagtgcct gaacaacgga ttgagctata tcagattctc 600

aagtccaaag atttaacatc tccaacccag cgctacatcg acagcaaagt tgtgaaaaca 660

agagcagaag gcgaatggct ctccttcgat gtaactgatg ctgttcatga atggcttcac 720

cataaagaca ggaacctggg atttaaaata agcttacact gtccctgctg cacttttgta 780

ccatctaata attacatcat cccaaataaa agtgaagaac tagaagcaag atttgcaggt 840

attgatggca cctccacata taccagtggt gatcagaaaa ctataaagtc cactaggaaa 900

aaaaacagtg ggaagacccc acatctcctg ctaatgttat tgccctccta cagacttgag 960

tcacaacaga ccaaccggcg gaagaagcgt gctttggatg cggcctattg ctttagaaat 1020

gtgcaggata attgctgcct acgtccactt tacattgatt tcaagaggga tctagggtgg 1080

aaatggatac acgaacccaa agggtacaat gccaacttct gtgctggagc atgcccgtat 1140

ttatggagtt cagacactca gcacagcagg gtcctgagct tatataatac cataaatcca 1200

gaagcatctg cttctccttg ctgcgtgtcc caagatttag aacctctaac cattctctac 1260

tacattggca aaacacccaa gattgaacag ctttctaata tgattgtaaa gtcttgcaaa 1320

tgcagctaa 1329

<210> 62

<211> 930

<212> DNA

<213> artificial sequence

<220>

<223> hTGF beta 3

<400> 62

atgaagatgc acttgcaaag ggctctggtg gtcctggccc tgctgaactt tgccacggtc 60

agcctctctc tgtccacttg caccaccttg gacttcggcc acatcaagaa gaagagggtg 120

gaagccatta ggggacagat cttgagcaag ctcaggctca ccagcccccc tgagccaacg 180

gtgatgaccc acgtccccta tcaggtcctg gccctttaca acagcacccg ggagctgctg 240

gaggagatgc atggggagag ggaggaaggc tgcacccagg aaaacaccga gtcggaatac 300

tatgccaaag aaatccataa attcgacatg atccaggggc tggcggagca caacgaactg 360

gctgtctgcc ctaaaggaat tacctccaag gttttccgct tcaatgtgtc ctcagtggag 420

aaaaatagaa ccaacctatt ccgagcagaa ttccgggtct tgcgggtgcc caaccccagc 480

tctaagcgga atgagcagag gatcgagctc ttccagatcc ttcggccaga tgagcacatt 540

gccaaacagc gctatatcgg tggcaagaat ctgcccacac ggggcactgc cgagtggctg 600

tcctttgatg tcactgacac tgtgcgtgag tggctgttga gaagagagtc caacttaggt 660

ctagaaatca gcattcactg tccatgtcac acctttcagc ccaatggaga tatcctggaa 720

aacattcacg aggtgatgga aatcaaattc aaaggcgtgg acaatgagga tgaccatggc 780

cgtggagatc tggggcgcct caagaagcag aaggatcacc acaaccctca tctaatcctc 840

atgatgattc ccccacaccg gctcgacaac ccgggccagg ggggtcagag gaagaagcgg 900

gctttggaca ccaattactg cttccggtga 930

<210> 63

<211> 537

<212> DNA

<213> artificial sequence

<220>

<223> hIL-10

<400> 63

atgcacagct cagcactgct ctgttgcctg gtcctcctga ctggggtgag ggccagccca 60

ggccagggca cccagtctga gaacagctgc acccacttcc caggcaacct gcctaacatg 120

cttcgagatc tccgagatgc cttcagcaga gtgaagactt tctttcaaat gaaggatcag 180

ctggacaact tgttgttaaa ggagtccttg ctggaggact ttaagggtta cctgggttgc 240

caagccttgt ctgagatgat ccagttttac ctggaggagg tgatgcccca agctgagaac 300

caagacccag acatcaaggc gcatgtgaac tccctggggg agaacctgaa gaccctcagg 360

ctgaggctac ggcgctgtca tcgatttctt ccctgtgaaa acaagagcaa ggccgtggag 420

caggtgaaga atgcctttaa taagctccaa gagaaaggca tctacaaagc catgagtgag 480

tttgacatct tcatcaacta catagaagcc tacatgacaa tgaagatacg aaactga 537

<210> 64

<211> 93

<212> PRT

<213> artificial sequence

<220>

<223> hSCGB3A2

<220>

<221> signal

<222> (1)..(21)

