CN117693361A

CN117693361A - Tolerance-inducing constructs and compositions and their use in the treatment of immune disorders

Info

Publication number: CN117693361A
Application number: CN202280043882.2A
Authority: CN
Inventors: 阿涅特·布伦斯维克·弗雷德里克森; 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: 2024-03-12

Abstract

The present disclosure relates to constructs and compositions for use in the treatment of conditions involving undesired immune responses, for example for prophylactic or therapeutic treatment of autoimmune diseases, allergic diseases and graft rejection.

Description

Tolerance-inducing constructs and compositions and their use in the treatment of immune disorders

Technical Field

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, which are characterized by an excessive immune response to substances that are generally harmless in the environment and may produce an inflammatory response that leads 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, recognizes alloantigens or xenogeneic antigens of the donor and causes destruction of the transplanted organs or tissues. Immune tolerance is the inability to obtain a specific immune response to a substance or tissue that is 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 for unwanted immune responses are immunosuppressive drugs based on a broad spectrum of actions. 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.

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

The scientific article "Schjetne K W et al, eur.J.Immunol.35 (11), 3142-3152,2005" discloses recombinant antibody constructs known as "Troybody". These troybodies are recombinant antibodies with a V region specific for APC surface molecules and T cell epitopes grafted in the loop between the β -strands of their C domains.

Summary of The Invention

The present disclosure relates to tolerance-inducing constructs (tolerance-inducing construct) comprising an antigenic unit and a targeting unit that interacts with a surface molecule on an APC (e.g., a dendritic cell) in a non-inflammatory or tolerogenic manner, resulting in presentation of the antigen without activation (e.g., inflammatory activation).

The inventors have surprisingly found that the vaccine platform can deliver disease-associated antigens to Antigen Presenting Cells (APCs) in an optimal manner for inducing a selected antigen-specific tolerance response by binding to and signaling selected surface receptors on the APCs, which internalize the construct and present the antigen in a tolerogenic manner (e.g., inducing regulatory T cells (tregs) and inhibiting memory and effector T cell responses).

The tolerance-inducing constructs of the present disclosure may have improved flexibility compared to known constructs, for example compared to "Troybody" as disclosed in Schjetne K W et al, eur.j. Immunol.35 (11), 3142-3152, 2005. For example, the targeting units of the constructs of the present disclosure are not limited to V regions derived from antibodies, but can be a variety of different units.

Another advantage of the tolerance-inducing constructs of the present disclosure may be that fewer doses (e.g., one dose) may be sufficient to achieve the same efficacy as known constructs. For example, a smaller dose (e.g., one dose) may be sufficient to reduce the level of an immune response, delay the onset or progression of an immune response, and/or reduce the risk of an immune response from onset or progression.

The vaccine construct is a multimeric protein consisting of a plurality of polypeptides, e.g. a dimeric protein consisting of two polypeptides, each comprising a targeting unit, a dimeric unit and an antigenic unit targeting antigen presenting cells-see e.g. WO2004/076489A1, WO2011/161244A1, WO2013/092875A1 or WO2017/118695A1. These constructs have been shown to be effective in generating an immune response against an antigen or epitope contained in an antigenic unit.

The vaccine or tolerance-inducing construct of the present disclosure may be administered to a subject in the form of a polynucleotide (e.g., a DNA plasmid) comprising a nucleotide sequence encoding a polypeptide. Upon administration to a host cell, such as a human muscle cell, the polypeptide is expressed and a multimeric protein is formed as a result of the multimerization unit; when dimerization units are used, the polypeptides form dimeric proteins upon expression.

The present disclosure provides tolerance-inducing constructs based on the structure of the vaccine for prophylactic or therapeutic treatment of immune disorders, such as autoimmune diseases, allergic diseases and graft rejection.

The tolerance-inducing constructs of the present disclosure comprise an antigenic unit comprising one or more T cell epitopes of a self-antigen, allergen or alloantigen/xenogeneic antigen, a multimerizing unit, e.g. a dimerization unit, and a targeting unit for targeting APCs. The targeting unit interacts with surface molecules on the APC such that the construct is internalized and the epitope in the antigenic unit is presented in a manner that induces tolerance.

Thus, in a first aspect, the present disclosure provides a tolerance-inducing construct comprising:

i) A polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, such as a dimerization unit, and an antigenic unit, that targets or is capable of targeting an antigen presenting cell; or alternatively

ii) a polypeptide encoded by a nucleic acid sequence as defined in (i); or alternatively

iii) A multimeric protein, e.g. a dimeric protein, consisting of a plurality of polypeptides, e.g. two polypeptides, as defined in (ii);

wherein the antigenic unit comprises one or more T cell epitopes of a self antigen, allergen, alloantigen or xenogeneic antigen.

In another aspect, the present disclosure provides a tolerance-inducing construct comprising:

i) A polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigenic unit that targets or is capable of targeting an antigen presenting cell; or alternatively

iii) A multimeric protein consisting of a plurality of polypeptides defined in (ii);

i) A polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit and an antigenic unit that targets or is capable of targeting an antigen presenting cell; or alternatively

iii) A dimeric protein consisting of two polypeptides defined in (ii);

In another aspect, the present disclosure provides a polynucleotide as defined herein.

In another aspect, the present disclosure provides a vector comprising a polynucleotide as defined herein.

In another aspect, the present disclosure provides a host cell comprising a polynucleotide as defined herein.

In another aspect, the present disclosure provides a dimeric protein consisting of two polypeptides as defined herein.

In another aspect, the present disclosure provides polypeptides encoded by a nucleic acid as defined herein.

In another aspect, the present disclosure provides a pharmaceutical composition comprising a tolerance-inducing construct as defined herein and a pharmaceutically acceptable carrier.

In another aspect, the present disclosure provides a method of preparing a pharmaceutical composition as defined herein, wherein the pharmaceutical composition comprises a polypeptide as defined herein or a multimeric protein as defined herein, such as a dimeric protein, wherein the method comprises:

a) Transfecting a cell with a polynucleotide as defined herein;

b) Culturing the cells;

c) Collecting and purifying multimeric proteins, such as dimeric proteins or polypeptides, expressed by the cells; and

d) Mixing the multimeric protein, e.g. a dimeric protein or polypeptide, obtained from step c) with a pharmaceutically acceptable carrier.

In another aspect, the present disclosure provides a method of preparing a pharmaceutical composition as defined herein, wherein the pharmaceutical composition comprises a polynucleotide as defined herein, the method comprising:

a) Preparing the polynucleotide;

b) Optionally cloning the polynucleotide into an expression vector; and

c) Mixing the polynucleotide obtained from step a) or the vector obtained from step b) with a pharmaceutically acceptable carrier.

In another aspect, the present disclosure provides a method of treating a subject suffering from a condition involving an undesired immune response, such as an autoimmune disease, allergic disease, or graft rejection, or in need of prevention of the condition, the method comprising administering to the subject a pharmaceutical composition as defined herein.

In another aspect, the present disclosure provides a pharmaceutical composition as defined herein for use in the treatment of a condition involving an undesired immune response, such as an autoimmune disease, allergic disease or graft rejection.

Drawings

Fig. 1: schematic representation of exemplary tolerance-inducible constructs

Fig. 1 shows an example of a tolerance-inducing construct of the present disclosure. Tolerance-inducing constructs of the present disclosure can be described as polypeptides having an N-terminal start end and a C-terminal end (shown in fig. 1). The elements of the polypeptide, the Targeting Unit (TU), the dimerization unit (DimU) and the antigenic unit, may be arranged in the polypeptide such that the antigenic unit is located at the C-terminal end of the polypeptide (fig. 1 a) or at the N-terminal beginning of the polypeptide (fig. 1 b).

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. The Unit Linker (UL) may connect the dimerization unit and the antigenic unit. Figure 1 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 multimeric proteins, dimeric proteins and polynucleotides.

Fig. 2: expression and secretion of tolerance-inducing constructs containing MOG

FIGS. 2A and 2B show the detection of protein expression and secretion levels of the tolerance-inducing construct containing MOG and the pro-inflammatory control constructs VB5002B and VB5052 by sandwich ELISA (capture antibody: anti-MOG antibody, detection antibody: anti-hIgG CH3 domain antibody) performed on: (A) Supernatant of HEK293 cells transiently transfected with DNA vectors VB5002B, VB5003B, VB5004B, VB5005B, VB5006B and VB5012B, (B) supernatant of Expi293F cells transiently transfected with DNA vectors VB5052, VB5046, VB5048, VB5058, VB5059, VB5060, VB5061 and VB 5071. All MOG-containing constructs were highly expressed and secreted. (A) The negative control in (a) was the supernatant of HEK293 cells treated with transfection reagent Lipofectamine alone, and the negative control in (B) was the supernatant of Expi293F cells treated with transfection reagent Lipofectamine alone.

Fig. 3: secretion of full-length MOG-containing tolerance-inducing constructs with different targeting units

Figure 3 shows high level secretion of full length tolerance inducing constructs with different targeting units and pro-inflammatory control construct VB5052 detected by sandwich ELISA on supernatants of HEK293 cells or Expi293F cells transiently transfected with vectors VB5005b, VB5006b (HEK 293 cells), VB5052, VB5058, VB5059, VB5060 and VB5061 (Expi 293F cells). Capture antibody: mouse anti-MOG antibody, 0.25. Mu.g/ml, 100. Mu.l/well, sc-73330,Santa Cruz Biotechnology. Detection of antibodies: (A) And (B) is 0.2 μg/ml goat anti-murine IL-10 biotinylated antibody, 100 μl/well, BAF417, R & D Systems; (C) 0.8 μg/ml chicken anti-human TGF-beta 1 biotinylated antibody, 100 μl/well, BAF240, RD Systems; (D) Is 0.83 μg/ml goat anti-murine SCGB3A2 biotinylated antibody, 100 μl/well, BAF3465, R & D Systems; (E) Is 0.8 μg/ml goat anti-murine CTLA-4 biotinylated antibody, 100 μl/well, BAF476, RD Systems; (F) Is 0.29 μg/ml goat anti-mouse PD-1 biotinylated antibody, 100 μl/well, DY1021, R & D System; (G) Is 0.2. Mu.g/ml goat anti-human CCL3 biotinylated antibody, 100. Mu.l/well, BAF270, R & D Systems. The negative control was supernatant from HEK293 cells treated with transfection reagent Lipofectamine alone or from Expi293F cells treated with ExpiFectamine alone.

Fig. 4: secretion of MOG (27-63) peptides

Secretion of MOG (27-63) peptide encoded in DNA vector VB5051 was verified by direct ELISA (detection antibody: mouse anti-MOG antibody, 3.3. Mu.g/ml, 100. Mu.l/well, sc-73330,Santa Cruz Biotechnology) of supernatant from Expi293F cells transiently transfected with DNA vector VB 5051. The negative control was the supernatant of the Expi293F cells treated with transfection reagent expictamine alone.

Fig. 5: expression and secretion of tolerance-inducing constructs containing Met e 1

FIG. 5 shows detection of antibodies CaptureStyle by sandwich ELISA (Capture antibody: anti-human IgG3 (CH 3 domain) antibody) on supernatants of Expi293F cells transiently transfected with Met e 1-containing DNA vectors VB5024, VB5030 and VB5079 ^TM Biotin anti-IgG-Fc (human) conjugate) to detect protein expression and secretion levels of the Met e 1-containing tolerance-inducing construct. All tolerance-inducing constructs containing Met e 1 were expressed and secreted. The negative control was the supernatant of the Expi293F cells treated with transfection reagent expictamine alone.

Fig. 6: tolerance-inducing constructs comprising scFv anti-DEC 205 as targeting units bind to recombinant DEC205 receptor

FIG. 6 shows that tolerance-inducing proteins containing scFv anti-DEC 205 targeting units bind to recombinant DEC205 receptor by direct ELISA (coating: recombinant DEC205 (216-503), detection antibodies: anti-MOG antibodies or anti-hIgG CH3 domain antibodies) of supernatants of HEK293 cells transiently transfected with scFv anti-DEC 205 containing DNA vector VB5004 b. Both antibodies demonstrated binding to the receptor, while anti-MOG antibodies demonstrated secretion of the full-length protein.

Fig. 7: tolerance-inducing constructs comprising IL-10 as targeting unit bind to recombinant IL-10 receptor

FIG. 7 shows that tolerance-inducing proteins comprising IL-10 as targeting unit bind to recombinant IL-10 receptor by direct ELISA (coating: recombinant IL-10 receptor, detection antibody: anti-MOG antibody or anti-hIgG CH3 domain antibody) of supernatant from HEK293 cells transiently transfected with IL-10-containing DNA vector VB5006 b. Both antibodies demonstrated binding to the receptor, while anti-MOG antibodies demonstrated secretion of the full-length protein.

Fig. 8: characterization of secreted tolerance-inducible constructs in size, protein integrity and dimer formation

FIG. 8 shows Western Blot (WB) analysis of supernatants from Expi293F cells transiently transfected with MOG-containing DNA vectors under reducing and non-reducing conditions. The negative control was supernatant from the Expi293F cells treated with the transfection reagent ExpiFectamine (transfection control).

Fig. 8A: western blot shows expression and full-length secretion of tolerance-inducing proteins. Reduced supernatant samples (25 μl loaded) from transfected Expi293F cells. An antibody: mice were anti-MOG (sc-73130). And (2) secondary antibody: donkey is resistant to mice, dylight 800 (SA 5-10172). Protein standards were detected in Chemidoc channel Dylight 650 (signal not shown) and Chemidoc channel Dylight 800.

Fig. 8B: western blot shows dimerization of tolerance-inducing proteins (black arrow). Unreduced supernatant samples (loaded with 25 μl) from transfected Expi293F cells. An antibody: mice were anti-MOG (sc-73130). And (2) secondary antibody: donkey is resistant to mice, dylight 800 (SA 5-10172). Chemidoc channel dlight 650 (for protein standards) and 800.

Fig. 8C: western blot shows expression and full-length secretion of tolerance-inducing proteins. Reduced supernatant samples (25 μl loaded) from transfected Expi293F cells. An antibody: rat anti-IL 10 (MAB 417). And (2) secondary antibody: donkey is resistant to rats, dylight 488 (SA 5-10026). Chemidoc channel Dylight 650 (for protein standards) and 488.

Fig. 8D: western blot shows expression and full-length secretion of tolerance-inducing proteins (black arrow). Reduced supernatant samples (35 μl loaded) from transfected Expi293F cells. An antibody: goat anti-CTLA-4 (AF 476). And (2) secondary antibody: donkey is against goat, dylight 800 (SA 5-10092). Chemidoc channel dlight 650 (for protein standards) and 800.

Fig. 9: bicolor IL-10/IFN gamma fluoroSpot

C57BL/6 mice were vaccinated once (day 0) with 50. Mu.g of the indicated DNA vectors (VB 5004b, VB5002b and VB5001 b) and spleens were harvested on day 7 post-vaccination. (A) After restimulation with MOG (35-55) peptide, mice spleen cells were tested for IFN-gamma and IL-10 secretion (SFU/106 spleen cells) with bicolor FluoSpot. Individual mice are shown, each group n=4 (VB 5001 b), 5 (VB 5004b and VB5002 b) or 2 (PBS). The IL-10/IFN-gamma ratio of (B) was plotted from the data in (A). Individual mice and mean ± range (p < 0.05) × (p < 0.01), two-tailed Mann-Whitney test are shown.

Fig. 10: the percentage of Foxp3+ producing CD4+ T cells, IFN-gamma producing CD4+ T cells and IL-17 producing CD4+ T cells were detected by flow cytometry

C57BL/6 mice were vaccinated once (day 0) with 50. Mu.g of the indicated DNA vectors (VB 5004b, VB5002b, VB5001 b) and spleens were harvested on day 7 post-vaccination. The percentages [% ] of (A) Foxp3+, (B) IFN-. Gamma. +, and (C) IL-17+ splenocytes in the total CD4+ T cell population after restimulation with MOG (35-55) peptide are shown. Data were obtained from pools of 4 mice/group (VB 5001 b), 5 mice/group (VB 5004b and VB5002 b) or 2 mice/group (PBS). Construct ID numbers are indicated at the x-axis.

Fig. 11: bicolor IL-10/IFN gamma fluoroSpot

C57BL/6 mice were vaccinated twice (day 0 and day 4) with 50 μg of the indicated DNA vector (VB 5012b, VB5052, VB 5051) and spleens were harvested on day 10 after priming. (A) After restimulation with MOG (35-55) peptide, mice spleen cells were tested for IFN-gamma and IL-10 secretion (SFU/10) with bicolor FluoSpot ⁶ Individual spleen cells). Individual mice are shown. (B) The IL-10/IFN-gamma ratio is plotted from the data in (A). Individual mice and mean ± ranges are shown, n=5 or n=2 (PBS) for each group, p < 0.01, two-tailed Mann-Whitney test.

Fig. 12: detection of MOG (38-49) specific Foxp3+ T cells

C57BL/6 mice were vaccinated twice (day 0 and day 4) with 50 μg of the indicated DNA vector (VB 5012b, VB5048, VB5006b, VB5046, VB 5051) and spleens were harvested on day 10 post priming. Percentage of splenic foxp3+ cells detected ex vivo by the H-2Iab/MOG (38-49) tetramer of the total cd4+ population. Data were obtained from pools of 5 mice or 2 mice (PBS) per group. Construct ID numbers are indicated at the x-axis.

Fig. 13: expression and secretion of tolerance-inducing constructs containing MOG

FIG. 13 shows protein expression and secretion levels of the MOG-containing tolerance-inducing construct VB5009 with TGF-beta 1 as targeting unit, detected by sandwich ELISA of supernatants of HEK293 cells transiently transfected with VB5009 (capture antibody: rabbit anti-human TGF-beta 1 (orb 77216, biorbyte), detection antibody: biotinylated mouse anti-human IgG (05-4240, invitrogen)). The negative control was the supernatant of HEK293 cells treated with transfection reagent Lipofectamine alone.

Detailed Description

iii) A dimeric protein consisting of two polypeptides defined in (ii);

Such constructs, once administered to a subject, will allow epitopes in the antigenic unit to be presented in a tolerogenic manner and are therefore suitable for use as a prophylactic or therapeutic treatment for immune diseases such as autoimmune diseases, allergic diseases and graft rejection.

Since the tolerance-inducing construct causes a down-regulation of the disease-specific cells of the immune system leading to the immune disease in question, it does not suppress the whole immune system. Thus, treatment of the immune disease in question with the constructs of the present disclosure will therefore not result in increased susceptibility to infection and decreased immune surveillance for cancer. However, bystander suppression of immune cells specific for the relevant disease antigen is expected due to the release of short-range inhibitory cytokines by intercellular contact with the induced antigen-specific regulatory cells (bystander suppression).

The tolerance-inducing constructs of the present disclosure may be administered in the form of a pharmaceutical composition comprising the constructs of the present disclosure and a pharmaceutically acceptable carrier for prophylactic or therapeutic treatment of immune disorders, such as autoimmune disorders, allergic disorders, and graft rejection.