<223> Signal peptide

<400> 64

Met Lys Leu Val Thr Ile Phe Leu Leu Val Thr Ile Ser Leu Cys Ser

1 5 10 15

Tyr Ser Ala Thr Ala Phe Leu Ile Asn Lys Val Pro Leu Pro Val Asp

20 25 30

Lys Leu Ala Pro Leu Pro Leu Asp Asn Ile Leu Pro Phe Met Asp Pro

35 40 45

Leu Lys Leu Leu Leu Lys Thr Leu Gly Ile Ser Val Glu His Leu Val

50 55 60

Glu Gly Leu Arg Lys Cys Val Asn Glu Leu Gly Pro Glu Ala Ser Glu

65 70 75 80

Ala Val Lys Lys Leu Leu Glu Ala Leu Ser His Leu Val

85 90

<210> 65

<211> 282

<212> DNA

<213> artificial sequence

<220>

<223> hSCGB3A2

<400> 65

atgaagctgg taactatctt cctgctggtg accatcagcc tttgtagtta ctctgctact 60

gccttcctca tcaacaaagt gccccttcct gttgacaagt tggcaccttt acctctggac 120

aacattcttc cctttatgga tccattaaag cttcttctga aaactctggg catttctgtt 180

gagcaccttg tggaggggct aaggaagtgt gtaaatgagc tgggaccaga ggcttctgaa 240

gctgtgaaga aactgctgga ggcgctatca cacttggtgt ga 282

<210> 66

<211> 241

<212> PRT

<213> artificial sequence

<220>

<223> hVSIG-3

<220>

<221> signal

<222> (1)..(22)

<223> Signal peptide

<220>

<221> Domain

<222> (23)..(241)

<223> extracellular Domain

<400> 66

Met Thr Ser Gln Arg Ser Pro Leu Ala Pro Leu Leu Leu Leu Ser Leu

1 5 10 15

His Gly Val Ala Ala Ser Leu Glu Val Ser Glu Ser Pro Gly Ser Ile

20 25 30

Gln Val Ala Arg Gly Gln Pro Ala Val Leu Pro Cys Thr Phe Thr Thr

35 40 45

Ser Ala Ala Leu Ile Asn Leu Asn Val Ile Trp Met Val Thr Pro Leu

50 55 60

Ser Asn Ala Asn Gln Pro Glu Gln Val Ile Leu Tyr Gln Gly Gly Gln

65 70 75 80

Met Phe Asp Gly Ala Pro Arg Phe His Gly Arg Val Gly Phe Thr Gly

85 90 95

Thr Met Pro Ala Thr Asn Val Ser Ile Phe Ile Asn Asn Thr Gln Leu

100 105 110

Ser Asp Thr Gly Thr Tyr Gln Cys Leu Val Asn Asn Leu Pro Asp Ile

115 120 125

Gly Gly Arg Asn Ile Gly Val Thr Gly Leu Thr Val Leu Val Pro Pro

130 135 140

Ser Ala Pro His Cys Gln Ile Gln Gly Ser Gln Asp Ile Gly Ser Asp

145 150 155 160

Val Ile Leu Leu Cys Ser Ser Glu Glu Gly Ile Pro Arg Pro Thr Tyr

165 170 175

Leu Trp Glu Lys Leu Asp Asn Thr Leu Lys Leu Pro Pro Thr Ala Thr

180 185 190

Gln Asp Gln Val Gln Gly Thr Val Thr Ile Arg Asn Ile Ser Ala Leu

195 200 205

Ser Ser Gly Leu Tyr Gln Cys Val Ala Ser Asn Ala Ile Gly Thr Ser

210 215 220

Thr Cys Leu Leu Asp Leu Gln Val Ile Ser Pro Gln Pro Arg Asn Ile

225 230 235 240

Gly

<210> 67

<211> 723

<212> DNA

<213> artificial sequence

<220>

<223> hVSIG-3 extracellular Domain

<220>

<221> misc_feature

<222> (1)..(723)

<223> extracellular Domain

<400> 67

atgacttctc agcgttcccc tctggcgcct ttgctgctcc tctctctgca cggtgttgca 60

gcatccctgg aagtgtcaga gagccctggg agtatccagg tggcccgggg tcagccagca 120

gtcctgccct gcactttcac taccagcgct gccctcatta acctcaatgt catttggatg 180

gtcactcctc tctccaatgc caaccaacct gaacaggtca tcctgtatca gggtggacag 240

atgtttgatg gtgccccccg gttccacggt agggtaggat ttacaggcac catgccagct 300

accaatgtct ctatcttcat taataacact cagttatcag acactggcac ctaccagtgc 360

ctggtcaaca accttccaga catagggggc aggaacattg gggtcaccgg tctcacagtg 420

ttagttcccc cttctgcccc acactgccaa atccaaggat cccaggatat tggcagcgat 480

gtcatcctgc tctgtagctc agaggaaggc attcctcgac caacttacct ttgggagaag 540

ttagacaata ccctcaaact acctccaaca gctactcagg accaggtcca gggaacagtc 600

accatccgga acatcagtgc cctgtcttca ggtttgtacc agtgcgtggc ttctaatgct 660

attggaacca gcacctgtct tctggatctc caggttattt caccccagcc caggaacatt 720

gga 723

<210> 68

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> 2A consensus sequence of self-cleaving peptide, X may be any amino acid

<220>

<221> site

<222> (2)..(2)

<223> X may be any amino acid

<220>

<221> site

<222> (4)..(4)

<223> X may be any amino acid

<400> 68

Asp Xaa Glu Xaa Asn Pro Gly Pro

1 5

<210> 69

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> self-cleaving peptides

<400> 69

Asp Val Glu Glu Asn Pro Gly Pro

1 5

<210> 70

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> self-cleaving peptides

<400> 70

Asp Val Glu Ser Asn Pro Gly Pro

1 5

<210> 71

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> self-cleaving peptides

<400> 71

Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu

1 5 10 15

Glu Asn Pro Gly Pro

20

<210> 72

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> self-cleaving peptides

<400> 72

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210> 73

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> self-cleaving peptides

<400> 73

Gly Ser Gly Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp

1 5 10 15

Val Glu Ser Asn Pro Gly Pro

20

<210> 74

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> self-cleaving peptides

<400> 74

Gly Ser Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala

1 5 10 15

Gly Asp Val Glu Ser Asn Pro Gly Pro

20 25

<210> 75

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 75

Gly Gly Gly Gly Ser Gly Gly Gly Ser Ser

1 5 10

<210> 76

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 76

Gly Gly Gly Ser Gly

1 5

<210> 77

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 77

Gly Gly Ser Gly Gly

1 5

<210> 78

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 78

Ser Gly Ser Ser Gly Ser

1 5

<210> 79

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 79

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 80

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<220>

<221> site

<222> (1)..(5)

<223> may be repeated m times, where m is an integer of 1 to 5

<400> 80

Gly Gly Gly Gly Ser

1 5

<210> 81

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<220>

<221> site

<222> (1)..(5)

<223> may be repeated m times, where m is an integer of 1 to 5

<400> 81

Gly Gly Gly Ser Ser

1 5

<210> 82

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<220>

<221> site

<222> (1)..(5)

<223> may be repeated m times, where m is an integer of 1 to 5

<400> 82

Gly Gly Ser Gly Gly

1 5

<210> 83

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<220>

<221> site

<222> (1)..(5)

<223> may be repeated m times, where m is an integer of 1 to 5

<400> 83

Gly Gly Gly Ser Gly

1 5

<210> 84

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> joint

<220>

<221> site

<222> (1)..(6)