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

A "tolerance-inducing construct" is one that does not elicit an immune response, such as an inflammatory immune response, against a T cell epitope contained in an antigenic unit, but instead 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 "tolerizing" as used herein refers to a reduction in the level of an immune response, such as an inflammatory immune response, a delay in the onset or progression of an immune response, such as an inflammatory immune response, and/or a reduction in the risk of an immune response, such as an onset or progression of an inflammatory immune response.

A "subject" is an animal or human. The subject may be a patient in need of therapeutic treatment, i.e., a person suffering from an immune disorder such as an autoimmune disorder, allergy or graft rejection. 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, disorder or disease involving an undesired immune response, including autoimmune disease, allergy or graft rejection, i.e., rejection of an allograft or xenograft, such as host rejection of cells, tissues or organs from the same species (allograft, allo) or a different species (xeno) that are transplanted to the host.

The term "alloantigen" or "allograft antigen" as used herein refers to an antigen derived from (shed from and/or present in) a cell or tissue that, when transferred from a donor to an recipient, is recognized and bound by antibodies of the recipient's B or T cell recipient. Alloantigens are typically the products of polymorphic genes. Alloantigens are proteins or peptides that show a slight structural difference when compared between a donor and an acceptor (belonging to the same species). The presence of such donor antigens in the recipient can elicit an immune response in the recipient. This alloreactive immune response is specific for the alloantigen.

The terms "mouse" and "mouse" are used interchangeably to refer to substances derived from mice, such as peptides, proteins, nucleic acids, and the like.

As used herein, the term "heterologous antigen" refers to an antigen derived from an individual of a different species.

"treatment" is prophylactic or therapeutic treatment.

"prophylactic treatment" is a treatment administered to a subject that does not exhibit signs or symptoms of an immune disease or exhibits only early signs or symptoms of an immune disease, the purpose of which is to prevent or at least reduce the risk of developing the 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.

"therapeutic treatment (therapeutic treatment)" is a treatment administered to a subject exhibiting symptoms or signs of an immune disease, the treatment being administered to the subject with the aim of attenuating or eliminating these signs or symptoms and/or with the aim of delaying or preventing the progression of the condition.

"part" refers to a portion or fragment of an antigen, i.e., a portion or fragment of the amino acid sequence of an antigen, or a nucleotide sequence encoding the same, e.g., an epitope; preferably, the portion or fragment of the antigen is immunogenic. These terms will be used interchangeably throughout.

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

The terms "vaccination" and "administration" are used interchangeably herein.

The term "minimal epitope" refers to a subsequence of an epitope predicted to bind to MHC I or MHC II. In other words, the minimal epitope may be immunogenic, i.e. capable of eliciting an immune response. The term minimal epitope may thus refer to a short subsequence of an epitope that is predicted to bind to MHC I or MHC II. Thus, a 27-mer epitope can comprise several smallest epitopes, each of which can be shorter than 27 amino acids in length, and each of which is immunogenic. For example, the smallest epitope may consist of the first 14 amino acids of the epitope as long as it is predicted to bind to MHC I or MHC II, or it may consist of amino acids 9 to 18 or amino acids 7 to 22 of the epitope as long as these sequences are predicted to bind to MHC I or MHC II.

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

Tolerance-inducing constructs

Fig. 1 shows an example of a tolerance-inducing construct of the present disclosure. Tolerance-inducing constructs of the present disclosure can be described as polypeptides having an N-terminal start end and a C-terminal end (shown in fig. 1). The elements of the polypeptide, the Targeting Unit (TU), the dimerization unit (DimU) and the antigenic unit, may be arranged in the polypeptide such that the antigenic unit is located at the C-terminal end of the polypeptide (fig. 1 a) or at the N-terminal beginning of the polypeptide (fig. 1 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 (TL). The Unit Linker (UL) may connect the dimerization unit and the antigenic unit. FIG. 1 shows an antigenic unit with 2T cell epitopes (T1, T2) separated by TL. The order and orientation of the above units and elements are the same in dimeric proteins and polynucleotides.

Hereinafter, various units of the construct will be discussed in detail. These units are present in the polynucleotide as nucleic acid sequences which encode the units which are present as amino acid sequences in polypeptides, multimeric proteins or dimeric proteins. For ease of reading, in the following, the units of the construct are mainly explained with respect to polypeptides, multimeric proteins or dimeric proteins, i.e. based on their amino acid sequences.

Targeting unit

The tolerance-inducing constructs of the present disclosure comprise targeting units that target Antigen Presenting Cells (APCs).

The term "targeting unit" as used herein refers to a unit that delivers a construct of the disclosure to an APC and interacts with surface molecules on the APC (e.g., binds to surface receptors on the APC) without activating the cell and/or 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 upregulating costimulatory signals and/or by upregulating inhibitory surface receptors and/or secreting inhibitory cytokines.

In some embodiments, the targeting unit comprises or consists of a moiety that binds to a receptor selected from the group consisting of: TGF-beta receptors such as TGF-beta R1, TGF-beta R2 or TGF-beta R3, IL10R such as IL-10RA and IL10-RB, IL2R, IL4R, IL6R, IL R and IL13R, IL27R, IL35R, IL R, GM-CSFR, FLT3, CCR7, CD11b, CD11c, CD103, CD14, CD36, CD205, CD109, VISTA, MARCO, MHCII, CD83, SIGLEC, MGL/Clec10A, ASGR (ASGR 1/ASGR 2), CD80, CD86, clec9A, clec12A, clec12B, DCIR, langerin, MR, DC-Sign, treml4, dectin-1, PDL2, HVEM, CD163 and CD141.

In some embodiments, the targeting unit comprises or consists of a moiety that binds to a human (h) receptor selected from the group consisting of: hTGFbeta receptors such as hTGFbeta R1, hTGFbeta R2 or hTGFbeta R3, hIL10R such as hIL-10RA and hIL10-RB, hIL2R, hIL4R, hIL6R, hIL R and hIL13R, hIL27R, hIL35R, hIL R, hGM-CSFR, hFLT3, hCR 7, hCD11b, hCD11c, hCD103, hCD14, hCD36, hCD205, hCD109, hVISTA, hMARCO, hMHCII, hCD83, hSIGLEC, hMGL/hCDec 10A, hASGR (hASGR 1/hASGR 2), hCD80, hCD86, hCDL 9A, hClec12A, hClec12B, hDCIR2, hLangerin, 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 some embodiments, the moiety is an antibody or portion thereof specific for any of the foregoing receptors, e.g., an scFv, whose binding to the receptor results in presentation of the antigen and/or T cell epitope in an anti-inflammatory tolerogenic manner.

In other embodiments, the moiety is a synthetic ligand specific for any of the foregoing receptors, the binding of which to the receptor results in presentation of the antigen and/or T cell epitope in an anti-inflammatory tolerogenic manner. Protein modeling can be used to design such synthetic ligands.

In other embodiments, the moiety is a natural ligand.

In some embodiments, the natural ligand is selected from: TGF-beta such as TGF-beta 1, TGF-beta 2 or TGF-beta 3, IL-10, IL2, IL4, IL6, IL11, IL13, IL27, IL35, IL37, GM-CSF, FLT3L, CCL19, CCL21, ICAM-1 (intercellular adhesion molecule 1, also known as CD 54), keratin, 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 other embodiments, the targeting unit is or comprises IL2, preferably human IL2. In other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of human IL2, e.g., an amino acid sequence having at least 80% sequence identity to SEQ ID NO. 33. In other embodiments, the targeting unit comprises, or consists of, a nucleotide sequence encoding human IL2, e.g., the nucleotide sequence of SEQ ID NO: 36.

In other embodiments, 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 other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of human TGF-beta, e.g., an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs 205-207.

In other embodiments, the targeting unit comprises or consists of: an amino acid sequence having at least 85% sequence identity to the amino acid sequence of human tgfβ, e.g., an amino acid sequence having at least 85% sequence identity to SEQ ID NO:205-207, e.g., having at least 85% sequence identity thereto, e.g., having at least 86%, e.g., at least 87%, e.g., at least 88%, e.g., at least 89%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, or e.g., 100% sequence identity thereto.

In another embodiment, the targeting unit comprises or consists of: an amino acid sequence of human tgfβ, e.g. selected from SEQ ID NO:205-207 except that up to 22 amino acids have been substituted, deleted or inserted, e.g. up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid has been substituted, deleted or inserted.

In other embodiments, the targeting unit comprises or consists of the amino acid sequence of human tgfβ or a nucleotide sequence encoding human tgfβ.

In other embodiments, the targeting unit comprises a nucleotide sequence encoding human tgfβ, e.g., a sequence selected from SEQ ID NOs: 208-210, or consists of a nucleotide sequence of seq id no.

In other embodiments, the targeting unit comprises, or consists of, an amino acid sequence that has at least 80% sequence identity to murine TGF-beta (e.g., murine TGF-beta as set forth in SEQ ID NO: 177).

In other embodiments, the targeting unit comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of human IL-10, e.g., with SEQ ID NO:211, or consists of an amino acid sequence having at least 80% sequence identity.

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

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

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

In other embodiments, the targeting unit comprises a nucleotide sequence encoding human IL-10, e.g., SEQ ID NO:212, or consists of a nucleotide sequence of seq id no.

In other embodiments, the targeting unit comprises, or consists of, an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of murine IL-10 (murine IL-10 as set forth in SEQ ID NO: 169).

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

In other embodiments, the targeting unit comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of human SCGB3A2, e.g. with SEQ ID NO:213, or consists of an amino acid sequence having at least 80% sequence identity.

In other embodiments, the targeting unit comprises or consists of: an amino acid sequence having at least 85% sequence identity to the amino acid sequence of human SCGB3A2, e.g. with SEQ ID NO:213, e.g., having at least 85% sequence identity thereto, e.g., having at least 86%, e.g., at least 87%, e.g., at least 88%, e.g., at least 89%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99%, or e.g., 100% sequence identity thereto.

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

In other embodiments, the targeting unit comprises or consists of the amino acid sequence of human SCGB3A2 or the nucleotide sequence encoding human SCGB3 A2.

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

In other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of murine SCGB3A2 (e.g., murine SCGB3A2 shown in SEQ ID NO: 171).

In other embodiments, the targeting unit comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of human VSIG-3, e.g., with SEQ ID NO:215, or consists of an amino acid sequence having at least 80% sequence identity.

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

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

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

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

In other embodiments, the targeting unit comprises, or consists of, an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of murine VSIG-3 (e.g., murine VSIG-3 shown in SEQ ID NO: 173).

In other embodiments, the targeting unit is or comprises an antibody or portion thereof (e.g., scFv) specific for CD205, e.g., scFv specific for human or murine CD205 or scFv anti-DEC 205. In some embodiments, the scFv specific for murine CD205 comprises the amino acid sequence of SEQ ID NO:49 or consist of SEQ ID NO: 49.

In other embodiments, the targeting unit comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of human CTLA4, e.g., as set forth in SEQ ID NO:217 has, or consists of, an amino acid sequence having at least 80% sequence identity.

In other embodiments, the targeting unit comprises or consists of: an amino acid sequence having at least 85% sequence identity to the amino acid sequence of human CTLA4, e.g., an amino acid sequence of SEQ ID NO:217 has at least 85% sequence identity, for example with its 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%, such as at least 99% or such as 100% sequence identity.

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

In other embodiments, the targeting unit comprises or consists of the amino acid sequence of human CTLA4 or a nucleotide sequence encoding human CTLA 4.

In other embodiments, the targeting unit comprises a nucleotide sequence encoding human CTLA4, e.g., SEQ ID NO:218, or consists of a nucleotide sequence of seq id no.

In other embodiments, the targeting unit comprises or consists of an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of murine CTLA4 (murine CTLA4 as shown in SEQ ID NO: 175).

In other embodiments, the targeting unit comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of human PD-1, e.g., with SEQ ID NO:219, or consists of an amino acid sequence having at least 80% sequence identity.

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

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

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

In other embodiments, the targeting unit comprises a nucleotide sequence encoding human PD-1, e.g., SEQ ID NO:220, or consists of a nucleotide sequence of 220.

In other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of murine PD-1 (e.g., murine PD-1 as set forth in SEQ ID NO: 179).

Antigenic unit

The antigenic units of the tolerance-inducing constructs of the present disclosure comprise one or more T cell epitopes of autoantigens, 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 have been published in the literature.

In some embodiments, 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 some embodiments, the plurality of T cell epitopes belong to the same autoantigen, i.e., are comprised in the same autoantigen. In other embodiments, the plurality of T cell epitopes belong to, i.e. are comprised in, a plurality of different autoantigens.

In some embodiments, the antigenic unit comprises one or more T cell epitopes of a self antigen, such as a T reg epitope or an inhibitory neoantigen.

In some embodiments, when the antigenic unit comprises more than one T cell epitope, the antigenic unit comprises one or more linkers separating the T cell epitopes. In some embodiments, the antigenic unit comprises a plurality of T cell epitopes of autoantigens, allergens, alloantigens or xenogeneic antigens, wherein the T cell epitopes are preferably separated by a linker. In other embodiments, the antigenic unit comprises a plurality of T cell epitopes of a self antigen, allergen, alloantigen or xenogeneic antigen, wherein each T cell epitope is separated from other T cell epitopes by a linker. An alternative way of describing the separation of each T cell epitope of a self-antigen, allergen, alloantigen or xenogeneic antigen from other T cell epitopes by means of a linker is that all T cell epitopes are arranged in subunits, with each subunit comprising or consisting of a T cell epitope as described herein, except for the terminal T cell epitope, i.e. except for the T cell epitope located at the beginning of the N-terminus of the polypeptide or at the end of the C-terminus of the polypeptide (i.e. at the end of an antigenic unit not linked to a dimerization unit).

Thus, an antigenic unit comprising n antigens comprises n-1 subunits, wherein each subunit comprises a T cell epitope and a linker of a self antigen, an allergen, an alloantigen or a xenogeneic antigen, and further comprises a terminal T cell epitope. In some embodiments, n is an integer from 1 to 50, such as an integer from 3 to 50 or 15 to 40 or 10 to 30 or 10 to 25 or 10 to 20 or 15 to 30 or 15 to 25 or 15 to 20.

The linker in the antigenic unit separates the antigens, e.g. epitopes, contained therein. As described above, all T cell epitopes of autoantigens, allergens, alloantigens or xenogeneic antigens may be separated from each other by linkers and arranged in subunits.

In some embodiments, the linker is designed to be non-immunogenic. It may be a rigid linker, which means that it does not allow the two amino acid sequences to which it is attached to move substantially freely 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 may be used. In one embodiment, the T cell epitope linker is a flexible linker that allows for the presentation of T cell epitopes to T cells in an optimal manner even though the antigenic unit comprises a large number of T cell epitopes.

Since the T cell epitopes are separated by a linker, each T cell epitope of the autoantigen, allergen, alloantigen or xenogeneic antigen is presented to the immune system in an optimal manner.

For example, myelin Basic Protein (MBP), proteolipid protein (PLP), myelin-related glycoprotein (MAG), myelin Oligodendrocyte Glycoprotein (MOG) and myelin-related basic oligodendrocyte protein (MOBP) have all been studied and proposed as autoantigens involved in Multiple Sclerosis (MS), and antigenic units may comprise, for example, 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. Furthermore, 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 some embodiments, the antigenic unit may comprise one or more T cell epitopes of MOG, for example one or more T cell epitopes of MOG comprising or consisting of a sequence selected from SEQ ID NOS: 180-182.

In other embodiments, 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 some embodiments, the plurality of T cell epitopes belong to the same allergen, i.e. are comprised in the same allergen. In other embodiments, the plurality of T cell epitopes belong to, i.e. are comprised in, a plurality of different allergens.

For example, fel d1, fel d4 and Fel d7 are three of the most predominant cat allergens, accounting for the majority of human allergy to cats, and an antigenic unit may comprise, for example, one or more T cell epitopes of Fel d1, i.e. one T cell epitope of Fel d1 or multiple T cell epitopes of Fel d 1. Furthermore, the antigenic unit may comprise a plurality of T cell epitopes, e.g. Fel d4 and Fel d7, e.g. one or more T cell epitopes of Fel d4 and one or more T cell epitopes of Fel d 7.

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

In other embodiments, 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 some embodiments, the plurality of T cell epitopes belong to the same alloantigen/xenogeneic antigen, i.e. are comprised in the same alloantigen/xenogeneic antigen. In other embodiments, the plurality of T cell epitopes belong to, i.e. are comprised in, a plurality of different alloantigens/xenogeneic antigens.

In some embodiments, the antigenic unit comprises a T cell epitope. In other embodiments, the antigenic unit comprises more than one T cell epitope, i.e., a plurality of T cell epitopes.

The tolerance-inducing constructs of the present disclosure may be personalized therapies, i.e., designed for a particular subject/patient. In other embodiments, the tolerance-inducing constructs of the present disclosure are used universally in patient populations or patients, i.e., in-stock treatment.

Individualized tolerance-inducing constructs

For an individualized tolerance-inducing construct, T cell epitopes are selected for inclusion in the antigenic unit, wherein the T cell epitopes are optimized for the patient to be treated with the construct. This will increase the therapeutic effect compared to spot-treatments comprising the tolerance-inducing construct.

Taking MS patients as an example, the antigenic units of the personalized tolerance-inducing construct 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 tolerance-inducing 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:

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

off-the-shelf tolerance-inducing constructs

The antigenic units of the ready tolerance-inducing constructs preferably comprise hot spots of minimal T cell epitopes, i.e. one or more regions of the antigen containing multiple minimal T cell epitopes (e.g. 7-15 amino acids in length) that are predicted to be presented by different HLA alleles so as to cover a range of numerous subjects (e.g. ethnic groups or even worldwide or global groups).

By including such hot spots, the chance that the construct will induce tolerance in a range of numerous subjects is maximized.

Further description of antigenic units

The T cell epitopes in the antigenic units of the constructs of the present disclosure have a length of 7 to about 200 amino acids, wherein longer T cell epitopes may comprise the hot spot of the smallest T cell epitope.

In some embodiments, the antigenic unit comprises a T cell epitope of 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 in length, e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids.

T cell epitopes of about 60 to 200 amino acids in length can be split into shorter sequences and incorporated into antigenic units separated by linkers 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 some embodiments, the length of one T cell epitope causes the protein to fold incorrectly. For example, the best known cat allergen Fel d 1 is a protein formed from 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.

If a longer T cell epitope is incorporated into the 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.

In some embodiments, 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 one 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 some embodiments, the T cell epitope has a length of 7 to 11 amino acids for MHC class I presentation. In other embodiments, the T cell epitope sequence has a length of 9 to 60 amino acids, e.g., 9 to 30 amino acids, such as 15 to 60 amino acids, e.g., 15 to 30 amino acids, for MHC class II presentation. In other embodiments, the T cell epitope has a length of 15 amino acids for MHC class II presentation.

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 epitope linkers as described herein).