<223> may be repeated m times, where m is an integer of 1 to 5

<400> 84

Ser Gly Ser Ser Gly Ser

1 5

<210> 85

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 85

Leu Gly Gly Gly Ser

1 5

<210> 86

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 86

Gly Leu Gly Gly Ser

1 5

<210> 87

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 87

Gly Gly Leu Gly Ser

1 5

<210> 88

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 88

Gly Gly Gly Leu Ser

1 5

<210> 89

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 89

Gly Gly Gly Gly Leu

1 5

<210> 90

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 90

Leu Gly Gly Ser Gly

1 5

<210> 91

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 91

Gly Leu Gly Ser Gly

1 5

<210> 92

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 92

Gly Gly Leu Ser Gly

1 5

<210> 93

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 93

Gly Gly Gly Leu Gly

1 5

<210> 94

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 94

Gly Gly Gly Ser Leu

1 5

<210> 95

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 95

Leu Gly Gly Ser Ser

1 5

<210> 96

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 96

Gly Leu Gly Ser Ser

1 5

<210> 97

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 97

Gly Gly Leu Ser Ser

1 5

<210> 98

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 98

Leu Gly Leu Gly Ser

1 5

<210> 99

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 99

Gly Leu Gly Leu Ser

1 5

<210> 100

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 100

Gly Leu Leu Gly Ser

1 5

<210> 101

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 101

Leu Gly Gly Leu Ser

1 5

<210> 102

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 102

Gly Leu Gly Gly Leu

1 5

<210> 103

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 103

Leu Gly Leu Ser Gly

1 5

<210> 104

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 104

Gly Leu Leu Ser Gly

1 5

<210> 105

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 105

Gly Gly Leu Ser Leu

1 5

<210> 106

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 106

Gly Gly Leu Leu Gly

1 5

<210> 107

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 107

Gly Leu Gly Ser Leu

1 5

<210> 108

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 108

Leu Gly Leu Ser Ser

1 5

<210> 109

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 109

Gly Gly Leu Leu Ser

1 5

<210> 110

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 110

Leu Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 111

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 111

Gly Leu Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 112

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 112

Gly Gly Leu Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 113

<211> 117

<212> PRT

<213> artificial sequence

<220>

<223> tetramerization Unit

<400> 113

Ala Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala Cys Gly Gly Ala Gly

1 5 10 15

Ala Gly Thr Ala Thr Thr Thr Cys Ala Cys Thr Cys Thr Cys Cys Ala

20 25 30

Gly Ala Thr Cys Cys Gly Gly Gly Gly Cys Cys Cys Cys Gly Ala Ala

35 40 45

Ala Gly Gly Thr Thr Cys Gly Ala Ala Ala Thr Gly Thr Thr Cys Cys

50 55 60

Gly Gly Gly Ala Gly Cys Thr Thr Ala Ala Cys Gly Ala Gly Gly Cys

65 70 75 80

Cys Thr Thr Gly Gly Ala Gly Cys Thr Gly Ala Ala Ala Gly Ala Cys

85 90 95

Gly Cys Ala Cys Ala Gly Gly Cys Cys Gly Gly Ala Ala Ala Gly Gly

100 105 110

Ala Ala Cys Cys Gly

115

<210> 114

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 114

Gly Gly Gly Gly Leu Gly Gly Gly Gly Ser

1 5 10

<210> 115

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 115

Leu Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10

<210> 116

<211> 180

<212> DNA

<213> artificial sequence

<220>

<223> trimerization unit

<400> 116

gctgggcagg tgaggatctg ggccacatac cagaccatgc tggacaagat ccgggaggtg 60

ccggagggct ggctcatctt tgtggccgag agggaagagc tctatgtacg cgttagaaat 120

ggcttccgga aggtgctgct ggaggcccgg acagccctcc cgagaggcac gggcaatgag 180

<210> 117

<211> 27

<212> PRT

<213> artificial sequence

<220>

<223> trimerization unit

<400> 117

Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys

1 5 10 15

Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

20 25

<210> 118

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 118

Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly

1 5 10

<210> 119

<211> 333

<212> DNA

<213> artificial sequence

<220>

<223> IgM hinge

<400> 119

gccgaactcc cgcccaaggt gtccgtgttc gtccctcccc gcgatgggtt cttcggcaat 60