In some embodiments, 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 some embodiments, 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 other embodiments, the antigenic unit comprises 1 to 3T cell epitopes, e.g., 1, 2, 3T cell epitopes, or 1 to 5T cell epitopes, such as 1, 2,3, 4, 5T cell epitopes, or 3 to 6T cell epitopes, e.g., 3, 4, 5, 6T cell epitopes, or 5 to 15T cell epitopes, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15T cell epitopes, or 7 to 17T cell epitopes, e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17T cell epitopes, or 9 to 19T cell epitopes, e.g., 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19T cell epitopes.

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

In some embodiments, the T cell epitopes are arranged in order of higher antigenicity to lower antigenicity in a direction from the multimerization unit (e.g., dimerization unit) to the end of the antigenic unit. Alternatively, especially if the hydrophilicity/hydrophobicity varies greatly between T cell epitopes, the most hydrophobic T cell epitope may be located substantially centrally to the antigenic unit, and the most hydrophilic T cell epitope is located closest to the multimerization unit (e.g., dimerization unit) or at the end of the antigenic unit.

In some embodiments, the T cell epitopes are arranged in order of higher antigenicity to lower antigenicity in the direction from the multimerization unit to the end of the antigenic unit. Alternatively, especially if the hydrophilicity/hydrophobicity varies greatly between T cell epitopes, the most hydrophobic T cell epitope may be located substantially centrally of the antigenic unit, and the most hydrophilic T cell epitope is located closest to the multimerization unit or at the end of the antigenic unit.

In some embodiments, the T cell epitopes are arranged in order of higher antigenicity to lower antigenicity in the direction from the dimerization unit to the end of the antigenic unit. Alternatively, especially if the hydrophilicity/hydrophobicity varies greatly between T cell epitopes, the most hydrophobic T cell epitope may be located substantially centrally of the antigenic unit, and the most hydrophilic T cell epitope is closest to the dimerization unit or the antigenic unit end.

Since it is actually centrally located in the antigenic unit that only an odd number of T cell epitopes is possible in the antigenic unit, the term "substantially" in this context refers to an antigenic unit comprising an even number of T cell epitopes, wherein the most hydrophobic T cell epitope is 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 T cell epitopes are arranged in a manner that avoids GC clusters. In a preferred embodiment, the GC-rich T cell epitopes are arranged in such a way that there is at least one non-GC-rich T cell epitope between them. In some embodiments, the GC-rich sequences encoding the T cell epitopes are arranged such that there is at least one non-GC-rich T cell sequence between them. GC-rich sequences are sequences having a GC content of 60% or higher, e.g., 65% or higher, e.g., 70% or higher, e.g., 75% or higher, e.g., 80% or higher.

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.

The T cell epitope linker is designed to be non-immunogenic and preferably also flexible, allowing presentation of T cell epitopes to the immune system in an optimal way even if the antigenic unit comprises a large number of T cell epitopes.

Preferably, the T cell epitope linker is a peptide consisting of 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, e.g. 8, 9, 10, 11, 12, 13, 14 or 15 amino acids or 10 to 15 amino acids or 8 to 12 amino acids, e.g. 8, 9, 10, 11 or 12 amino acids. In a particularly preferred embodiment, the T cell epitope linker consists of 10 amino acids.

Preferably, 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 T cell epitope/linker junctions constitute epitopes, it is preferred to use T cell epitope linkers of different sequences.

In one embodiment, the T cell epitope linker is designed to be non-immunogenic. It may be a rigid linker, which means that it does not allow the two amino acid sequences to which it is attached to move substantially freely 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 T cell epitope linker is a flexible linker that allows for optimal presentation of T cell epitopes to T cells even though the antigenic unit comprises a large number of T cell epitopes.

Preferably, the T cell epitope linker is a serine (S) and/or glycine (G) rich linker, i.e. a linker comprising several serine and/or several glycine residues. Preferred examples are GGGGSGGGSS (SEQ ID NO: 51), GGGSG (SEQ ID NO: 52), GGGGS (SEQ ID NO: 53), SGSSGS (SEQ ID NO: 54), GGSGG (SEQ ID NO: 55) or variants thereof, such as GGGGSGGGGS (SEQ ID NO: 56), (GGGGS) m (SEQ ID NO:53 and 56-59), (GGGSS) m (SEQ ID NO: 60-64), (GGGSG) m (SEQ ID NO:52 and 65-68) or (SGSSGS) m (SEQ ID NO:54 and 69-72), 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 other preferred embodiments, the serine and/or glycine rich linker further comprises at least one leucine (L) residue, e.g. at least 1 or at least 2 or at least 3 leucine residues, e.g. 1, 2, 3 or 4 leucine residues.

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

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

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

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

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

In other embodiments, the T cell epitope linker is a rigid linker. Such rigid linkers can be used to effectively separate (larger) antigens and prevent them from interfering with each other. In one embodiment, the T cell epitope linker comprises or consists of: KPEPKPAPAPKP (SEQ ID NO: 142), AEAAAKEAAAKA (SEQ ID NO: 143), (EAAAK) m (SEQ ID NO: 144-148), PSRLEEELRRRLTEP (SEQ ID NO: 149) or SACCCELS (SEQ ID NO: 150).

In other embodiments, the T cell epitope linker comprises or consists of sequence TQKSLSLSPGKGLGGL (SEQ ID NO: 151). In other embodiments, the T cell epitope linker comprises or consists of sequence SLSLSPGKGLGGL (SEQ ID NO: 152). In other embodiments, the T cell epitope linker comprises or consists of AAY or GPGPG (SEQ ID NO: 153).

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

In other embodiments, the T cell epitope linker is a cleavable linker, e.g., a linker comprising one or more endopeptidase recognition sites, e.g., furin, caspase, cathepsin, and the like. Cleavable linkers can be introduced to release free functional protein domains (e.g., encoded by larger antigens), which can overcome steric hindrance between these domains or other drawbacks due to interference of these domains, such as reduced bioactivity, altered biodistribution.

Examples of T cell epitope linkers are disclosed in paragraphs [0098] - [0099] and the recited sequences (particularly SEQ ID NOs: 37 to 65 and 67 to 76) of WO 2020/176797A1 (incorporated by reference), and paragraphs [0135] to [0139] of US2019/0022202A1 (incorporated by reference).

Allergen

The tolerance-inducing constructs described herein can be used to induce tolerance to a range of different protein allergens, such as allergens that can be encoded by nucleic acid sequences contained in the polynucleotides of the constructs of the present disclosure, including protein allergens that undergo post-translational modification.

In some embodiments, the allergen is a food allergen. In some embodiments, the allergen is a shellfish allergen. In some embodiments, the allergen is tropomyosin, and in other embodiments, the allergen is arginine kinase, a myosin light chain, a troponin, troponin C, or a trisaccharide phosphate isomerase, or actin. In some embodiments, the allergen is Pan b 1. In some embodiments, the antigenic unit is a Pan b 1T cell epitope (251-270).

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 allergen. In some embodiments, the egg allergen is an ovomucoid, in other embodiments, the egg allergen is ovalbumin, ovotransferrin, conalbumin, gal 3 3, egg lysozyme, or ovomucoid.

One T cell epitope known in the art and studied in the context of egg allergy is OVA (257-264) having the amino acid sequence SIINFEKL (SEQ ID NO: 45).

In some embodiments, the antigenic unit of the constructs of the present disclosure comprises the T cell epitope OVA (257-264). Pharmaceutical compositions comprising the T cell epitopes are useful in the treatment of 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 pre-fibrin (profilin), a nsLTP, a sweet protein-like protein (thaumatin-like protein), a gibberellin-regulating protein, an isoflavone reductase-related protein, a chitinase class 1, a beta 1,3 glucanase, a germinating element-like protein (germin like protein), an alkaline serine protease, a pathogenic related protein 1, actinidin, a phytocystatin, 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 pathogenic related protein 10, pre-fibrin, nsLTP type 1, nsLTP type 2, osmorexin-like protein, isoflavone reductase-like protein, β -fructofuranosidase, PR protein TSI-1, cyclophilin, or 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 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 pre-fibrin.

In other embodiments, the food allergen is or is derived from buckwheat, celery, a pigment additive, garlic, gluten, oat, beans, corn, mustard, poultry, meat, rice, sesame.

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/lcarapin, 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. 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, hev b 15.

In some embodiments, the allergen is a dust mite allergen. In some embodiments, the allergen is a house dust mite allergen (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 is 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 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, blag 2, blag 3, blag 4, blag 5, blag 6, blag 7, blag 8, or Blag 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 allergen. In some embodiments, the A.fumigatus allergen is Asp f 1, asp f2, 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 f2, 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 dog allergen. In some embodiments, the dog allergen is Can f 1, can f 2, can f 3, can f 4, can f 5, or Can f 6. In some embodiments, the allergen is a equine allergen. In some embodiments, the horse allergen is ecl c 1, ecl c 2, ecl c 3, or ecl c 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 (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 (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. 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, birch, hornbeam (hornbeam), hazel, elnut (European hophornbeam), chestnut (chestnut), beech (European beech), white oak, white wax, glossy privet, olive, clove, cypress, or 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, cora 2, cora 6, cora 8, cora 9, cora 10, cora 11, cora 12, cora 13, cora 14, ost c 1, cas 5, cas 8, or Cas 9, fag s 1, que a 1, fre 1, lig v 1, ole e 1 the method comprises the steps of 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 a 1, pla a 2 or Pla a 3 or Pla or 1, pla or 2 or Pla or 3. In some embodiments, the antigenic unit is 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 ragweed, mugwort, sunflower, parthenote, pellitory, british plantain, arnebia, quinoa, russia Luo Siji, or amaranth pollen allergen. In some embodiments, the ragweed pollen allergen is Amb a 1, amb a 4, amb a 6, amb a 8, amb a 9, amb a 10, or Amb a 11. In some embodiments, the 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 other embodiments, the allergen is selected from environmental allergens, such as insects, cockroaches, house dust mites, or molds.

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

More than 7% of the general population is allergic to the drug. Constructs of the present disclosure induce tolerance to immunogenic epitopes present in such drugs and will therefore allow the affected patient to continue and benefit from treatment with the drug.

Thus, in some embodiments, the allergen is comprised in a medicament having undesired immunogenicity. In some embodiments, the allergen is factor VIII. In some embodiments, the allergen is insulin. In some embodiments, the allergen is one or more monoclonal antibodies for use in therapy.

Autoantigens

In other embodiments, the tolerance-inducing constructs of the invention contain T cell epitopes contained in an autoantigen involved in an autoimmune disease. This allows antigen-specific downregulation of the part of the immune system responsible for autoimmune diseases without inhibiting the immune system as a whole.

In some embodiments, the autoimmune disease is Multiple Sclerosis (MS). In some embodiments, the autoantigen is Myelin Oligodendrocyte Glycoprotein (MOG). In other embodiments, the autoantigen is MAG, MOBP, CNPase, S100 β or transaldolase. In some embodiments, the autoantigen is Myelin Basic Protein (MBP). In some embodiments, the autoantigen is myelin proteolipid protein (PLP).

In an example, we provide constructs for multiple sclerosis that include short (35-55 amino acids) or longer (27-63 amino acids) T cell epitopes from Myelin Oligodendrocyte Glycoprotein (MOG). MOGs are members of the immunoglobulin superfamily, which are expressed only in the central nervous system. MOG (35-55) is capable of inducing autoantibody production and relapse remitting neurological disease, leading to extensive plaque-like demyelination. Autoantibody responses to MOG (35-55) have been observed in MS patients and in MOG (35-55) -induced Experimental Autoimmune Encephalomyelitis (EAE) in C57/BL6 mice and Lewis rats.

Other MS-associated T cell epitopes known and studied in the art include the following:

* T cell epitope-induced EAE was observed

In preferred embodiments, the antigenic units of the constructs of the present disclosure 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). Pharmaceutical compositions comprising such constructs are useful for the treatment of 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 still other embodiments, the autoantigen is IGRP, chgA, IAPP, a peripherin, a tetraspanin-7, a GRP78, a uremic acid-3, or an insulin gene enhancer protein isl-1.

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

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 Bank 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 autoimmune disease is Chronic Inflammatory Demyelinating Polyneuropathy (CIDP) and the autoantigen is clusterin 155. In other embodiments, the autoimmune disease is Hashimoto Thyroiditis (HT) and the autoantigen is thyroid peroxidase and/or thyroglobulin. In other embodiments, the autoimmune disease is pemphigus larum and the autoantigen is desmosome-associated glycoprotein. In other embodiments, the autoimmune disease is pemphigus vulgaris and the autoantigen is desmosome 3. In other embodiments, the autoimmune disease is Thyroiditis (TED) and the autoantigen is a calbindin (calpain). In other embodiments, the autoimmune disease is Grave's disease and the autoantigen is a thyroid stimulating hormone receptor. In other embodiments, 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 other embodiments, the autoimmune disease is myasthenia gravis and the autoantigen is an acetylcholine receptor. In other embodiments, the autoimmune disease is insulin resistant diabetes and the autoantigen is an insulin receptor. In other embodiments, the autoimmune disease is autoimmune hemolytic anemia and the autoantigen is red blood cells. In other embodiments, the autoimmune disease is rheumatoid arthritis and the autoantigen is citrullinated protein, homocysteine protein, and/or Fc portion of IgG.

In other embodiments, the autoimmune disease is psoriasis and the autoantigens are cathelicidin (LL-37), depolymerized protein-like metalloprotease domain containing thrombin-sensitive protein type 1 motif 5 (ADAMTS L5), phospholipase A2 group IVD (PLA 2G 4D), nuclear heterogeneous ribonucleoprotein A1 (hnRNP-A1) and keratin 17.

Unit joint

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

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

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

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

Multimerization units and dimerization units

Constructs of the disclosure comprise multimerization units, e.g., dimerization units.

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

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

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 facilitates multimerization/linking of multiple polypeptides, e.g., two, three, four or more polypeptides, into a multimeric protein, e.g., a dimeric protein, a trimeric protein or a tetrameric protein. Multimerization units also provide flexibility to multimeric proteins to allow optimal binding of the targeting units to surface molecules on APCs even though they are located at different distances. 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, e.g., a collagen-derived trimerization unit, e.g., a human collagen-derived trimerization domain, e.g., a human collagen-derived XVIII trimerization domain (see, e.g., a. Alvarez-cienfugos et al, sci Rep 6,28643 (2016)) or a human collagen XV trimerization domain. Thus, in one embodiment, the multimerization unit is a trimerization unit comprising or consisting of: has the sequence of SEQ ID NO:42, or an amino acid sequence encoded by said nucleotide sequence. In another embodiment, the trimerization unit is the C-terminal domain of T4 fibritin. Thus, in one embodiment, the multimerization unit is a trimerization unit comprising or consisting of: has the sequence of SEQ ID NO:43, or a nucleotide sequence encoding said amino acid sequence.

In another embodiment, the multimerization unit is a tetramerization unit, e.g., 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 having SEQ ID NO. 43 or an amino acid sequence encoded by said nucleic acid sequence optionally further comprises a hinge region as described below.

In the context of a multimerization unit, the term "hinge region" refers to an amino acid sequence comprised in the multimerization unit that facilitates the joining of two or more polypeptides, e.g. three or four polypeptides, i.e. facilitates the formation of multimeric or dimeric proteins and/or acts as a flexible spacer, allowing the targeting unit of the multimeric protein to bind to multiple surface molecules on the APC simultaneously, even if these surface molecules are located at different distances.

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 polypeptides into a dimeric protein. The dimerization unit also provides flexibility for the dimeric protein to allow optimal binding of the targeting unit to surface molecules on the APC even though they are located at different distances. The dimerization unit may be any unit that meets one or more of these requirements.

Thus, in some embodiments, the constructs of the present disclosure comprise a dimerization unit comprising a hinge region. In other embodiments, the dimerization unit comprises a hinge region and another domain that promotes dimerization. In other embodiments, 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 to the other dimerization promoting domain. In other embodiments, 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 to the other dimerization promoting domain. Dimerization unit linkers are described further below.

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

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

Furthermore, the hinge region acts as a flexible spacer, allowing two targeting units of the dimeric protein to bind simultaneously to two surface molecules on APC, even if they are located at different distances. The hinge region may be Ig derived, e.g. derived from IgG1, igG2 or IgG3. In one embodiment, the hinge region is derived from IgM, e.g., comprises a polypeptide having the amino acid sequence of SEQ ID NO:47 or an amino acid sequence encoded by said nucleic acid sequence, or consists of the same. The hinge region may promote dimerization (or multimerization) by forming covalent bonds such as disulfide bridges between cysteines. Thus, in some embodiments, the hinge region has the ability to form one or more covalent bonds. Preferably, the covalent bond is a disulfide bridge.

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

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 with amino acid sequences 1-27 of SEQ ID No. 1, 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.

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

In a preferred embodiment, the dimerization unit comprises or consists of: hinge exon h1 and hinge exon h4 having amino acid sequences 1-27 of SEQ ID NO. 1, except that at most ten amino acids have been substituted, deleted or inserted, for example at most nine amino acids, for example at most eight amino acids, for example at most seven amino acids, for example at most six amino acids, for example at most five amino acids, for example at most four amino acids, for example at most three amino acids, for example at most two amino acids or for example at most one amino acid have been substituted, deleted or inserted.

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

In other embodiments, the dimerization unit comprises another domain that promotes dimerization; preferably, the further domain is an immunoglobulin domain, e.g. an immunoglobulin constant domain (C domain), e.g. a CH1 domain, a CH2 domain or a carboxy-terminal C domain (i.e. a CH3 domain), or a sequence substantially identical to such a C domain or a variant thereof. 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 some embodiments, the dimerization unit comprises or consists of: a carboxy-terminal C domain derived from IgG3 having an amino acid sequence having at least 80% sequence identity to amino acid sequence 39-144 of SEQ ID No. 1, or a nucleotide sequence encoding said amino acid sequence.

In a preferred embodiment, the dimerization unit comprises or consists of: a carboxy-terminal C domain derived from IgG3 having an amino acid sequence having at least 85% sequence identity to amino acid sequences 39-144 of SEQ ID No. 1, 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.

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

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

Immunoglobulin domains promote dimerization through non-covalent interactions, such as hydrophobic interactions. Thus, in some embodiments, 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 further preferred embodiment, the dimerization unit comprises a polypeptide consisting of hinge exon h1, hinge exon h4, a dimerization unit linker and the CH3 domain of human IgG 3.

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

In preferred embodiments, the dimerization unit comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO. 1, such as at least 86%, 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.

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

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

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

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

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

Signal peptides

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

Any suitable signal peptide may be used. Examples of suitable peptides are human Ig VH signal peptides or signal peptides naturally occurring at the N-terminus of any of the targeting units described herein, e.g.human signal peptide of human IL-10 or human signal peptide of human TGF-beta.

Thus, in some embodiments, the polynucleotide comprises a nucleotide sequence encoding a human IL-10 signal peptide, and preferably comprises a nucleotide sequence encoding a human IL-10 targeting unit. In other embodiments, the polynucleotide comprises a nucleotide sequence encoding a human Ig VH signal peptide, and preferably comprises a nucleotide sequence encoding an scFv (e.g., human anti-DEC 205).