ccacgaaaat ccaaactgat ttgtcaggcc accggcttct ccccccgaca gatccaggtg 120

agttggctac gagagggtaa acaggtgggg agcggagtga ccactgacca ggtgcaggcc 180

gaggccaagg aaagcggacc cacaacatac aaagtgacaa gcactctgac gattaaggag 240

tcagactggc tcggccaatc catgtttaca tgccgggttg atcacagagg gttgaccttc 300

caacagaacg catccagtat gtgcgttcca gat 333

<210> 120

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> t cell epitope

<400> 120

Ser Ile Ile Asn Phe Glu Lys Leu

1 5

<210> 121

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 121

Leu Gly Gly Ser Ser Gly Gly Gly Ser Ser

1 5 10

<210> 122

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 122

Gly Leu Gly Ser Ser Gly Gly Gly Ser Ser

1 5 10

<210> 123

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 123

Gly Gly Leu Ser Ser Gly Gly Gly Ser Ser

1 5 10

<210> 124

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 124

Gly Gly Gly Leu Ser Gly Gly Gly Ser Ser

1 5 10

<210> 125

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 125

Gly Gly Gly Ser Leu Gly Gly Gly Ser Ser

1 5 10

<210> 126

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 126

Leu Gly Gly Gly Ser Leu Gly Gly Gly Ser

1 5 10

<210> 127

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 127

Gly Leu Gly Gly Ser Gly Leu Gly Gly Ser

1 5 10

<210> 128

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 128

Gly Gly Leu Gly Ser Gly Gly Leu Gly Ser

1 5 10

<210> 129

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 129

Gly Gly Gly Leu Ser Gly Gly Gly Leu Ser

1 5 10

<210> 130

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 130

Gly Gly Gly Gly Leu Gly Gly Gly Gly Leu

1 5 10

<210> 131

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 131

Leu Gly Gly Ser Gly Leu Gly Gly Ser Gly

1 5 10

<210> 132

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 132

Gly Leu Gly Ser Gly Gly Leu Gly Ser Gly

1 5 10

<210> 133

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 133

Gly Gly Leu Ser Gly Gly Gly Leu Ser Gly

1 5 10

<210> 134

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 134

Gly Gly Gly Leu Gly Gly Gly Gly Leu Gly

1 5 10

<210> 135

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 135

Gly Gly Gly Ser Leu Gly Gly Gly Ser Leu

1 5 10

<210> 136

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 136

Leu Gly Gly Ser Ser Leu Gly Gly Ser Ser

1 5 10

<210> 137

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 137

Gly Leu Gly Ser Ser Gly Leu Gly Ser Ser

1 5 10

<210> 138

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 138

Gly Gly Leu Ser Ser Gly Gly Leu Ser Ser

1 5 10

<210> 139

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 139

Gly Ser Gly Gly Gly Ala

1 5

<210> 140

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 140

Gly Ser Gly Gly Gly Ala Gly Ser Gly Gly Gly Ala

1 5 10

<210> 141

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 141

Gly Ser Gly Gly Gly Ala Gly Ser Gly Gly Gly Ala Gly Ser Gly Gly

1 5 10 15

Gly Ala

<210> 142

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 142

Gly Ser Gly Gly Gly Ala Gly Ser Gly Gly Gly Ala Gly Ser Gly Gly

1 5 10 15

Gly Ala Gly Ser Gly Gly Gly Ala

20

<210> 143

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 143

Gly Glu Asn Leu Tyr Phe Gln Ser Gly Gly

1 5 10

<210> 144

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 144

Ser Gly Gly Gly Ser Ser Gly Gly Gly Ser

1 5 10

<210> 145

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 145

Ser Ser Gly Gly Gly Ser Ser Gly Gly Gly

1 5 10

<210> 146

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 146

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

1 5 10

<210> 147

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 147

Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser

1 5 10

<210> 148

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 148

Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly

1 5 10

<210> 149

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 149

Gly Gly Gly Ser Ser Ser

1 5

<210> 150

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 150

Gly Gly Gly Ser Ser Gly Gly Gly Ser Ser Gly Gly Gly Ser Ser

1 5 10 15

<210> 151

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 151

Gly Leu Gly Gly Leu Ala Ala Ala

1 5

<210> 152

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 152

Lys Pro Glu Pro Lys Pro Ala Pro Ala Pro Lys Pro

1 5 10

<210> 153

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 153

Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala

1 5 10

<210> 154

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<220>

<221> site

<222> (1)..(5)