In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a signal peptide comprising an amino acid sequence having at least 85%, such as at least 86%, 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. 6 or SEQ ID NO. 48.

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

In other embodiments, the polynucleotide comprises a nucleotide sequence encoding a signal peptide consisting of an amino acid sequence having 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. 6 or SEQ ID NO. 48.

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

In other embodiments, the polynucleotide comprises a nucleotide sequence encoding a signal peptide comprising or consisting of: the amino acid sequence of SEQ ID No. 6 or SEQ ID No. 48, except that at most five amino acids have been substituted, deleted or inserted, for example at most four amino acids, for example at most three amino acids, for example at most two amino acids or for example at most one amino acid have been substituted, deleted or inserted.

In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a murine IL-10 signal peptide (e.g., the IL-10 signal peptide set forth in SEQ ID NO: 50), and preferably comprises a nucleotide sequence encoding a murine IL-10 targeting unit (e.g., the murine IL-10 targeting unit set forth in SEQ ID NO: 169).

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

Sequence identity

Sequence identity can be determined as follows: a high level of sequence identity indicates the likelihood that the second sequence is derived from the first sequence. Amino acid sequence identity requires 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 means of 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 choice.nucleic Acids Res.22:4673-4680), and default parameters suggested therein. Using this procedure and its default settings, the mature (biologically active) portions of the query and reference polypeptides can be aligned. The number of fully conserved residues is counted and divided by the length of the reference polypeptide. In this process, any tag or fusion protein sequences that form part of the query sequence are ignored 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 similar manner as shown for the amino acid sequence.

Another preferred mathematical algorithm for comparing sequences is the algorithm of Myers and Miller, 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 penalty gaps at the end of the sequence. When comparing amino acid sequences using the ALIGN and ALIGN0 programs, the BLOSUM50 substitution matrix with a gap opening/extension penalty of-12/-2 is preferably used.

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

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 secondary binding activity of the substance is 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 (like-for-like) substitutions, such as basic substitution basic, acidic substitution acidic, polar substitution polar, etc., are contemplated herein, as well as non-conservative substitutions, i.e., from one type of residue to another or alternatively include the incorporation of unnatural amino acids, such as ornithine, diaminobutyrate, norleucine, ornithine, pyridylalanine, thienylalanine, naphthylalanine, and phenylglycine. Conservative substitutions that may be made within, for example, the following groups: basic amino acid groups (arginine, lysine and histidine), acidic amino acid groups (glutamic acid and aspartic acid), aliphatic amino acid groups (alanine, valine, leucine, isoleucine), polar amino acid groups (glutamine, asparagine, serine, threonine), aromatic amino acid groups (phenylalanine, tryptophan, tyrosine), hydroxyl amino acid groups (serine, threonine), large amino acid groups (phenylalanine, tryptophan) and small amino acid groups (glycine, alanine).

Substitutions may also be made by unnatural amino acids, and the substitution residues include: α and α -disubstituted amino acids, N-alkylamino acids, lactic acid, halide derivatives of natural amino acids such as trifluorotyrosine, p-chloro-phenylalanine, p-bromo-phenylalanine, p-L-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, methyl derivatives of p-nitro-L-phenylalanine, L-hydroxyproline, L-thioproline, phenylalanine (Phe) such as 4-methyl-Phe, pentamethyl-Phe, L-Phe (4-amino), L-Tyr (methyl), L-Phe (4-isopropyl) L-3, 3-tetrahydrophe (3, 3-phenylic acid) and 4-benzyl-amino-3-isopropyl-3, 3-amino-propanic acid.

In the above paragraph, # denotes the hydrophobicity of the substituted residue, while # denotes the hydrophilicity of the substituted residue, and # denotes the amphipathic nature of the substituted residue. The variant amino acid sequence may include a suitable spacer group that may be inserted between any two amino acid residues of the sequence, including an alkyl group such as methyl, ethyl or propyl in addition to an amino acid spacer such as glycine or β -alanine residue. Another form of variation involves the presence of one or more amino acid residues in the peptoid (pepoid) form.

Polynucleotide

The tolerance-inducing constructs of the present disclosure may be in the form of polynucleotides.

Another aspect of the disclosure is a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigenic unit that targets or is capable of targeting an antigen presenting cell, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, allergen, alloantigen, or xenogeneic antigen.

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

Preferably, the polynucleotide is optimised for the species to which it is applied. Thus, for human administration, preferably the polynucleotide sequence is human codon optimized.

Polypeptides and multimeric/dimeric proteins

The constructs of the present disclosure may be in the form of a polypeptide encoded by a nucleotide sequence comprised in a polynucleotide as described above.

Another aspect of the disclosure is a polypeptide comprising a targeting unit that targets or is capable of targeting an antigen presenting cell, 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.

Another aspect of the invention is a polypeptide comprising a targeting unit that targets or is capable of targeting an antigen presenting cell, a multimerizing unit, and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of a self antigen, allergen, alloantigen, or xenogeneic antigen.

Another aspect of the disclosure is a polypeptide comprising a targeting unit that targets or is capable of targeting an antigen presenting cell, a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of a self antigen, an allergen, an alloantigen, or a xenogeneic antigen.

The polypeptide may be expressed in vitro to produce a tolerance-inducing construct, e.g., for producing a pharmaceutical composition comprising the construct, or the polypeptide may be expressed in vivo as a result of administering the polynucleotide to a subject, as described above. Due to the presence of multimerization/dimerization units, polypeptides form multimerization/dimerization proteins upon expression, i.e., by linking multiple polypeptides via their respective multimerization/dimerization units.

Another aspect of the disclosure is a multimeric protein, e.g., a dimeric protein, comprising a plurality of polypeptides, e.g., two polypeptides, each comprising a targeting unit that targets or is capable of targeting an antigen presenting cell, a multimerizing unit, e.g., a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of a self antigen, an allergen, an alloantigen, or a heterologous antigen.

Another aspect of the disclosure is a multimeric protein comprising a plurality of polypeptides, each polypeptide comprising a targeting unit that targets or is capable of targeting an antigen presenting cell, a multimerizing unit, and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of a self antigen, allergen, alloantigen, or xenogeneic antigen.

Another aspect of the disclosure is a dimeric protein comprising a plurality of polypeptides, each polypeptide comprising a targeting unit that targets or is capable of targeting an antigen presenting cell, a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of a self antigen, an allergen, an alloantigen, or a xenogeneic antigen.

The multimeric protein may be a homomultimer, i.e., a multimeric protein in which multiple polypeptide chains are identical and thus comprise the same units and thus comprise the same antigen sequence, or the multimeric protein may be a heteromultimer comprising multiple polypeptide chains, each of which may comprise a different antigen sequence in its antigenic unit. The dimeric protein may be a homodimer, i.e. a dimeric protein in which the two polypeptide chains are identical and thus comprise the same units and thus comprise the same antigen sequence, 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 the case if the number of T cell epitopes comprised in the antigenic unit exceeds the upper size limit of the antigenic unit. Preferably, the multimeric/dimeric protein is a homomultimeric/homodimeric protein.

Carrier body

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

Another aspect of the disclosure is a vector comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerizing unit, such as a dimerization unit, and an antigenic unit that comprises one or more T cell epitopes of an autoantigen, allergen, alloantigen, or xenogeneic antigen that targets or is capable of targeting an antigen presenting cell.

Another aspect of the disclosure is a vector comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerizing unit, and an antigenic unit that targets or is capable of targeting an antigen presenting cell, wherein the antigenic unit comprises one or more T cell epitopes of a self antigen, an allergen, an alloantigen, or a xenogeneic antigen.

Another aspect of the disclosure is a vector comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigenic unit that targets or is capable of targeting an antigen presenting cell, wherein the antigenic unit comprises one or more T cell epitopes of a self antigen, an allergen, an alloantigen, or a xenogeneic antigen.

Preferably, the carrier allows easy exchange of the individual units described above, in particular antigenic units.

In some embodiments, the vector may be pALD-CV77 or any other vector that does not comprise a bacterial nucleotide sequence that is known to trigger an immune response in an adverse manner upon introduction into a subject. The antigenic unit may be exchanged with an antigenic unit cassette, e.g.a SfiI restriction enzyme cassette, which is restricted by a convenient restriction enzyme, wherein the 5 'site is incorporated in the nucleotide sequence encoding the GLGGL (SEQ ID NO: 90) and/or GLSGL (SEQ ID NO: 163) unit linker and the 3' site is comprised after the stop codon in the vector.

The vector of the present disclosure may be any molecule suitable for carrying and expressing an exogenous nucleic acid sequence (e.g., DNA or RNA) into a cell, i.e., an expression vector.

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

In other embodiments, the vector is an RNA vector, such as an RNA plasmid or an RNA viral vector, such as 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. In a preferred embodiment, the vector is a DNA plasmid and the polynucleotide is DNA.

Plasmid(s)

A plasmid is a small extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and capable of independent replication. Plasmids are usually present in bacteria as small circular double stranded DNA molecules; however, plasmids are sometimes present in archaebacteria and eukaryotic organisms. Artificial plasmids are widely used as vectors in molecular cloning for delivery and to ensure high expression of recombinant DNA sequences in host organisms. Plasmids contain several important features, including features for selecting cells containing the plasmid (e.g., antibiotic resistance genes), origins of replication, multiple Cloning Sites (MCSs), and promoters for driving expression of the inserted gene of interest.

In general, a promoter is a sequence that attracts an initiation factor and a polymerase to the promoter to transcribe a gene. The promoter is located upstream of the transcription initiation site of the gene on the DNA. The length of the promoter is about 100-1000 base pairs. 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 plasmid DNA, the RNA molecule is transcribed. After processing, when the ribosome translates the mRNA into a protein, the mRNA will be able to be translated multiple times, producing many copies of the protein encoded by the gene of interest. In general, ribosomes facilitate decoding by inducing binding of complementary tRNA anticodon sequences to mRNA codons. tRNA carries specific amino acids that link together to form a polypeptide when mRNA passes through and is "read" by a ribosome. Translation proceeds in three stages: initiation, extension, and termination. Following the translation process, the polypeptide folds into an active protein and performs its function in the cell, or is exported from the cell and performs its function elsewhere, sometimes after a considerable amount of post-translational modification.

When a protein is designated to be exported outside the cell, the signal peptide directs the protein into the endoplasmic reticulum, where the signal peptide is cleaved, and the protein is transferred to the periphery after translation is terminated.

The DNA plasmids of the present disclosure are not limited to any particular plasmid, and those skilled in the art will appreciate that any plasmid having a suitable backbone may be selected and engineered to contain the elements and units of the present disclosure by methods known in the art.

Host cells

Another aspect of the disclosure is a host cell comprising

i) A polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerizing unit, such as a dimerization unit, and an antigenic unit, which comprises one or more T cell epitopes of an autoantigen, allergen, alloantigen or xenogeneic antigen, which targets or is capable of targeting an antigen presenting cell; or alternatively

iii) A multimeric protein, e.g. a dimeric protein, which consists of a plurality of polypeptides, e.g. two polypeptides, as defined in (ii).

Another aspect of the disclosure is a host cell comprising

i) A polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigenic unit that targets or is capable of targeting an antigen presenting cell, wherein the antigenic unit comprises one or more T cell epitopes of a self antigen, an allergen, an alloantigen, or a heterologous antigen; or alternatively

iii) A multimeric protein consisting of two polypeptides as defined in (ii).

Another aspect of the disclosure is a host cell comprising

i) A polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigenic unit that targets or is capable of targeting an antigen presenting cell, wherein the antigenic unit comprises one or more T cell epitopes of a self antigen, an allergen, an alloantigen, or a xenogeneic antigen; or alternatively

iii) A dimeric protein consisting of two polypeptides as defined in (ii).

Suitable host cells include prokaryotes, yeast, insects, or higher eukaryotic cells. In a preferred embodiment, the host cell is a human cell, preferably a cell of a human individual suffering from an immune disease and in need of prophylactic or therapeutic treatment with the constructs of the present disclosure.

Pharmaceutical composition

The constructs of the present disclosure may be administered to a subject as a pharmaceutical composition comprising the construct (e.g., in the form of a polynucleotide or a multimeric/dimeric protein) and a pharmaceutically acceptable carrier.

Another aspect of the present disclosure is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and

wherein the antigenic unit comprises one or more T cell epitopes of autoantigens, allergens, alloantigens or xenogeneic antigens.

iii) A multimeric protein consisting of a plurality of polypeptides as defined in (ii);

i) A polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigenic unit that targets or is capable of targeting an antigen presenting cell; or alternatively

iii) A dimeric protein consisting of two polypeptides as defined in (ii);

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

In some embodiments, the composition may comprise one or more adjuvants. Suitable adjuvants include, but are not limited to, dexamethasone, the B subunit of the enterotoxin Cholera Toxin (CTB), TLR2 ligands, helminth-derived excretion/secretion (ES) products, rapamycin or vitamin D3 analogs, and aryl hydrocarbon receptor ligands.

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

As used herein, an "amphiphilic block copolymer" is a linear or branched copolymer comprising or consisting of blocks of poly (ethylene oxide) ("PEO") and blocks of poly (propylene oxide) ("PPO"). Typical examples of useful PEO-PPO amphiphilic block copolymers have the following general structure: PEO-PPO-PEO (poloxamer), PPO PEO PPO, (PEO PPO-) 4ED (poloxamine) and (PPO PEO-) 4ED (reverse poloxamine), wherein "ED" is an ethylenediamido group.

"poloxamer" is a linear amphiphilic block copolymer consisting of a 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, coupled with a poly (ethylene oxide) block coupled with a polyethylene oxide block coupled with a PEO block. Poloxamers are typically named using a 3-digit identifier, where the first 2 digits multiplied by 100 provide an approximate molecular weight of the PPO content, and 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 about 80% (w/w) PEO (corresponding to a of about 82). However, these values are known to vary to some extent, and are of research grade F68 and clinical grade->Commercial products such as P188 (both poloxamers 188 according to the manufacturer's data sheet) exhibit large molecular weight variations (between 7,680 and 9,510) and provide a and b values of about 79 and 28, respectively, for these particular products. This reflects the heterogeneity of the block copolymer, indicating that the values of a and b are the average values present in the final formulation.

"poloxamine" or "sequential poloxamine (sequential poloxamine)" (under the trade name "poloxamineCommercially available) is an X-block copolymer that carries 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 the ethylenediamine moiety.

Preferred amphiphilic block copolymers are poloxamers or poloxamines. Preferred are poloxamers 407 and 188, in particular poloxamer 188. The preferred poloxamine is the sequential poloxamine of formula (PEO-PPO) 4-ED. Particularly preferred poloxamines are under the registered trade marks respectively 904. 704 and 304. These poloxamines are characterized as follows: />904 has a total average molecular weight of 6700, a total average weight of ppo units of 4020, and a peo percentage of about 40%.

704 has a total average molecular weight of 5500, a total average weight of ppo units of 3300, a peo percentage of about 40%; />304 has a total average molecular weight of 1650, a total average weight of ppo units of 990, and a peo percentage of about 40%.

In some embodiments, the composition comprises amphiphilic block copolymer in an amount of 0.2% w/v to 20% w/v, e.g., 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 other embodiments, the composition comprises the amphiphilic block copolymer in an amount of 2% w/v to 5% w/v, for example about 3% w/v.

For pharmaceutical compositions comprising polynucleotides, the composition may further comprise molecules that facilitate cell transfection.

The pharmaceutical composition may be formulated in any manner suitable for administration to a subject (e.g., a patient suffering from or suspected of suffering from an autoimmune disease, allergic disease, or graft rejection), e.g., for injection, such as intradermal or intramuscular injection, of a liquid formulation.

In some embodiments, the pharmaceutical compositions comprising the polynucleotides described herein (e.g., contained in a carrier) may be administered in any manner suitable for administration to a subject (e.g., transdermal, intramuscular, or subcutaneous administration, or administration via mucous membranes or epithelium, e.g., intranasal or oral administration).

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

The pharmaceutical compositions of the present disclosure typically comprise polynucleotides in the range of 0.1 μg to 10mg, for example about 0.2 μg, 0.3 μg, 0.4 μg, 0.5 μg, 0.75 μg, 1 μg, 5 μg, 10 μg, 25 μg, 50 μg, 75 μg or more; for example 0.1 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 for example 2, 3, 4, 5, 6, 7, 8, 9 or 10mg of polynucleotide. The pharmaceutical compositions of the present disclosure typically comprise polypeptide/dimer protein in the range of 5 μg to 5 mg.

The amount of polynucleotide/polypeptide/dimer protein/multimer protein may vary depending on whether the pharmaceutical composition is administered for prophylactic or therapeutic treatment, the severity of the immune disorder in an individual suffering from the immune disorder, and parameters such as age, weight, sex, medical history, and past conditions.

Preparation of the medicamentMethod of making a composition

Suitable methods for preparing the pharmaceutical compositions or vaccines of the present disclosure are disclosed in WO 2004/076489A1, WO 2011/161244A1, WO 2013/092875A1 and WO 2017/118695A1, which are incorporated herein by reference.

In one aspect, the present disclosure relates to a method for preparing a pharmaceutical composition comprising a multimeric/dimeric protein or polypeptide as defined above by in vitro production of the polypeptide. In vitro synthesis of polypeptides and proteins may be performed by any suitable method known to those skilled in the art, for example by peptide synthesis or expression of the polypeptide in a variety of expression systems followed by purification.

In one aspect, the present disclosure relates to a method for preparing a pharmaceutical composition comprising a multimeric protein or polypeptide as defined above by in vitro production of the polypeptide. In vitro synthesis of polypeptides and proteins may be performed by any suitable method known to those skilled in the art, for example by peptide synthesis or expression of the polypeptide in a variety of expression systems followed by purification.

In one aspect, the present disclosure relates to a method for preparing a pharmaceutical composition comprising a dimeric protein or polypeptide as defined above by in vitro production of the polypeptide. In vitro synthesis of polypeptides and proteins may be performed by any suitable method known to those skilled in the art, for example by peptide synthesis or expression of the polypeptide in a variety of expression systems followed by purification.

Thus, another aspect of the invention is a method of preparing a pharmaceutical composition comprising a multimeric or dimeric protein consisting of a plurality of polypeptides, such as two, three, four or more polypeptides, or comprising a polypeptide,

wherein the method comprises the following steps:

a) Transfecting a cell with a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, e.g., a dimerization unit, and an antigenic unit that targets or is capable of targeting an antigen presenting cell, wherein the antigenic unit comprises one or more T cell epitopes of a self antigen, allergen, alloantigen, or xenogeneic antigen;

b) Culturing the cells;

c) Collecting and purifying multimeric or dimeric proteins or polypeptides expressed by the cells; and

d) Mixing the multimeric or dimeric protein or polypeptide obtained from step c) with a pharmaceutically acceptable carrier.