<223> may be repeated m times, where m is an integer of 1 to 5

<400> 154

Glu Ala Ala Ala Lys

1 5

<210> 155

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 155

Gly Gly Gly Leu Ser Gly Gly Gly Gly Ser

1 5 10

<210> 156

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 156

Gly Leu Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10

<210> 157

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 157

Gly Gly Leu Ser Gly Gly Gly Gly Ser Gly

1 5 10

<210> 158

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 158

Gly Gly Gly Leu Gly Gly Gly Gly Ser Gly

1 5 10

<210> 159

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 159

Gly Gly Gly Ser Leu Gly Gly Gly Ser Gly

1 5 10

<210> 160

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 160

Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu Pro

1 5 10 15

<210> 161

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 161

Ser Ala Cys Tyr Cys Glu Leu Ser

1 5

<210> 162

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 162

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly Gly Leu

1 5 10 15

<210> 163

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 163

Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly Gly Leu

1 5 10

<210> 164

<211> 36

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 164

Gly Gly Ser Ala Gly Gly Ser Gly Ser Gly Ser Ser Gly Gly Ser Ser

1 5 10 15

Gly Ala Ser Gly Thr Gly Thr Ala Gly Gly Thr Gly Ser Gly Ser Gly

20 25 30

Thr Gly Ser Gly

35

<210> 165

<211> 36

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 165

Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

1 5 10 15

Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser

20 25 30

Gly Gly Gly Ser

35

<210> 166

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 166

Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

1 5 10 15

<210> 167

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> T cell epitope

<400> 167

His Cys Leu Gly Lys Trp Leu Gly His Pro Asp Lys Phe

1 5 10

<210> 168

<211> 29

<212> PRT

<213> artificial sequence

<220>

<223> T cell epitope

<400> 168

Ala His Ser Leu Glu Arg Val Cys His Cys Leu Gly Lys Trp Leu Gly

1 5 10 15

His Pro Asp Lys Phe Val Gly Ile Thr Tyr Ala Leu Thr

20 25

<210> 169

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> T cell epitope

<400> 169

Asn Thr Trp Thr Thr Cys Gln Ser Ile Ala Phe Pro Ser Lys

1 5 10

<210> 170

<211> 30

<212> PRT

<213> artificial sequence

<220>

<223> T cell epitope

<400> 170

Ala Val Pro Val Tyr Ile Tyr Phe Asn Thr Trp Thr Thr Cys Gln Ser

1 5 10 15

Ile Ala Phe Pro Ser Lys Thr Ser Ala Ser Ile Gly Ser Leu

20 25 30

<210> 171

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> T cell epitope

<400> 171

Val His Phe Phe Lys Asn Ile Val Thr Pro Arg Thr Pro Pro Pro Ser

1 5 10 15

Gln Gly Lys Gly Arg

20

<210> 172

<211> 36

<212> PRT

<213> artificial sequence

<220>

<223> T cell epitope

<400> 172

Arg Thr Gln Asp Glu Asn Pro Val Val His Phe Phe Lys Asn Ile Val

1 5 10 15

Thr Pro Arg Thr Pro Pro Pro Ser Gln Gly Lys Gly Arg Gly Leu Ser

20 25 30

Leu Ser Arg Phe

35

<210> 173

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 173

His Glu Tyr Gly Ala Glu Ala Leu Glu Arg Ala Gly

1 5 10

<210> 174

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 174

Gly Leu Ser Gly Leu

1 5

<210> 175

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 175

Gly Gly Gly Gly Ser

1 5

<210> 176

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 176

Glu Ala Ala Ala Lys

1 5

<210> 177

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> joint

<220>

<221> site

<222> (1)..(5)

<223> may be repeated m times, where m is an integer of 1 to 5

<400> 177

Glu Ala Ala Ala Lys Gly Ser

1 5

<210> 178

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> joint

<220>

<221> site

<222> (1)..(4)

<223> may be repeated m times, where m is an integer of 1 to 5

<400> 178

Glu Ala Ala Lys Gly Ser

1 5

<210> 179

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> joint

<400> 179

Gly Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu Pro

1 5 10 15

Gly

Claims

1. A carrier, comprising:

(a) A first nucleic acid sequence encoding a first polypeptide, wherein the first polypeptide comprises a targeting unit that targets antigen presenting cells, a multimerizing unit such as a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, allergen, alloantigen, or xenogeneic antigen; and

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

3. The vector according to any one of claims 1 to 2, wherein the one or more immunosuppressive compounds are extracellular portions, such as extracellular domains, of an inhibitory checkpoint molecule.

4. A vector according to claim 3, wherein the inhibitory checkpoint molecule is selected from the group consisting of: CLTA-4, PD-1, BTLA, LAG3, NOX2, SIGLEC7, SIGLEC9 and TIM-3, preferably human inhibitory checkpoint molecules selected from the group consisting of: hCLTA-4, hPD-1, hBTLA, hLAG3, hNOX2, hSIGLEC7, hSIGLEC9 and hTIM-3.

5. The vector according to any one of claims 1 to 2, wherein the immunosuppressive 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, preferably human cytokines 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.

6. The vector according to any one of the preceding claims, wherein the vector comprises a plurality of other nucleic acid sequences encoding more than one immunosuppressive compound, such as 2, 3, 4, 5, 6, 7 or 8 immunosuppressive compounds, such as 2, 3, 4, 5, 6, 7 or 8 different immunosuppressive compounds.

7. The vector according to any one of the preceding claims, wherein the vector comprises one or more co-expression elements.

8. The vector of claim 7, wherein the one or more co-expression elements cause transcription of the first polypeptide and the one or more immunosuppressive compounds on a single transcript and independent translation into the first polypeptide alone and the one or more immunosuppressive compounds alone.

9. The vector of any one of claims 7 to 8, wherein the one or more co-expression elements are IRES elements or nucleic acid sequences encoding a 2A self-cleaving peptide.

10. The vector of claim 9, wherein the one or more co-expression elements cause transcription of the first polypeptide and the one or more immunosuppressive compounds as separate transcripts.

11. The vector of claim 10, wherein the one or more co-expression elements are a) a bi-directional promoter or b) a promoter, wherein the vector comprises a separate promoter for each of the nucleic acid sequences encoding the first polypeptide and the one or more immunosuppressive compounds.

12. The vector according to any one of the preceding claims, wherein the antigenic unit comprises one or more T cell epitopes of a self antigen.

13. The vector of claim 12, wherein the antigenic unit comprises a plurality of T cell epitopes of one or more autoantigens.

14. The vector of claim 13, wherein the one or more autoantigens are selected from the group consisting of: autoantigens associated with multiple sclerosis, autoantigens associated with type 1 diabetes, autoantigens associated with celiac disease, autoantigens associated with rheumatoid arthritis, autoantigens associated with chronic inflammatory demyelinating multiple radiculoneuropathy, autoantigens associated with hashimoto's thyroiditis, autoantigens associated with pemphigus largehead, autoantigens associated with pemphigus vulgaris, autoantigens associated with thyroiditis, autoantigens associated with Grave's disease, autoantigens associated with primary biliary cirrhosis, autoantigens associated with myasthenia gravis, autoantigens associated with insulin-resistant diabetes, autoantigens associated with hemolytic anemia, and autoantigens associated with psoriasis.