Thus, another aspect of the invention is a method of preparing a pharmaceutical composition comprising a multimeric protein consisting of a plurality of polypeptides, such as two, three, four or more polypeptides, or comprising a polypeptide,

wherein the method comprises the following steps:

a) Transfecting a cell with a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigenic unit that targets or is capable of targeting an antigen presenting cell, wherein the antigenic unit comprises one or more T cell epitopes of an autoantigen, allergen, alloantigen, or xenogeneic antigen;

b) Culturing the cells;

c) Collecting and purifying multimeric proteins or polypeptides expressed by the cells; and

d) Mixing the multimeric protein or polypeptide obtained from step c) with a pharmaceutically acceptable carrier.

Thus, another aspect of the invention is a method for preparing a pharmaceutical composition comprising a dimeric protein consisting of two polypeptides, or comprising a polypeptide,

wherein the method comprises the following steps:

b) Culturing the cells;

c) Collecting and purifying the dimeric protein or polypeptide expressed by the cells; and

d) Mixing the dimeric protein or polypeptide obtained from step c) with a pharmaceutically acceptable carrier.

In a preferred embodiment, the multimeric protein, dimeric protein or polypeptide obtained from step c) is dissolved in said pharmaceutically acceptable carrier.

In a preferred embodiment, the multimeric protein or polypeptide obtained from step c) is dissolved in said pharmaceutically acceptable carrier.

In a preferred embodiment, the dimeric protein or polypeptide obtained from step c) is dissolved in said pharmaceutically acceptable carrier.

Purification may be performed according to any suitable method, such as chromatography, centrifugation or differential solubility.

In another aspect, the present disclosure relates to a method of preparing a pharmaceutical composition comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerizing unit, e.g., a dimerization unit, and an antigenic unit, wherein the antigenic unit comprises one or more T cell epitopes of a self antigen, allergen, alloantigen, or xenogeneic antigen, wherein the method comprises:

a) Preparing the polynucleotide;

b) Optionally cloning the polynucleotide into an expression vector; and

In another aspect, the present disclosure relates to a method of preparing a pharmaceutical composition comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerizing unit, and an antigenic unit that targets or is capable of targeting an antigen presenting cell, wherein the antigenic unit comprises one or more T cell epitopes of a self antigen, an allergen, an alloantigen, or a heterologous antigen, wherein the method comprises:

a) Preparing the polynucleotide;

b) Optionally cloning the polynucleotide into an expression vector; and

In another aspect, the present disclosure relates to a method of preparing a pharmaceutical composition comprising a polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigenic unit that targets or is capable of targeting an antigen presenting cell, wherein the antigenic unit comprises one or more T cell epitopes of a self antigen, an allergen, an alloantigen, or a heterologous antigen, wherein the method comprises:

a) Preparing the polynucleotide;

b) Optionally cloning the polynucleotide into an expression vector; and

Polynucleotides may be prepared by any suitable method known to the skilled artisan. For example, polynucleotides can be prepared by chemical synthesis using an oligonucleotide synthesizer.

In particular, the nucleotide sequences encoding the targeting unit and/or dimerization unit may be synthesized separately and then ligated into the vector backbone to produce the final polynucleotide by ligating the nucleic acid sequences encoding the antigenic units into the vector.

In one aspect, the disclosure relates to the use of a construct, polynucleotide, polypeptide or multimeric protein described herein, e.g., a dimeric protein, as a medicament.

In one aspect, the disclosure relates to the use of a construct, polynucleotide, polypeptide or multimeric protein described herein as a medicament.

In one aspect, the disclosure relates to the use of a construct, polynucleotide, polypeptide or dimeric protein as described herein as a medicament.

Treatment of

The constructs or pharmaceutical compositions of the present disclosure may be used to treat autoimmune diseases, allergic diseases, or graft rejection, and the treatment may be for prophylactic or therapeutic purposes.

Administration of the construct/pharmaceutical composition is such that tolerance is induced in the individual to whom such pharmaceutical composition is administered. Tolerance is induced by a single administration and preferably by multiple administrations sufficiently spaced apart in time.

In another aspect, the present disclosure provides a method for treating a subject having or suspected of having an immune disease selected from the group consisting of autoimmune disease, allergic disease, and graft rejection, or in need of prevention of such disease, the method comprising administering to the subject a pharmaceutical composition comprising:

i) A polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, such as a dimerization unit, and an antigenic unit, that targets or is capable of targeting an antigen presenting cell; or (b)

ii) a polypeptide encoded by a nucleic acid sequence as defined in (i); or (b)

iii) A multimeric protein, such as a dimeric protein, consisting of a plurality of polypeptides as defined in (ii);

i) A polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, and an antigenic unit that targets or is capable of targeting an antigen presenting cell; or (b)

ii) a polypeptide encoded by a nucleic acid sequence as defined in (i); or (b)

i) A polynucleotide comprising a nucleotide sequence encoding a targeting unit, a dimerization unit, and an antigenic unit that targets or is capable of targeting an antigen presenting cell; or (b)

ii) a polypeptide encoded by a nucleic acid sequence as defined in (i); or (b)

iii) A dimeric protein consisting of two polypeptides as defined in (ii);

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.

In some embodiments, one dose of the pharmaceutical composition is administered to the subject. In some embodiments, multiple doses of the pharmaceutical composition are administered to a subject.

In another aspect, the present disclosure provides a pharmaceutical composition for the prophylactic or therapeutic treatment of an immune disorder selected from the group consisting of autoimmune disorders, allergic disorders, and graft rejection, the pharmaceutical composition comprising:

ii) a polypeptide encoded by a nucleic acid sequence as defined in (i); or (b)

iii) A dimeric protein consisting of two polypeptides as defined in (ii);

In another aspect, the present disclosure provides the use of a pharmaceutical composition for the prophylactic or therapeutic treatment of a subject suffering from or suspected of suffering from an immune disease selected from the group consisting of autoimmune disease, allergic disease and graft rejection, the method comprising administering to the subject a pharmaceutical composition comprising:

ii) a polypeptide encoded by a nucleic acid sequence as defined in (i); or (b)

iii) A multimeric protein, e.g. a dimeric protein, consisting of a plurality of polypeptides, e.g. as defined in (ii);

ii) a polypeptide encoded by a nucleic acid sequence as defined in (i); or (b)

iii) A dimeric protein consisting of two polypeptides as defined in (ii);

In another aspect, the present disclosure provides the use of a pharmaceutical composition in the manufacture of a medicament for the prophylactic or therapeutic treatment of an immune disease selected from the group consisting of autoimmune disease, allergic disease and graft rejection, or in a subject in need of prevention of such disease, the medicament comprising:

ii) a polypeptide encoded by a nucleic acid sequence as defined in (i); or (b)

iii) Multimeric proteins, such as dimeric proteins, which consist of a plurality of polypeptides, such as defined in (ii);

ii) a polypeptide encoded by a nucleic acid sequence as defined in (i); or (b)

iii) A dimeric protein consisting of two polypeptides defined in (ii);

In another aspect, the present disclosure provides the use of a pharmaceutical composition for the prophylactic or therapeutic treatment of a subject suffering from or suspected of suffering from an immune disease selected from the group consisting of autoimmune disease, allergic disease and graft rejection, or in need of prevention of said immune disease, the treatment comprising administering to the subject a pharmaceutical composition comprising:

ii) a polypeptide encoded by a nucleic acid sequence as defined in (i); or (b)

iii) A dimeric protein consisting of two polypeptides defined in (ii);

In another aspect, the present disclosure provides a medicament for prophylactic or therapeutic treatment of a subject having or suspected of having an immune disease selected from the group consisting of autoimmune disease, allergic disease, and graft rejection, or in need of prevention of such immune disease, the medicament comprising:

ii) a polypeptide encoded by a nucleic acid sequence as defined in (i); or (b)

In another aspect, the present disclosure provides a medicament for prophylactic or therapeutic treatment of a subject suffering from or suspected of suffering from an immune disease selected from the group consisting of autoimmune disease, allergic disease and graft rejection, or in need of prevention of such immune disease, the medicament comprising

ii) a polypeptide encoded by a nucleic acid sequence as defined in (i); or (b)

iii) A dimeric protein consisting of two polypeptides defined in (ii);

In another aspect, the present disclosure provides a pharmaceutical composition comprising:

ii) a polypeptide encoded by a nucleic acid sequence as defined in (i); or (b)

wherein the antigenic unit comprises one or more T cell epitopes of a self antigen, allergen, alloantigen or xenogeneic antigen,

when used for prophylactic or therapeutic treatment of an immune disorder selected from autoimmune disorders, allergic disorders and graft rejection.

ii) a polypeptide encoded by a nucleic acid sequence as defined in (i); or (b)

iii) A dimeric protein consisting of two polypeptides as defined in (ii);

In some embodiments, one dose of the drug is administered to the subject. In some embodiments, multiple doses of the pharmaceutical composition are administered to a subject.

In another aspect, the invention provides methods of improving tolerance to self-antigens, allergens, alloantigens, or xenogeneic antigens using the tolerance-inducing constructs of the present disclosure.

In another aspect, the present disclosure provides a method for improving tolerance of a subject to an autoantigen, allergen, alloantigen or xenogeneic antigen, the method comprising administering to the subject a tolerance-inducing construct or pharmaceutical composition of the present disclosure.

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

Indicators of treatment success are known in the art and include increased antigen-specific regulatory T cell levels, decreased antigen-specific effector T cell levels (and increased regulatory T cell levels), decreased effector T cell levels, decreased levels of T cell activation in ELISPOT when stimulated with an antigenic unit/T cell epitope in an antigenic unit, decreased levels of basophil activation in a basophil activation assay (BAT).

Radioallergen adsorption detection (RAST) can also be used to compare allergen-specific IgE antibody levels in a subject's blood sample before and after administration of the immunotherapeutic construct, wherein lower allergen-specific IgE antibody levels are indicative of successful tolerance induction.

Examples

Example 1: design and production of the vectors of the present disclosure

All the gene sequences described in examples 1a, 1b and 1c were ordered from GenScript (New Jersey, US) and cloned into the expression vector pALD-CV 77.

Example 1a: design and production of vectors of the present disclosure for the treatment of multiple sclerosis

Myelin Oligodendrocyte Glycoprotein (MOG) is a protein located in the central nervous system. Immunodominant (immunodomino) epitope 35-55 of MOG (MOG 35-55) is a major 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 (Hunterman, H.et al 2022).

A DNA vector was designed comprising a nucleotide sequence encoding the units/portions described in table 1 below.

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TABLE 1

* Murine MOG (27-63) sequences obtained from Krienke et al 2021 and U.S. patent application US2020061166A 1.

* Extracellular domain

DNA vectors VB5003b, VB5004b, VB5005b, VB5006b, VB5012b, VB5046, VB5048, VB5058, VB5059, VB5060, VB5061 and VB5071 encode a tolerance-inducing construct comprising a targeting unit, a dimerization unit and an antigenic unit as shown in table 1. The murine MOG (27-63) antigenic unit comprises the T cell epitope MOG (35-55).

DNA vectors VB5002b and VB5052 encode constructs comprising human CCL3L1 targeting units ("vaccine") known to target APCs in a pro-inflammatory manner, i.e., constructs comprising such targeting units will induce an enhanced immune response in subjects to which they are administered, and the compounds are expected to induce an activated immune response, wherein production of IFN- γ is increased (see, e.g., WO2011161244 A1).

The DNA vectors VB5001b and VB5051 do not encode targeting units or dimerization units, but only MOGs (27-63) as antigenic units, i.e. single proteins/peptides.

Example 1b: design and production of vectors of the present disclosure for the treatment of diabetes

Glutamate decarboxylase 65 (GAD 65) is considered to be the major autoantigen in diabetes. After immunization with GAD65, the peptide GAD65 (201-220) produced the greatest T cell response in transgenic mice expressing major histocompatibility complex class II allele HLA-DQ 8. GAD65 (206-220) is an immunodominant T cell epitope of GAD65 in NOD mice (Liu, J.et al, 1999).

A DNA vector comprising a nucleotide sequence encoding the unit/portion described in table 2 below was designed.

TABLE 2

The DNA vector VB5016b encodes a tolerance-inducing construct comprising a targeting unit, a dimerization unit and an antigenic unit as described in table 2.

The DNA vector VB5015b encodes a construct comprising a human CCL3L1 targeting unit ("vaccine") which is known to target APCs in a pro-inflammatory manner, i.e. the construct comprising this targeting unit will induce an inflammatory immune response in the subject to which they are administered, and the compound is expected to induce IFN- γ production (see for example WO2011161244 A1).

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

The murine glutamate decarboxylase 65 (GAD 65) 202-221 (SEQ ID NO: 183) antigenic unit comprises the known T cell epitopes GAD65 (206-220) and GAD65 (202-221).

Example 1c: the design and production of the vectors of the present disclosure for the treatment of shrimp allergy

Tropomyosin is the major allergen in shellfish. Six major T cell epitopes were identified for tropomyosin from penaeus vannamei (Metapenaeus ensis, met e 1) in the Balb/c mouse model for Met e 1 hypersensitivity. Oral immunotherapy using peptides of the six T cell epitopes effectively reduced the allergic response to shrimp tropomyosin (Wai, c.y.y et al 2015).

A DNA vector comprising a nucleotide sequence encoding the unit/portion described in table 3 below was designed.

TABLE 3 Table 3

The sequence of the antigenic units is further detailed in table 3a below.

Met e 1	SEQ ID NO:184
		Met e 1(16-35)	SEQ ID NO:185
Met e 1(46-65)	SEQ ID NO:186
		Met e 1(76-95)	SEQ ID NO:187
Met e 1(136-155)	SEQ ID NO:188
		Met e 1(210-230)	SEQ ID NO:189
Met e 1(241-260)	SEQ ID NO:190

TABLE 3a

DNA vectors VB5024, VB5030 and VB5079 encode a tolerance-inducing construct comprising a targeting unit, a dimerization unit and an antigenic unit as described in table 3.

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

Example 1d: design and production of DNA constructs of the present disclosure for the treatment of multiple sclerosis

All the gene sequences of the test constructs described in examples 1d and 1e were ordered from Genscript (860 Centennial Ave,Piscataway,NJ 08854,USA) and cloned into the expression vectors pUMVC4a and/or pALD-CV77, whereas construct VB5017 was cloned into the expression vector pALD-CV77 only.

Eight constructs (DNA plasmids) were designed for mice, comprising a plurality of murine signal peptides and targeting units, identical murine dimerization units and antigenic units, comprising short T cell epitopes of the murine Multiple Sclerosis (MS) autoantigen Myelin Oligodendrocyte Glycoprotein (MOG), MOG (35-55) or longer T cell epitopes of the murine MOG, MOG (27-63).

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* Extracellular domain

Example 1e: DNA constructs of the present disclosureDesigned and produced for the treatment of diabetes

A construct (DNA plasmid) was designed for mice comprising the T cell epitope from murine glutamate decarboxylase, GAD65 (202-221). GAD65 is an important diabetes autoantigen. The construct also comprises the elements shown in the following table (all murine proteins):

example 1f: design and production of the DNA constructs of the present disclosure for the treatment of shrimp allergy

Constructs (DNA plasmids) were designed for mice, comprising T cell epitopes from tropomyosin (Pan b 1 epitopes). This epitope is an important shellfish allergen. The construct also comprises the elements shown in the following table (all murine proteins):

example 2a: in vitro characterization of protein expression and secretion of MOG-containing constructs

The aim of this study was to characterize the protein expression and secretion of the protein encoded by the MOG-containing DNA vector following transient transfection of mammalian cells.

HEK293 cells were obtained from ATCC and transiently transfected with MOG-containing DNA vectors (VB 5002b, VB5003b, VB5004b, VB5005b, VB5006b and VB5012 b). Briefly, 2X 10 ⁵ Individual cells/wells were plated in 24-well tissue culture plates with growth medium of 10% FBS and used 2000 reagents were transfected with 1. Mu.g of the corresponding DNA vector under the conditions recommended by the manufacturer (Thermo Fischer Scientific). Transfected cells were maintained at 37℃in 5% CO2 for 5 days, and cell supernatants were collected.

Expi293F cells were obtained from Thermo Fisher, and transiently transfected with DNA vectors (VB 5052, VB5046, VB5048, VB5058, VB5059, VB5060, VB5061 and VB 5071) containing MOG (27-63). Briefly, expi293F cells (1.7X10 ⁶ Individual cells/ml, 1 ml) were inoculated in 96-well culture plates. Cells were transfected with 0.64. Mu.g/ml plasmid DNA using the Expiefectamine 293 reagent (Thermo Fisher Sci) and incubated in a humidified CO2 cell incubator (8% CO ₂ Plates were incubated on a rotary shaker (3 mm diameter, 900 rpm) at 37 ℃. Supernatants were harvested 72 hours post transfection.

The supernatant was subjected to sandwich ELISA using antibodies against MOG (capture antibody, mouse anti-MOG antibody, 0.25 μg/ml,100 μl/well, sc-73330,Santa Cruz Biotechnology) and hIgG CH3 domain (detection antibody, mouse anti-human IgG Fc secondary antibody, biotin, 0.1 μg/ml,100 μl/well, 05-4240, invitrogen) to characterize the expression and secretion of proteins encoded by the MOG-containing vector. Figures 2A and 2B show that all 14 MOG-containing constructs were expressed and secreted at high levels.

Secretion of full-length tolerance-inducing proteins with MOG antigenic units and six different targeting units was confirmed by sandwich ELISA of supernatants with antibodies directed against MOG and targeting units with murine sequences of IL-10, tgfβ1, SCGB3A2, CTLA-4, PD-1 and CCL1L3, respectively. The results for tolerance-inducing proteins are shown in figures 3A-3F and the pro-inflammatory controls are shown in figure 3G.

Detection of IL-10 (VB 5005b, VB5006b and VB 5058), TGF-beta 1 (VB 5059), SCGB3A2 (VB 5060), CTLA-4 (VB 5061), PD-1 (VB 5071) and CCL3L1 (VB 5052), respectively, combined with detection of MOG, showed high levels of secretion of full-length tolerance-inducible proteins and pro-inflammatory controls.

To evaluate secretion and expression of the individual MOG (27-63) antigen control peptides encoded by vector VB5051, expi293F cells were transiently transfected with VB5051 and supernatants were harvested after 3 days. Secretion and expression of VB5051 was assessed by direct ELISA using supernatant as coating and detection using anti-MOG antibodies (mouse anti-MOG antibody, 3.3. Mu.g/ml, 100. Mu.l/well, sc-73330,Santa Cruz Biotechnology). FIG. 4 shows that MOG (27-63) peptide is secreted after transfection of mammalian cells with VB 5051.