15. The vector of any one of claims 1 to 12, wherein the antigenic unit comprises one or more T cell epitopes of an allergen.

16. The vector of claim 15, wherein the antigenic unit comprises a plurality of T cell epitopes of one or more allergens.

17. The vector of claim 16, wherein the one or more allergens are selected from the group consisting of: shellfish allergen, cow's milk allergen, egg allergen, fish allergen, fruit allergen, wheat allergen, peanut allergen, woody nut allergen, soybean allergen, seed allergen, buckwheat allergen, celery allergen, garlic allergen, gluten allergen, oat allergen, legume allergen, corn allergen, 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, fungus allergen, hair animal allergen, pollen allergen and drug allergen.

18. The vector of any one of claims 15 to 17, 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.

19. The vector of any one of claims 1 to 12, wherein the antigenic unit comprises one or more T cell epitopes of an alloantigen or a xenogeneic antigen.

20. The vector of claim 12, wherein the antigenic unit comprises a plurality of T cell epitopes of one or more alloantigens or a plurality of T cell epitopes of one or more alloantigens.

21. The vector of any one of the preceding claims, wherein the antigenic unit comprises a plurality of discrete T cell epitopes separated by a T cell epitope linker.

22. The vector according to any one of the preceding claims, wherein the antigenic unit comprises a plurality of T cell epitopes, the plurality of T cell epitopes being the smallest T cell epitope comprised in one or more hotspots.

23. The vector of any one of the preceding claims, wherein the targeting unit is or comprises a moiety that interacts with a surface molecule on an antigen presenting cell without activating the cell.

24. The vector according to any one of the preceding claims, 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.

25. The vector according to any one of claims 23 to 24, wherein the surface molecule is selected from the group consisting of: TGF-beta receptors such as TGF-beta R1, TGF-beta R2 and TGF-beta R3, IL-10R such as IL-10RA and IL-10RB, IL-2R, IL-4R, IL-6R, IL-11R, IL-13R, IL-27R, IL-35R, IL-37-R, GM-CSFR, FLT3, CCR7, CD11b, CD11c, CD103, CD14, CD36, CD205, CD109, VISTA, MARCO, MHCII, CD83, SIGLEC, clec10A (MGL), ASGR (ASGR 1/ASGR 2), CD80, CD86, clec9A, clec12A, clec B, DCIR2, langerin, MR, DC-Sign, treml4, dectin-1, PDL2, HVEM, CD163 and CD141.

26. The vector of claim 25, wherein the surface molecule is a surface molecule present on a human antigen presenting cell, and wherein the surface molecule is selected from the group consisting of: hTGFbeta receptors such as hTGFbeta R1, hTGFbeta R2 and hTGFbeta R3, hIL-10R such as hIL-10RA and hIL-10RB, hIL-2R, hIL-4R, hIL-6R, hIL-11R, hIL-13R, hIL-27R, hIL-35R, hIL-37-R, hGM-CSFR, hFLT3, hCR 7, hCD11b, hCD11c, hCD103, hCD14, hCD36, hCD205, hCD109, hVISTA, hMARCO, hMHCII, hCD83, hSIGLEC, hClec A (hMGL), hASGR (hASGR 1/hASGR 2), hCD80, hCD86, hlec 9A, hClec12A, hClec12B, hDCIR2, langerin, hMR, hDC-Sign, hTreml4, hDen-1, hPDL2, hHVEM, hCD163 and hCD141.

27. The vector according to any one of claims 23 to 26, wherein the moiety is a natural ligand, an antibody or a part thereof such as an scFv or a synthetic ligand.

28. The vector of claim 27, wherein the moiety is a natural ligand selected from the group consisting of: TGF-beta such as TGF-beta 1, TGF-beta 2 and TGF-beta 3, 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, the extracellular domain of VSIG-3 preferably VSIG-3, SCGB3A2, the extracellular domain of CTLA-4 preferably CTLA-4, the extracellular domain of PD-1 preferably PD-1, and the extracellular domain of BTLA preferably BTLA.

29. The vector of claim 28, wherein the moiety is a human natural ligand selected from the group consisting of: hTGF beta, hIL-10, hIL-2, hIL-4, hIL-6, hIL-11, hIL-13, hIL-27, hIL-35, hIL-37, hGM-CSF, hFLT3L, hCCL, hCL 21, hICAM-1, human keratin, the extracellular domain of hVSIG-3, preferably hVSIG-3, hSCGB3A2, the extracellular domain of hCDLA-4, preferably hCDLA-4, hPD-1, preferably PD-1, and the extracellular domain of hBTLA, preferably hBTLA.