Example 2b: in vitro characterization of protein expression and secretion of constructs of the present disclosure

The aim of this study was to measure the presence of protein in the cell supernatant and characterize the protein expression level after transient transfection of mammalian cells with DNA plasmids by ELISA assay using specific antibodies binding to the targeting, dimerization and antigenic units of the protein. HEK293 cells were obtained from ATCC. HEK293 cells were transiently transfected with the construct. Briefly, 2X 10 ⁵ Individual cells/wells were plated in 24-well tissue culture plates with 10% FBS growth medium and used under conditions recommended by the manufacturer (Invitrogen, thermo Fischer Scientific)2000 reagents were transfected with 1. Mu.g DNA plasmid. Transfected cells were then maintained at 37 ℃ for 6 days at 5% co2, and cell supernatants were harvested for characterization of protein expression.

ELISA was performed to verify the amount of protein produced by HEK293 cells and secreted into the cell supernatant. MaxiSorp Nunc-immune plates were coated with 0.06. Mu.g/ml rabbit anti-human TGF-beta 1 (orb 77216, biorbyte) as capture antibody in 1 XPBS at 100. Mu.l/well and the plates were incubated overnight at 4 ℃. The microtiter wells were blocked by adding 200. Mu.l/well of 1 XPBS with 4% BSA. Mu.l of cell supernatant from transfected HEK293 cells containing proteins expressed by DNA plasmids was added to the plates.

For detection of antibodies, 1 μg/ml biotinylated mouse anti-human IgG (HP 6017, invitrogen, CH3 domain of dimerization unit contained in the binding construct) was added and incubated. SA-HRP (streptavidin horseradish peroxidase, S2438-250UG, sigma-Aldrich, 1:3000) was then added and incubated. All incubations were performed at 37℃for 1 hour, followed by 3 washes with PBS-Tween, unless otherwise indicated. Then, 100. Mu.l/well of TMB solution was added, and after 5-15 minutes, 100. Mu.l/well of 1M HCl was added to terminate the color development. The optical density at 450nm was measured on an automated plate reader (Thermo Scientific Multiscan GO).

Fig. 13 shows that construct VB5009 is expressed and secreted as a protein.

Example 3: in vitro characterization of protein expression and secretion of Met e 1-containing constructs

The aim of this study was to characterize the protein expression and secretion of tolerance-inducing proteins encoded by Met e 1-containing DNA vectors following transient transfection of mammalian cells (see table 3).

Briefly, expi293F cells (1.7X10 ⁶ Individual cells/ml, 1ml,Thermo Fisher Sci.) were inoculated in 96-well culture plates. Cells were transfected with 0.64 μg/ml plasmid DNA using the ExpiFectamine 293 reagent (Thermo Fisher Sci.) and plates were incubated on a rotary shaker (3 mm diameter, 900 rpm) in a humidified CO2 cell incubator (8% CO2, 37 ℃). Supernatants were harvested 72 hours post transfection.

With antibodies against hIgG CH3 domain (capture antibody, 0.5. Mu.g/ml mouse anti-human IgG3 (CH 3 domain), 100. Mu.l/well, MCA878G, bioRad) and hIgG Fc domain (detection antibody, 0.250. Mu.g/ml CaptureStylelect) ^TM Biotin anti-IgG-Fc (human) conjugate, 100 μl/well, 710326220, invitrogen) was used to characterize secreted proteins encoded by Met e 1-containing vectors by sandwich ELISA of the supernatants. The results are shown in fig. 5.

As is apparent from FIG. 5, all tolerance-inducing proteins (VB 5024, VB5030 and VB 5079) containing Met e 1 were expressed and secreted.

Example 4: in vitro characterization of binding of tolerance-inducing constructs to DEC205 receptor

The purpose of this experiment was to characterize the functional binding of scFv anti-DEC 205 targeting units of VB5004 (see table 1) to the recombinant DEC205 receptor. Functional binding of the targeting units was assessed by coating ELISA plates with recombinant DEC205 receptor and using antibodies against the antigenic or dimerizing units as detection antibodies, ELISA of supernatants of HEK293 cells transiently transfected with tolerance-inducing DNA vectors encoding scFv as targeting units against DEC 205.

HEK293 cells were obtained from ATCC and transiently transfected with DNA vector VB5004b encoding scFv anti-DEC 205 Transfection at that time. Briefly, 2X 10 ⁵ Individual HEK293 cells/well were plated in 24-well tissue culture plates with 10% fbs growth medium and used2000 reagents were transfected with 1. Mu.g of the corresponding DNA vector under the conditions recommended by the manufacturer (Invitrogen, thermo Fischer Scientific). Transfected cells were maintained at 37℃in 5% CO2 for 5 days and cell supernatants were collected. The secreted protein encoded by the scFv-anti-DEC 205-containing vector was characterized by direct ELISA of the supernatant. ELISA plates were coated and blocked with 100. Mu.l/well of 5. Mu.g/ml recombinant DEC205 (216-503) (OPCD 05072, aviva Systems Biology) and then the supernatant was added. Binding to recombinant receptors was detected by antibodies against MOG (100. Mu.l/well, 1. Mu.g/ml mouse anti-MOG antibody, sc-73330,Santa Cruz Biotechnology) or antibodies against hIgG CH3 domain (100. Mu.l/well, 0.1. Mu.g/ml mouse anti-human IgG Fc secondary, biotin, 05-4240, invitrogen). The results are shown in fig. 6.

FIG. 6 demonstrates binding of the tolerance-inducing protein VB5004b containing scFv anti-DEC 205 to the DEC205 receptor and secretion of the full-length tolerance-inducing protein.

Example 5: in vitro characterization of tolerance-inducible constructs binding to IL10 receptor

The purpose of this experiment was to characterize the functional binding of the IL-10 targeting unit of VB5006b (see Table 1) to the recombinant IL-10 receptor (IL-10R). Functional binding of the targeting unit was assessed by coating ELISA plates with recombinant IL-10 receptor and using antibodies against the antigenic or dimeric units as detection antibodies, ELISA supernatants from HEK293 cells transiently transfected with DNA vectors encoding IL-10 as the targeting unit.

Briefly, HEK293 cells were obtained from ATCC and transiently transfected with the IL-10-containing DNA vector VB5006 b. Briefly, 2X 10 ⁵ Individual cells/well were plated in 24-well tissue culture plates with 10% FBS growth medium and used2000 reagents were transfected with 1. Mu.g of the corresponding DNA vector under the conditions recommended by the manufacturer (Invitrogen, thermo Fischer Scientific). Transfected cells were maintained at 37℃in 5% CO2 for 5 days, and cell supernatants were collected. Secreted proteins encoded by IL-10-containing vectors were characterized by direct ELISA of the supernatant. ELISA plates were coated and blocked with 100. Mu.l/well of 2.5. Mu.g/ml recombinant IL-10 receptor, and then the supernatant was added. Binding to recombinant receptors was detected by antibodies against MOG (100. Mu.l/well, 1. Mu.g/ml mouse anti-MOG antibody, sc-73330,Santa Cruz Biotechnology) or antibodies against hIgG CH3 domain (100. Mu.l/well, 0.1. Mu.g/ml mouse anti-human IgG Fc secondary, biotin, 05-4240, invitrogen). The results of VB5006b are shown in FIG. 7.

FIG. 7 demonstrates binding of IL 10-containing tolerance-inducing protein VB5006b to the IL-10 receptor and secretion of the full-length tolerance-inducing protein.

Example 6: in vitro characterization of tolerance-inducible construct size and protein integrity

Western blot analysis was performed on supernatant samples from transfected Expi293F cells to further characterize the proteins encoded by VB5046, VB5052, VB5058, VB5059, VB5061 and VB 5071.

Briefly, expi293F cells (1.7X10 ⁶ Individual cells/ml, 1 ml) were inoculated in 96-well culture plates. Cells were transfected with 0.64 μg/ml plasmid DNA using the ExpiFectamine 293 reagent (Thermo Fisher Sci.) and humidified CO ₂ Cell culture apparatus (8% CO) ₂ Plates were incubated on a rotary shaker (3 mm diameter, 900 rpm) at 37 ℃. Supernatants were harvested 72 hours post transfection.

Samples were prepared by mixing 14 μl of supernatant from transfected Expi293F cells with 5 μl of 4x Laemmli sample buffer (Bio-Rad) and 1 μl of DTT (Cayman Chemical) or 1 μl of ultrapure water (for reducing and non-reducing conditions, respectively) (total sample volume was amplified at a given ratio). The sample (reduced or unreduced) was heated at 70℃for 10 minutes, and then 4% -20% Criterion TGX Stain-Free pre-gel (Bio-Rad) was added. SDS-PAGE in 1 XTris/glycine/SDS running buffer (Bio-Rad) Line, precision Plus Protein All Blue pre-stained protein standard (Bio-Rad) was used. 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 as mouse anti-MOG (sc-73330,Santa Cruz Biotechnology), rat anti-mouse IL-10 (MAB 417, R)&D Systems) or goat anti-murine CTLA-4 (AF 467, R&D Systems) to detect MOG, IL-10 or CTLA-4, respectively. The membrane was incubated with the fluorescent dye conjugated species-specific secondary antibody for 1 hour at room temperature, then washed and dried. For IL-10 detection in the Dyight 488 channel, 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 to Dyight 488 was incubated for 1 hour at room temperature, then washed and dried. Image by using ChemiDoc ^TM MP imaging system.

Western blot analysis with anti-MOG antibodies showed full-length secretion of tolerance-inducing protein and VB5052 (fig. 8A). All proteins except VB5059 appear to have higher molecular weights than expected based on the protein sequence, possibly due to post-translational modifications. VB5059 uses TGF-beta 1 as a targeting unit that is known to decrease in size by 28kDa upon cleavage into latency-related peptides and mature TGF-beta 1. FIG. 8B shows the formation of dimers of proteins under non-reducing conditions. Figure 8B also shows the presence of monomeric protein under non-reducing conditions. The membranes probed with anti-IL-10 and anti-CTLA-4 (FIGS. 8C and 8D, respectively) showed bands of the same molecular weight corresponding to the bands detected on the membranes probed with anti-MOG (FIG. 8A). Thus, FIGS. 8C and 8D demonstrate that MOG and IL-10 or CTLA-4, respectively, are part of the same fusion protein. VB5048 was included as a control in FIGS. 8C and 8D to show that anti-IL-10 and anti-CTLA-4 each did not bind non-specifically to the protein.

Example 7: evaluation of tolerance-inducing ability of the DNA vector of the present disclosure

Tolerance induction capacity of VB5004b (described in table 1) was evaluated in the spleen of mice vaccinated with VB5004b and determined by calculating the induced IL-10/IFN- γ ratio. IL-10 (non-inflammatory cytokine associated with immune tolerance) and IFN-gamma (marker for inducing inflammatory immune response) signals were measured in a two-color FluoSpot assay after restimulation of spleen cells harvested from vaccinated mice with MOG (35-55) peptide. The IL-10/IFN-gamma ratio indicates the extent to which the immune response induced by the DNA vector is biased towards a tolerogenic response. Tolerogenic characteristics were further assessed by the frequency of the induced foxp3+ cells and by the lack of IFN- γ+ and IL-17+ production of cd4+ T cells after VB5004b vaccination. The results obtained were compared with the response induced by the pro-inflammatory control vaccine VB5002b (described in table 1) and the tolerance induction capacity of VB5001b (described in table 1).

Mouse inoculation and fluorospot

The following study design was applied:

female 6 week old C57BL/6 mice were obtained from Janvier Labs (France). All animals were housed in radio Hospital (Oslo, norway) animal facilities. All animal protocols were approved by the norway food safety agency (oslo, norway). 4-5 mice/group were used to test VB5004b, VB5002b and VB5001b, while 2 mice/group were used for negative control (PBS only). VB5002b is included as a pro-inflammatory form of the MOG (27-63) construct comprising a human CCL3L1 targeting unit known to target APCs and induce an inflammatory immune response, i.e., constructs comprising the targeting unit will induce a pro-inflammatory immune response in subjects to which they are administered, and the compound is expected to induce IFN-gamma production in T cells specific for the encoded antigen. A DNA vector VB5001b encoding only MOG (27-63) peptide was included as a comparison with VB5004 b.

A dose of 50 μg VB5004b or control DNA vectors VB5001b and VB5002b dissolved in sterile PBS was applied to each tibialis anterior (2X 25 μl,1000 μg/ml) by intramuscular needle injection followed by electroporation using the AgilPulse in vivo electroporation system (BTX, USA).

Spleens were harvested 7 days after inoculation and triturated in a cell filter to obtain a single cell suspension. The erythrocytes were lysed using ammonium-potassium chloride (ACK) lysis buffer. After washing, spleen fines were counted with a NucleoCoulter NC-202 (ChemoMetec, denmark)Cells were resuspended to a final concentration of 6X 10 ⁶ Individual cells/ml, at 6X 10 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, and then tested for IFN- γ and IL-10 cytokine production in a two-color FluoSpot assay according to the manufacturer's protocol (Mabtech AB, sweden). Spot forming cells were measured in IRIS fluoro 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.

It can be seen from FIG. 9A that VB5004b induced a higher level of IL-10 than VB5001b induced. In addition, low background levels of IFN-gamma in response to VB5004b vaccination were detected as compared to VB5002b inducing elevated levels of IFN-gamma. FIG. 9B shows the IL-10/IFN- γ ratio calculated from the values shown in FIG. 9A. The high IL-10/IFN-gamma ratio of VB5004b indicates that the level of VB5004 b-induced immunosuppressive cytokine IL-10 is significantly higher than the Yu Yan cytokine IFN-gamma. In contrast, spleen cells of mice administered VB5002b showed an IL-10/IFN-gamma ratio of about 1, indicating that both cytokines were produced at equal levels after restimulation with MOG (35-55) peptide. In order to avoid excessive inflammation and ensure final inflammatory resolution, it is important that the production of pro-inflammatory cytokines such as IFN-gamma is regulated by negative feedback mechanisms, including the production of anti-inflammatory cytokines such as IL-10 (Sugimoto MA et al 2016). Thus, the observed increase in IL-10 levels in response to VB5002b can be explained by this feedback mechanism controlling the induced inflammatory response. A significant increase in IL-10/IFN- γ ratio was detected for VB5004b compared to VB5001b, indicating that VB5004b has a higher tolerance induction potential compared to both VB5002b and VB5001 b.

Flow cytometry

Spleen cells were further analyzed for expression of Foxp3, IFN- γ and IL-17 by flow cytometry. Foxp3 acts as the primary regulator of the inhibitory pathway in the development and function of regulatory T cells (tregs).

Briefly, after 16 hours of restimulation with MOG (35-55) peptide, harvestCells were counted and washed and then incubated with Fixable Vital Dye (FVD) for 10 minutes in the dark. The cells were then centrifuged and washed, and then the surface stain mixture was added: anti-CD 3 (BUV 395), anti-CD 4 (BV 785) and anti-CD 8 and incubated in the dark at 4 ℃ for 30 min. After incubation, the cells were centrifuged, resuspended in running buffer (PBS, 10% FBS and 2mM EDTA) and the cell pellet was washed. Foxp3 fixation/permeabilization solution was added to the cells and incubated at 4 ℃ in the dark for 60 min. After the centrifugation step, the cells are resuspended and washed in permeabilization buffer, then centrifuged and the intracellular antibody mixture is added: anti-Foxp 3 (Alexa fluor 700), anti-IFN-gamma (APC), anti-IL-17 (Alexa fluor 488) and incubated in the dark at 4℃for 30 minutes. The cells were then washed and resuspended in running buffer until obtained. Compensation for fixable vital dyes was set using single stain Ultra comp eBeads and ArC reactive beads. To evaluate the quality of staining, we designed a gating strategy that excluded fluid instability, cell debris, doublets (douplets), and dead cells. We further define T cells based on CD3 expression. CD4 expression of cd3+ T cells was detected and then the cells were analyzed for Foxp3 and cytokine expression. The unstimulated cells from each group were used to evaluate the background level of cytokine production in the assay. Flow cell File (FCS) from FACSDiva ^TM Derived in software and analyzed using FlowJo. Data obtained from the flow cytometry analysis was analyzed using GraphPad prism 9.

As shown in fig. 10A, the percentage of foxp3+ cells to cd4+ T cells was greatly increased in mice vaccinated with VB5004b compared to the levels detected in mice vaccinated with VB5002b, VB5001b or PBS. IFN-gamma and IL-17 are both pro-inflammatory cytokines that contribute to the pathogenesis of chronic inflammatory and autoimmune diseases, including Experimental Autoimmune Encephalomyelitis (EAE) and multiple sclerosisK et al 2018). Thus, tolerogenic vaccines must reliably induce tolerance without inadvertently sensitizing the autoimmune response to the autoantigen, e.g., by inducing a potential exacerbation of autoimmunityIs a pro-inflammatory cytokine of (a). As shown in fig. 10B and 10C, elevated IFN- γ and IL-17 expression was detected in response to VB5002B, whereas the lack of these pro-inflammatory cytokines was demonstrated in mice vaccinated with VB 5004B.

Thus, example 7 shows that VB5004b, which encodes both the scFv anti-DEC 205 targeting unit and the MOG (27-63) antigenic unit, induces a higher non-inflammatory factor to inflammatory cytokine ratio (IL-10/IFN-gamma) and shows a lack of inflammatory cytokine production compared to its pro-inflammatory version VB5002 b. Example 7 further shows that VB5004b induced foxp3+cd4+ T cells at higher frequencies than VB5001b, VB5002b and background levels in PBS vaccinated mice, indicating the presence of Treg induced by VB5004b higher than control.

Example 8: assessment of tolerance-inducing ability of DNA vectors of the present disclosure

The tolerance-inducing capacity of VB5012b (described in Table 1) was determined by calculating the IL-10/IFN- γ ratio as described in example 7. Tolerogenic characteristics of VB5012b, VB5048, VB5058 and VB5046 (all constructs described in Table 1) were further assessed by the percentage of MOG (38-49) -specific Foxp3+ T cells induced in response to vaccination.

Mouse inoculation and fluorospot

The following study design was applied:

female 6 week old C57BL/6 mice were obtained from Janvier Labs (France). All animals were housed in radio Hospital (Oslo, norway) animal facilities. All animal protocols were approved by the norway food safety agency (oslo, norway). 5 mice/group were used for testing VB5012b, VB5048, VB5006b, VB5046, VB5052 and VB5051, while 2 mice/group were used for negative control (PBS-vaccinated mice only). As in example 7, VB5052 was included as a construct encoding pro-inflammatory MOG (27-63), while VB5051 was included as a control for only antigens encoding MOG (27-63) for comparison with VB5012b, VB5048, VB5006b and VB 5046.

A 50 μg dose of DNA vector VB5012b, VB5048, VB5006b, VB5046, VB5051 or VB5052 was administered intramuscularly twice (day 0 and day 4), then electroporated, the spleens harvested 10 days after the first inoculation and triturated in a cell filter to obtain a single cell suspension as described in example 7. Splenocytes were re-stimulated with MOG (35-55) peptide for 44 hours and tested for IFN-gamma and IL-10 cytokine production in a two-color FluoSpot assay as described in example 7.