30. The vector according to any one of claims 23 to 29, wherein the targeting unit is or comprises an extracellular domain of hIL1-10, tgfβ such as tgfβ -1, tgfβ -2 and tgfβ -3, hSCGB3A2 or hvig-3, preferably hvig-3.

31. The vector according to any one of the preceding claims, 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 minor fibrous egg, and a tetramerization unit, such as a domain derived from p53, and wherein the multimerization unit optionally comprises hinge regions, such as hinge exon h1 and hinge exon h4.

32. The vector according to claim 31, wherein the vector comprises a hinge region capable of forming one or more covalent bonds, and is preferably Ig-derived.

33. The vector according to any one of claims 31 to 32, wherein the multimerization unit is a dimerization unit and the dimerization unit further comprises another dimerization promoting domain, preferably wherein the other domain is an immunoglobulin domain, more preferably an immunoglobulin constant domain.

34. The vector according to claim 33, wherein the further domain is a carboxy terminal C domain derived from IgG, preferably from IgG 3.

35. The vector according to any one of claims 33 to 34, wherein the dimerization unit further comprises a dimerization unit linker, such as a glycine-serine rich linker, such as GGGSSGGGSG, and preferably wherein the dimerization unit linker connects the hinge region and another domain that facilitates dimerization.

36. The vector according to any one of claims 33 to 35, wherein the dimerization unit comprises hinge exon h1 and hinge exon h4, a dimerization unit linker and a CH3 domain of human IgG 3.

37. The vector according to any one of the preceding claims, wherein the first nucleic acid sequence encodes a first polypeptide further comprising a unit linker connecting the antigenic unit to the multimerization unit, and wherein the unit linker is a non-immunogenic linker and/or a flexible or rigid linker.

38. The vector according to any one of the preceding claims, wherein the first nucleic acid sequence encodes a first polypeptide further comprising a signal peptide, and preferably wherein the one or more further nucleic acid sequences also further encode a signal peptide.

39. The vector according to any of the preceding claims, 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.

40. A method of producing a vector as defined in any one of the preceding claims, the method comprising:

a) Transfecting cells with the vector in vitro;

b) Culturing the cells;

c) Optionally, lysing the cells to release the vector from the cells; and

d) The vector is collected and optionally purified.

41. A host cell comprising a vector as defined in any one of claims 1 to 39, such as a host cell selected from the group consisting of a prokaryotic cell, a yeast cell, an insect cell, a higher eukaryotic cell, such as a cell from an animal or a human.

42. A vector as defined in any one of claims 1 to 39 for use as a medicament.

43. A pharmaceutical composition comprising a carrier as defined in any one of claims 1 to 39 and a pharmaceutically acceptable carrier or diluent.

44. The pharmaceutical composition of claim 43, wherein the composition further comprises a transfection agent.

45. The pharmaceutical composition according to any one of claims 43 to 44, wherein the composition further comprises an adjuvant, such as an adjuvant selected from dexamethasone, enterotoxin cholera toxin B subunit (CTB), TLR2 ligand, worm-derived excretion/secretion (ES) product, rapamycin vitamin D3 analogue and aryl hydrocarbon receptor ligand.

46. The pharmaceutical composition according to any one of claims 43 to 45, wherein composition comprises 0.1 to 10mg of the vector, such as the DNA plasmid.

47. A method of treating a subject suffering from or in need of prophylaxis of an immune disorder selected from the group consisting of autoimmune disorders, allergic disorders and graft rejection, comprising administering to said subject a vector as defined in any one of claims 1 to 39 or a pharmaceutical composition as defined in any one of claims 43 to 46.

48. A method of treating a subject suffering from or in need of prophylaxis of an autoimmune disease, said method comprising administering to said subject a vector as defined in any one of claims 1 to 14 and 21 to 39 or a pharmaceutical composition comprising such a vector as defined in any one of claims 43 to 46.

49. A method for treating a subject suffering from or in need of prevention of an allergic disease, said method comprising administering to said subject a vector as defined in any one of claims 1 to 11, 15 to 18 and 21 to 39 or a pharmaceutical composition comprising such a vector as defined in any one of claims 43 to 46.

50. A method for treating a subject suffering from or in need of prophylaxis of graft rejection, said method comprising administering to said subject a vector as defined in any one of claims 1 to 11 and 19 to 39 or a pharmaceutical composition comprising such a vector as defined in any one of claims 43 to 46.

51. The method according to any one of claims 47 to 50, wherein the carrier or pharmaceutical composition is administered in a therapeutically or prophylactically effective amount, for example by intradermal, intramuscular or subcutaneous injection or by mucosal or epithelial administration, such as intranasal or oral administration.