FIG. 11A shows that IL-10 levels produced in spleen of mice vaccinated with VB5012b were elevated while lacking IFN-gamma production upon restimulation with MOG (35-55) peptide. This is in contrast to VB5052, which shows an increased IFN-gamma level in spleen cells of vaccinated mice. As shown in fig. 11B, a significantly higher IL-10/IFN- γ ratio was detected for VB5012B compared to VB5052, and a trend of higher IL-10/IFN- γ ratio was detected compared to VB 5051.

MOG (38-49) specificity in spleen cells of vaccinated mice using tetramer (H-2 IAb/GWYRSPFSRVVH) ⁺ Flow cytometry analysis of sex CD 4T cells

The production of MOG-specific foxp3+ cells, which showed Treg cells that inhibited and controlled the MOG-specific inflammatory immune response and thereby maintained self-tolerance, was identified by MOG-specific tetramer staining and flow cytometry (cd4+foxp3+mog (38-49) -tet+ cells).

Briefly, 2×10 will be pooled from each group ⁶ Individual spleen cells were transferred to a 96-well V-bottom plate. Tetramers and abs were diluted with PBS containing 5% fbs and protected from light prior to use. Unless otherwise indicated, all steps requiring cell washing were performed with PBS containing 5% fbs. First, by a method specific to MOG (38-49) MHC class II tetramer (1. Mu.g/ml, H-2IAb-GWYRSPFSRVVH->Tetrameric PE,2958, proimmue) staining cells and plating the plate on humidified CO ₂ Cell incubator (5% CO) ₂ Incubate for 2 hours at 37 ℃). Without washing the cells, the FC receptor was blocked on ice for 5 minutes to prevent flowCytometry antibodies (Ab) and Fc receptor (0.25. Mu.g/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, thermofischer/eBioscience), anti-mouse CD25 PerCP-Cy5.5 (0.25. Mu.g/ml, clone: PC61, 102030, biolegend). Cells were washed twice with PBS. Cells were then stained with fixable vital dye (150 μl per well, diluted 1:8000 in PBS, fixable Viability Stain 780,565388,BD biosciences) for 10 min on ice. Cells were washed twice with PBS only and fixed and permeabilized using Foxp3/Transcription Factor Staining Buffer Set according to manufacturer's instructions (200. Mu.l/well, 00-5523-00, thermofischer/eBioscience). Cells were washed and stained with an intracellular Ab mixture containing anti-mouse FOXP3 eFluor 660 (0.25. Mu.g/ml, clone: FJK-16s,50-5773-82, thermofischer/eBioscience), anti-mouse Ki-67 Alexa Fluor 488 (0.25. Mu.g/ml, clone: 11F6, 151204, bioleged) for 30 min on ice. Cells were washed and resuspended in 150 μl of 5% FBS-containing PBS and used BD FACSymphosy ^TM A3 cell analyzer was used for analysis. Using FlowJo ^TM v10.8 software (BD Life Sciences), the following controls were used as guidelines for gating the target population: unstained control (=cells did not receive any Ab) and Fluorescence Minus One (FMO) control (=samples stained with all fluorophore-labeled abs), one of which was subtracted to accurately distinguish positive from negative signals).

As shown in fig. 12, a higher percentage of MOG (38-49) -specific foxp3+ cells could be observed after two dose vaccination regimen (day 0 + day 4) with tolerance-inducing constructs VB5012b, VB5048, VB5006b and VB5046 compared to vaccination with VB5051 or PBS.

Thus, example 8 shows that VB5012b, which encodes the MARCO ligand SCGB3A2 as targeting unit and the MOG (27-63) as antigenic unit, induced a higher ratio of non-inflammatory cytokines to inflammatory cytokines (IL-10/IFN-gamma) than the pro-inflammatory version VB 5052. Example 8 further shows that vaccination with tolerance-inducing constructs VB5012b, VB5048, VB5006b or VB5046 induced a higher percentage of MOG (38-49) -specific foxp3+ cells than the background levels of mice vaccinated with VB5051 and PBS, indicating that higher levels of Treg were induced. These results indicate that DNA vectors VB5048, VB5012b, VB5006b and VB5046 are capable of eliciting tolerogenic responses.

Sequence(s)

SEQ ID NO:1

ELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

SEQ ID NO:2

VB5003

Murine IgVH signal peptide (1-19), murine anti-DEC 205 scFv (20-265), dimerization unit (266-442),unit joint Head(443-447), murine MOG 35-55 (448-468)

M ¹ NFGLRLIFLVLTLKGVQC ¹⁹ D ²⁰ IQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLELKGGGGSGGGGSGGGGSEVKLLESGGGLVQPGGSLRLSCAASGFTFNDFYMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISRDNTQNILYLQMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSS ²⁶⁵ P ²⁶⁶ SVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK ⁴⁴² G ⁴⁴³ LGGL ⁴⁴⁷ M ²⁴⁸ EVGWYRSPFSRVVHLYRNGK ⁴⁶⁸

SEQ ID NO:3

VB5004

Murine IgVH signal peptide (1-19), murine anti-DEC 205 scFv (20-265), dimerization unit (266-442),unit joint Head(443-447), murine MOG 27-63 (448-484)

M ¹ NFGLRLIFLVLTLKGVQC ¹⁹ DIQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLELKGGGGSGGGGSGGGGSEVKLLESGGGLVQPGGSLRLSCAASGFTFNDFYMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISRDNTQNILYLQMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSS ²⁶⁵ P ²⁶⁶ SVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK ⁴ ⁴² G ⁴⁴³ LGGL ⁴⁴⁷ S ⁴⁴⁸ PGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEAQP ⁴⁸⁴

SEQ ID NO:4

VB5012

Murine SCGB3A2 signal peptide (1-21), murine SCGB3A2 (22-91), dimerization unit (92-268),unit joint(269-273), murine MOG 35-55 (274-294)

M ¹ KLVSIFLLVTIGICGYSATA ²¹ L ²² LINRLPVVDKLPVPLDDIIPSFDPLKMLLKTLGISVEHLVTGLKKCVDELGPEASEAVKKLLEALSHLV ⁹¹ P ⁹² SVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK ²⁶⁸ G ²⁶⁹ LGGL ²⁷³ M ²⁷⁴ EVGWYRSPFSRVVHLYRNGK ²⁹⁴

SEQ ID NO:5

VB5013

Murine VSIG-3 signal peptide (1-22), murine VSIG-3 (23-428), dimerization unit (429-605),unit joint(606-610), murine MOG 35-55 (611-631)

M ¹ TRRRSAPASWLLVSLLGVATS ²² L ²³ EVSESPGSVQVARGQTAVLPCAFSTSAALLNLNVIWMVIPLSNANQPEQVILYQGGQMFDGALRFHGRVGFTGTMPATNVSIFINNTQLSDTGTYQCLVNNLPDRGGRNIGVTGLTVLVPPSAPQCQIQGSQDLGSDVILLCSSEEGIPRPTYLWEKLDNTLKLPPTATQDQVQGTVTIRNISALSSGLYQCVASNAIGTSTCLLDLQVISPQPRSVGVIAGAVGTGAVLIVICLALISGAFFYWRSKNKEEEEEEIPNEIREDDLPPKCSSAKAFHTEISSSENNTLTSSNTYNSRYWNNNPKPHRNTESFNHFSDLRQSFSGNAVIPSIYANGNHLVLGPHKTLVVTANRGSSPQVLPRNNGSVSRKPWPQHTHSYTVSQMTLERIGAVPVMVPAQSRAGSLV ⁴²⁸ P ⁴²⁹ SVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKG ⁶⁰⁶ LGGL ⁶¹⁰ M ⁶¹¹ EVGWYRSPFSRVVHLYRNGK ⁶³¹

SEQ ID NO:6

Murine IgVH signal peptide (1-19)

MNFGLRLIFLVLTLKGVQC

SEQ ID NO:7

VB5016

Murine IgVH signal peptide (1-19), murine anti-DEC 205 scFv (20-265), dimerization unit (266-442),unit joint Head(443-447), murine GAD65 202-221 (448-467)

M ¹ NFGLRLIFLVLTLKGVQC ¹⁹ D ²⁰ IQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLELKGGGGSGGGGSGGGGSEVKLLESGGGLVQPGGSLRLSCAASGFTFNDFYMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISRDNTQNILYLQMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSS ²⁶⁵ P ²⁶⁶ SVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK ⁴⁴² G ⁴⁴³ LGGL ⁴⁴⁷ T ⁴⁴⁸ NMFTYEIAPVFVLLEYVTL ⁴⁶⁷

SEQ ID NO:8

VB5005

Murine IL10 signal peptide (1-18), murine IL10 (2-178), dimerization unit (179-355),unit joint(356-360), murine MOG 35-55 (361-381)

M ¹ PGSALLCCLLLLTGMRI ¹⁸ SRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS ¹⁷⁸ P ¹⁷⁹ SVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK ³⁵⁵ G ³⁵⁶ LGGL ³⁶⁰ M ³⁶¹ EVGWYRSPFSRVVHLYRNGK ³⁸¹

SEQ ID NO:9

VB5006

Murine IL10 signal peptide (1-18), murine IL10 (2-178), dimerization unit (179-355),unit joint(356-360), murine MOG 27-63 (361-397)

M ¹ PGSALLCCLLLLTGMRI ¹⁸ SRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS ¹⁷⁸ P ¹⁷⁹ SVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK ³⁵⁵ ³ G ⁵⁶ LGGL ³⁶⁰ SPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEAQP ³⁹⁷

SEQ ID NO:10

VB5009

Murine TGF-beta 1 signal peptide (1-29), murine TGF-beta 1-390), dimerization unit (391-567),unit joint(568-572) murine MOG 35-55 (573-593)

MPPSGLRLLPLLLPLPWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKLRLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESADPEPEPEADYYAKEVTRVLMVDRNNAIYEKTKDISHSIYMFFNTSDIREAVPEPPLLSRAELRLQRLKSSVEQHVELYQKYSNNSWRYLGNRLLTPTDTPEWLSFDVTGVVRQWLNQGDGIQGFRFSAHCSCDSKDNKLHVEINGISPKRRGDLGTIHDMNRPFLLLMATPLERAQHLHSSRHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASASPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCSPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLMEVGWYRSPFSRVVHLYRNGK

SEQ ID NO:11

VB5017

Murine CTLA4 signal peptide (1-35), murine CTLA4 (36-161), dimerization unit (162-338),unit joint (339-343), murine MOG 35-55 (344-364)

M ¹ ACLGLRRYKAQLQLPSRTWPFVALLTLLFIPVFS ³⁵ EAIQVTQPSVVLASSHGVASFPCEYSPSHNTDEVRVTVLRQTNDQMTEVCATTFTEKNTVGFLDYPFCSGTFNESRVNLTIQGLRAVDTGLYLCKVELMYPPPYFVGMGNGTQIYVIDPEPCPDSD ¹⁶¹ P ¹⁶² SVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK ³³⁸ G ³³⁹ LGGL ³⁴³ M ³⁴⁴ EVGWYRSPFSRVVHLYRNGK ³⁶⁴

SEQ ID NO:12

VB5022

Murine IgVH signal peptide (1-19), murine anti-DEC 205 scFv (20-265), dimerization unit (266-442),unit joint Head(443-447), the murine Pan b 1 peptide (448-468)

M ¹ NFGLRLIFLVLTLKGVQC ¹⁹ D ²⁰ IQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLELKGGGGSGGGGSGGGGSEVKLLESGGGLVQPGGSLRLSCAASGFTFNDFYMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISRDNTQNILYLQMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSS ²⁶⁵ P ²⁶⁶ SVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK ⁴⁴² G ⁴⁴³ LGGL ⁴⁴⁷ A ⁴⁴⁸ YKEQIKTLTNKLKAAEARAE ⁴⁶⁸

Description of the embodiments

1. A tolerance-inducing construct comprising:

i) A polynucleotide comprising a nucleotide sequence encoding a targeting unit, a multimerization unit, such as a dimerization unit, and an antigenic unit, that targets or is capable of targeting an Antigen Presenting Cell (APC); or alternatively

iii) A multimeric protein, e.g. a dimeric protein, consisting of a plurality of polypeptides as defined in (ii), e.g. two polypeptides;

2. The tolerance-inducing construct of embodiment 1, wherein the construct comprises multimerization units.

3. The tolerance-inducing construct according to embodiment 1, wherein the construct comprises dimerization units and/or wherein the multimeric protein is a dimeric protein.

4. The tolerance inducing construct according to any one of the preceding embodiments, wherein the targeting unit is capable of delivering the construct to an antigen presenting cell and interacting with a surface molecule on an APC without activating the APC.

5. The tolerance inducing construct according to any one of the preceding embodiments, wherein the targeting unit is capable of binding to a surface molecule on an APC.

6. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the targeting unit comprises or consists of a moiety that binds to a receptor selected from the group consisting of: TGF-beta receptors such as TGF-beta R1, TGF-beta R2 or TGF-beta R3, IL10R such as IL-10RA and IL10-RB, IL2R, IL4R, IL6R, IL R and IL13R, IL27R, IL35R, IL37R, CCR7, CD11b, CD11c, CD103, CD14, CD36, CD205, CD109, VISTA, MARCO, MHCII, MHCII, CD, SIGLEC, MGL, CD80, CD86, clec9A, clec12A, clec B, DCIR2, langerin, MR, DC-Sign, treml4, dectin-1, PDL2 and HVEM.

7. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the moiety is selected from the group consisting of a natural ligand, a synthetic ligand and an antibody or a part thereof, e.g. scFv.

8. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the moiety is an antibody or a part thereof, e.g. an scFv, specific for the receptor, and wherein binding to the receptor results in presentation of the antigen in an anti-inflammatory tolerogenic manner.

9. The tolerance inducing construct according to any one of the preceding embodiments, wherein the moiety is a synthetic ligand specific for the receptor, and wherein binding to the receptor results in presentation of the antigen in an anti-inflammatory tolerogenic manner.

10. The tolerance inducing construct according to any one of the preceding embodiments, wherein the moiety is a natural ligand.

11. The tolerance inducing construct according to any one of the preceding embodiments, wherein the natural ligand is selected from the group consisting of: TGF-beta such as TGF-beta 1, TGF-beta 2 or TGF-beta 3, IL-10, IL2, IL4, IL6, IL11, IL13, IL27, IL35, IL37, CCL19, CCL21, ICAM-1 (intercellular adhesion molecule 1, also known as CD 54), keratin, VSIG-3, SCGB3A2, extracellular domains of CTLA-4 such as CTLA-4, extracellular domains of PD-1 such as PD-1, and BTLA.

12. The tolerance inducing construct according to any one of the preceding embodiments, wherein the natural ligand is selected from the group consisting of: tgfβ, IL-10, SCGB3A2, CTLA-4, e.g., extracellular domain of CTLA-4.

13. The tolerance inducing construct according to any one of the preceding embodiments, wherein the natural ligand is an extracellular domain of BTLA.

14. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the targeting unit consists of or comprises: IL-10 or TGF-beta, preferably human IL-10 or human TGF-beta.

15. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the targeting unit comprises or consists of: an amino acid sequence having at least 80% sequence identity to an amino acid sequence of human TGF-beta, such as an amino acid sequence selected from the group consisting of SEQ ID NOS: 205-207.

16. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the targeting unit comprises or consists of: 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%, such as at least 99% or such as 100% sequence identity, to an amino acid sequence of human TGF beta, such as an amino acid sequence selected from SEQ ID NOs 205-207.

17. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the targeting unit comprises or consists of: the amino acid sequence of human TGF-beta, e.g. the amino acid sequence selected from SEQ ID NO 205-207, except that up to 22 amino acids have been substituted, deleted or inserted, e.g. up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid has been substituted, deleted or inserted.

18. The tolerance-inducing construct according to any one of the preceding embodiments, wherein 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, e.g., the amino acid sequence of SEQ ID NO: 211.

19. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the targeting unit comprises or consists of: 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%, such as at least 99% or such as 100% sequence identity to the amino acid sequence of human IL-10, such as the amino acid sequence of SEQ ID NO: 211.

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

21. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the targeting unit is or comprises: SCGB3A2 or VSIG-3, such as human VSIG-3 or human SCGB3A2.

22. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the targeting unit comprises or consists of: an amino acid sequence having at least 80% sequence identity to the amino acid sequence of human VSIG-3, such as the amino acid sequence of SEQ ID No. 215.

23. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the targeting unit comprises or consists of: 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%, such as at least 99%, or such as 100% sequence identity to the amino acid sequence of human VSIG-3, such as the amino acid sequence of SEQ ID No. 215.

24. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the targeting unit comprises or consists of: the amino acid sequence of human VSIG-3, e.g., the amino acid sequence of SEQ ID No. 215, except that up to 22 amino acids have been substituted, deleted or inserted, e.g., up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid has been substituted, deleted or inserted.

25. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the targeting unit comprises or consists of: an amino acid sequence having at least 80% sequence identity to the amino acid sequence of human SCGB3A2, e.g. the amino acid sequence of SEQ ID NO: 213.

26. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the targeting unit comprises or consists of: 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%, such as at least 99% or such as 100% sequence identity to the amino acid sequence of human SCGB3A2, such as the amino acid sequence of SEQ ID NO: 213.

27. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the targeting unit comprises or consists of: the amino acid sequence of human SCGB3A2, for example the amino acid sequence of SEQ ID NO:213, has been substituted, deleted or inserted except that up to 22 amino acids have been substituted, deleted or inserted, for example up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid has been substituted, deleted or inserted.

28. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the targeting unit consists of or comprises: an antibody or portion thereof specific for CD 205.

29. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the targeting unit consists of or comprises: an scFv specific for CD205, for example an anti-DEC 205 scFv, for example an scFv comprising or consisting of SEQ ID NO: 49.

30. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the antigenic unit comprises one or more T cell epitopes of a self antigen, e.g. 1T cell epitope of a self antigen or a plurality of T cell epitopes of a self antigen.

31. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the plurality of T cell epitopes belong to the same autoantigen or to a plurality of different autoantigens.

32. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the antigenic unit comprises one or more linkers separating the T cell epitopes.

33. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the antigenic unit comprises a plurality of T cell epitopes of autoantigens, allergens, alloantigens or xenogeneic antigens, and wherein the T cell epitopes are separated by a linker.

34. The tolerance-inducing construct according to any one of the preceding embodiments, wherein all T cell epitopes except the terminal T cell epitope are arranged in subunits, wherein each subunit comprises or consists of a T cell epitope and a linker.

35. The tolerance-inducing construct according to any one of the preceding embodiments, wherein all T cell epitopes except the N-terminal T cell epitope are arranged in subunits, wherein each subunit comprises or consists of a T cell epitope and a linker.

36. The tolerance-inducing construct according to any one of the preceding embodiments, wherein all T cell epitopes except the C-terminal T cell epitope are arranged in subunits, wherein each subunit comprises or consists of a T cell epitope and a linker.

37. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the antigenic units comprise n antigens and n-1 subunits, wherein each subunit comprises T cell epitopes and linkers of autoantigens, allergens, alloantigens or xenogeneic antigens, and further comprises terminal T cell epitopes.

38. The tolerance-inducing construct according to any one of the preceding embodiments, wherein n is an integer from 1 to 50, such as from 3 to 50 or 15 to 40 or 10 to 30 or 10 to 25 or 10 to 20 or 15 to 30 or 15 to 25 or 15 to 20.

39. The tolerance inducing construct according to any one of the preceding embodiments, wherein the linker is non-immunogenic.

40. The tolerance inducing construct according to any one of the preceding embodiments, wherein the linker is a rigid linker or a flexible linker.

41. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the antigenic unit comprises one or more T-cell epitopes of Myelin Basic Protein (MBP), e.g. one T-cell epitope of MBP or a plurality of T-cell epitopes of MBP, one or more T-cell epitopes of Myelin Oligodendrocyte Glycoprotein (MOG), e.g. one T-cell epitope of MOG or a plurality of T-cell epitopes of MOG, or one or more T-cell epitopes of proteolipid protein (PLP), e.g. one T-cell epitope of PLP or a plurality of T-cell epitopes of PLP.

42. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the antigenic unit comprises one or more T cell epitopes of MOG, e.g. one or more T cell epitopes of MOG comprising or consisting of a sequence selected from SEQ ID NOs 180-182.

43. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the antigenic unit comprises one or more T cell epitopes of an allergen, e.g. 1T cell epitope of an allergen or a plurality of T cell epitopes of an allergen.

44. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the plurality of T cell epitopes belong to the same allergen or to a plurality of different allergens.

45. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the antigenic unit comprises one or more T-cell epitopes of Fel d1, e.g. one T-cell epitope of Fel d1 or a plurality of T-cell epitopes of Fel d 1.

46. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the antigenic unit comprises one or more T cell epitopes of Fel d4 and/or Fel d7, e.g. one or more T cell epitopes of Fel d4 and/or one or more T cell epitopes of Fel d 7.

47. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the antigenic unit comprises one or more T cell epitopes of an alloantigen/xenogeneic antigen, e.g. one T cell epitope of an alloantigen/xenogeneic antigen or a plurality of T cell epitopes of an alloantigen/xenogeneic antigen.

48. The tolerance inducing construct according to any one of the preceding embodiments, wherein the plurality of T cell epitopes belong to the same alloantigen/xenogeneic antigen or to a plurality of different alloantigens/xenogeneic antigens.

49. The tolerance inducing construct according to any one of the preceding embodiments, wherein the one or more T cell epitopes are 7 to about 200 amino acids in length.

50. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the one or more T cell epitopes are 7-150 amino acids, preferably 7-100 amino acids, such as 9-100 amino acids or 15-100 amino acids or 9-60 amino acids or 9-30 amino acids or 15-60 or 15-30 or 20-75 amino acids or 25-50 amino acids in length.

51. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the one or more T cell epitopes are 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids in length.

52. The tolerance inducing construct according to any one of the preceding embodiments, wherein the antigenic unit comprises one or more T cell epitopes of 7-11 amino acids in length.

53. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the antigenic unit comprises one or more T cell epitopes of 9 to 60 amino acids, such as 9 to 30 amino acids, such as 15 to 60 amino acids, such as 15 to 30 amino acids in length.

54. The tolerance inducing construct according to any one of the preceding embodiments, wherein the antigenic unit comprises one or more T cell epitopes of 15 amino acids in length.

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

56. The tolerance-inducing construct according to any one 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.

57. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the antigenic unit comprises 1 to 3T cell epitopes, such as 1, 2, 3T cell epitopes, or 1 to 5T cell epitopes, such as 1, 2, 3, 4, 5T cell epitopes, or 3 to 6T cell epitopes, such as 3, 4, 5, 6T cell epitopes, or 5 to 15T cell epitopes, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15T cell epitopes, or 7 to 17T cell epitopes, such as 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17T cell epitopes, or 9 to 19T cell epitopes, such as 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19T cell epitopes.

58. The tolerance inducing construct according to any one of the preceding embodiments, wherein the one or more T cell epitopes are randomly arranged in the antigenic unit.

59. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the one or more T cell epitopes are arranged in a higher to lower antigenicity order in the direction from the multimerization unit to the end of the antigenic unit.

60. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the most hydrophobic T cell epitope is located substantially in the middle of the antigenic unit, and the most hydrophilic T cell epitope is located closest to the multimerization unit or at the end of the antigenic unit.

61. The tolerance inducing construct according to any one of the preceding embodiments, wherein the T cell epitopes are arranged in order of higher antigenicity to lower antigenicity in the direction from the dimerization unit to the end of the antigenic unit.

62. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the most hydrophobic T cell epitope is located substantially in the middle of the antigenic unit, the most hydrophilic T cell epitope is closest to the dimer unit or is located at the end of the antigenic unit.

63. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the one or more T cell epitopes alternate between hydrophilic T cell epitopes and hydrophobic T cell epitopes.

64. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the GC-rich T cell epitopes are arranged such that there is at least one non-GC-rich T cell epitope in between.

65. The tolerance inducing construct according to any one of the preceding embodiments, wherein a plurality of T cell epitopes are separated by a T cell epitope linker.

66. The tolerance inducing construct according to any one of the preceding embodiments, wherein the antigenic unit comprises n T cell epitopes and n-1T cell epitope linkers.

67. The tolerance inducing construct according to any one of the preceding embodiments, wherein the T cell epitope linker is non-immunogenic.

68. The tolerance inducing construct according to any one of the preceding embodiments, wherein the T cell epitope linker is a flexible linker.

69. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the T cell epitope linker is a peptide consisting of 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, e.g. 8, 9, 10, 11, 12, 13, 14 or 15 amino acids or 10 to 15 amino acids or 8 to 12 amino acids, e.g. 8, 9, 10, 11 or 12 amino acids.

70. The tolerance inducing construct according to any one of the preceding embodiments, wherein the T cell epitope linker consists of 10 amino acids.

71. The tolerance-inducing construct according to any one of the preceding embodiments, wherein all T cell epitope linkers comprised in the antigenic unit are preferably identical.

72. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the T cell epitope linker is a serine (S) and/or glycine (G) rich linker.

73. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the serine and/or glycine rich T cell epitope tag further comprises at least one leucine (L) residue, e.g. at least 1 or at least 2 or at least 3 or at least 4 leucine residues.

74. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the T cell epitope linker is a serine-glycine linker of 10 amino acids in length and comprising 1 or 2 leucine residues.

75. The tolerance inducing construct according to any one of the preceding embodiments, wherein the T cell epitope linker is a GSAT linker or a SEG linker.

76. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the T cell epitope linker comprises or consists of GLGGL (SEQ ID NO: 90).

77. The tolerance inducing construct according to any one of the preceding embodiments, wherein the T cell epitope linker is a cleavable linker.

78. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the allergen is a food allergen, e.g. a shellfish allergen, e.g. tropomyosin, arginine kinase, myosin light chain, troponin C, triose phosphate isomerase, or actin.

79. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the allergen is Pan b 1, and optionally wherein the antigenic unit consists of Pan b 1T cell epitope (251-270) or comprises Pan b 1T cell epitope (251-270).

80. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the cow's milk allergen, such as Bos d 4, bos d 5, bos d 6, bos d 7, bos d 8, bos d 9, bos d 10, bos d 11 or Bos d 12.

81. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the allergen is an egg allergen, such as ovomucoid (ovicoud), ovalbumin (ovalbumin), ovotransferrin (ovatranferrin), conalbumin (conalbumin), gal 3 3, egg lysozyme (egg lyaozyme) or ovomucoin (ovamin).

82. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the allergen is OVA (257-264) comprising the amino acid sequence SIINFEKL (SEQ ID NO: 45).

83. The tolerance inducing construct according to any one of the preceding embodiments, wherein the antigenic unit comprises the T cell epitope OVA (257-264).

84. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the allergen is a fish allergen, such as parvalbumin, enolase, aldolase or vitellogenin.

85. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the allergen is a fruit allergen such as pathogenic related protein 10, pre-fibrin (profilin), nsLTP, sweet protein-like protein (thaumatin-like protein), gibberellin-regulating protein, isoflavone reductase-related protein, class 1 chitinase, beta-1, 3 glucanase, germinating element-like protein (germin like protein), alkaline serine protease, pathogenic related protein 1, actinidin (actinin), plant cystatin (phytocystatin), kiwellin, major latex protein (major latex protein), cupin or 2S albumin.

86. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the allergen is a plant allergen, such as a pathogenic related protein 10, a pre-fibrin, a nsLTP type 1, a nsLTP type 2, an osmorexin-like protein, an isoflavone reductase-like protein, a β -fructofuranosidase, a PR protein TSI-1, a cyclophilin (cyclophilin) or a FAD-containing oxidase.

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

88. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the allergen is a soybean allergen, such as 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, gly m lectin, gly m Bd28K, gly m 30kD, gly m CPI or Gly m TI.

89. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the allergen is a peanut allergen, e.g. Ara h 1, ara h 2, ara h 3, 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.

90. The tolerance inducing construct according to any one of the preceding embodiments, wherein the allergen is a woody nut or seed allergen.

91. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the allergen is 11S globulin, 7S globulin, 2S globulin, PR10, PR-14nsLTP, oleosin (oleosin) or pre-fibrin.

92. The tolerance inducing construct according to any one of the preceding embodiments, wherein the food allergen is buckwheat, celery, a pigment additive, garlic, gluten (gluten), oat, beans, corn, mustard, poultry, meat, rice or sesame.

93. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the allergen is a bee venom allergen, such as phospholipase A2, hyaluronidase, acid phosphatase, melittin (melittin), allergen C/DPP, CRP/lcarpin or vitellogenin.

94. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the allergen is a wasp allergen (vespid allergy), such as phospholipase A1, hyaluronidase, protease, antigen 5, DPP IV or vitellogenin.

95. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the allergen is a latex allergen, e.g. 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.

96. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the allergen is a dust mite allergen, such as house dust mite (house dust mite) allergen or a storage dust allergen (storage dust allergen), such as 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, der p 23, der f 1, der f 2, der f 3, der f 7, der f 8, der f 10, blot t 1, blot t 2, blot 3, blot 4, blot 5, blot t 8, blot t 10, blot 12 or Blot 21.

97. The tolerance inducing construct according to any one of the preceding embodiments, wherein the antigenic unit consists of Der p 1T cell epitope (111-139) or comprises Der p 1T cell epitope (111-139).

98. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the allergen is a cockroach allergen, e.g. Bla g 1, bla g 2, bla g 3, bla g 4, bla g 5, bla g 6, bla g 7, bla g 8, bla g 11, per a 1, per a 2, per a 3, per a 6, per a 7, per a 9 or Per a 10.

99. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the allergen is a mould allergen, e.g. an aspergillus fumigatus (Aspergillus fumigatus) allergen, e.g. 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.

100. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the allergen is a fungal allergen, e.g. a Malassezia (Malassezia) allergen, such as 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.

101. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the allergen is a furred animal allergen, such as a dog allergen, such as Can f 1, can f 2, can f 3, can f 4, can f 5 or Can f 6, or the allergen is a horse allergen, such as ecl c 1, ecl c 2, ecl c 3 or ecl c 4, or the allergen is a cat allergen, such as 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, or the allergen is a laboratory animal allergen, such as lipocalin (urinary prealbumin), pre-urinary albumin (secretoglobulin) or serum albumin.

102. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the allergen is a pollen allergen, such as a pasture pollen allergen (grass pollen allergen), such as moxiella tikoua (timothy grass), festuca arundinacea (orcard grass), kentucky blue grass (Kentucky bluegrass), perennial rye (perennial rye), festuca arundinacea (sweet vernal grass), bahia grass (bahia grass), johnson grass (johnson grass) or bermula grass (bertruda grass) allergen, 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.

103. A tolerance-inducing construct according to any one of the preceding embodiments, wherein the allergen is a tree pollen allergen, such as an alder, birch, hornbeam, hazel, elnut (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, for example 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, cora 2, cora 6, cora 8, cora 9, cora 10, cora 11, cora 12, cora 13, cora 14, ost c 1, cas 5, cas 8 or Cas 9, fag s 1, que a 1, fra 1 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 2 or Pla 3 or Pla 1, pla 2 or Pla 3.

104. The tolerance inducing construct according to any one of the preceding embodiments, wherein the antigenic unit consists of the Bet v 1T cell epitope (139-152) or comprises the Bet v 1T cell epitope (139-152).

105. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the allergen is a weed pollen allergen (weed pollen allergen), such as ragweed (ragweed), mugwort (mugwort), sunflower (sunflower), parthenon (feverfew), pellitory (pellitory), british plantain (engish plantain), arnica (annual mercry), quinoa (goose) or Russian thistle (Russian thistle) or amaranth pollen allergen, such as Amb a 1, amb a 4, amb a 6, amb a 8, amb a 9, amb a 10 or Amb a 11, art v 1, art v 3, art v 4, art v 5 or Art v 6, hel a 1 or hela 2, park 1, park 2, park 3 or park 4, pla 1, sal 2 or Sal 2, chek 1, sal 4 or Sal 1, sal 2.

106. The tolerance inducing construct according to any one of the preceding embodiments, wherein the allergen is selected from environmental allergens, such as insects, cockroaches, house dust mites or moulds.

107. The tolerance inducing construct according to any one of the preceding embodiments, wherein the allergen causes an allergic disease selected from the group consisting of: allergic rhinitis, asthma, atopic dermatitis, allergic gastroenteropathy, contact dermatitis, drug allergy, or a combination thereof.

108. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the allergen is comprised in a medicament having undesired immunogenicity.

109. The tolerance inducing construct according to any one of the preceding embodiments, wherein the allergen is factor VIII.

110. The tolerance inducing construct according to any one of the preceding embodiments, wherein the allergen is insulin.

111. The tolerance inducing construct according to any one of the preceding embodiments, wherein the allergen is one or more monoclonal antibodies for use in therapy.

112. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the construct contains a T cell epitope comprised in an autoantigen associated with an autoimmune disease.

113. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the autoantigen is associated with Multiple Sclerosis (MS).

114. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the autoantigen is Myelin Oligodendrocyte Glycoprotein (MOG), MAG, MOBP, CNPase, S beta, transaldolase (transaldolase), myelin Basic Protein (MBP), myelin proteolipid protein (PLP).

115. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the antigenic unit comprises 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).

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

117. The tolerance inducing construct according to any one of the preceding embodiments, wherein the autoantigen is associated with type 1 diabetes.

118. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the autoantigen is a 65 kilodaltons isoform of glutamate decarboxylase (GAD 65), insulin, IA-2 or ZnT8, IGRP, chgA, IAPP, peripheral protein (periherein), tetraspanin-7 (tetraspin-7), GRP78, urotensin-3 (urosporin-3) or insulin gene enhancer protein isl-1.

119. The tolerance inducing construct according to any one of the preceding embodiments, wherein the autoantigen is associated with celiac disease.

120. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the autoantigen is alpha-gliadin, gamma-gliadin, omega-gliadin, low molecular weight glutenin, high molecular weight glutenin, hordein (hordei), secalin (secalin) or avenin b (avenin b).

121. The tolerance inducing construct according to any one of the preceding embodiments, wherein the antigenic unit comprises the T cell epitope α -gliadin (76-95).

122. The tolerance inducing construct according to any one of the preceding embodiments, wherein the autoantigen is associated with rheumatoid arthritis.

123. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the autoantigen is collagen, heat shock protein 60 (HSP 60), bank 3, ribonucleoprotein D1 (SmD 1), acetylcholine receptor (AChR), or myelin protein zero (P0).

124. The tolerance inducing construct according to any one of the preceding embodiments, wherein the autoantigen is associated with Chronic Inflammatory Demyelinating Polyneuropathy (CIDP) and the autoantigen is neurofascin 155.

125. The tolerance inducing construct according to any one of the preceding embodiments, wherein the autoantigen is associated with Hashimoto Thyroiditis (HT) and the autoantigen is thyroid peroxidase and/or thyroglobulin.

126. The tolerance inducing construct according to any one of the preceding embodiments, wherein the autoantigen is associated with pemphigus larum and the autoantigen is a desmosome associated glycoprotein.

127. The tolerance inducing construct according to any one of the preceding embodiments, wherein the autoantigen is associated with pemphigus vulgaris and the autoantigen is desmoglein3 (desmoglein 3).

128. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the self-antigen is associated with Thyroid Eye Disease (TED), and the self-antigen is a calbindin (calequestrin).

129. The tolerance inducing construct according to any one of the preceding embodiments, wherein the autoantigen is associated with Grave's disease and the autoantigen is a thyroid stimulating hormone receptor.

130. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the autoantigen is associated with Primary Biliary Cirrhosis (PBC), and the autoantigen is an anti-mitochondrial antibody (AMA), an anti-nuclear antibody (ANA), a Rim-like/membrane (RL/M) and/or a polynucleic spot (MND).

131. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the autoantigen is associated with myasthenia gravis and the autoantigen is an acetylcholine receptor.

132. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the autoantigen is associated with insulin resistant diabetes and the autoantigen is an insulin receptor.

133. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the autoantigen is associated with autoimmune hemolytic anemia and the autoantigen is a red blood cell.

134. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the autoantigen is associated with rheumatoid arthritis and the autoantigen is a citrullinated protein, a homocrullinated protein and an Fc portion of IgG.

135. The tolerance inducing construct according to any one of the preceding embodiments, wherein the antigenic unit and the multimerization unit are connected by a unit linker.

136. The tolerance inducing construct according to any one of the preceding embodiments, wherein the antigenic unit and the dimerization unit are connected by a unit linker.

137. The tolerance inducing construct according to any one of the preceding embodiments, wherein the unit linker comprises a restriction site.

138. The tolerance inducing construct according to any one of the preceding embodiments, wherein the unit linker is a GLGGL linker (SEQ ID NO: 90) or a GLSGL linker (SEQ ID NO: 163).

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

140. The tolerance inducing construct according to any one of the preceding embodiments, wherein the construct comprises multimerization units.

141. The tolerance inducing construct according to any one of the preceding embodiments, wherein the construct comprises a dimerization unit.

142. The tolerance inducing construct according to any one of the preceding embodiments, wherein the multimerization unit is a trimerization unit or a tetramerization unit.

143. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the multimerization unit is a trimerization unit, e.g. a collagen-derived trimerization unit, such as a human collagen-derived trimerization domain, e.g. a human collagen-derived XVIII trimerization domain or a human collagen XV trimerization domain.

144. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the multimerization unit is a trimerization unit comprising or consisting of: the nucleotide sequence of SEQ ID NO. 42 or the amino acid sequence encoded by said nucleotide sequence.

145. The tolerance inducing construct according to any one of the preceding embodiments, wherein the multimerization unit is a trimerization unit that is the C-terminal domain of T4 fibritin.

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

147. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the multimerization unit is a tetramerization unit, e.g. a domain derived from p 53.

148. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the multimerization unit is a tetramerization unit comprising or consisting of: the nucleic acid sequence of SEQ ID NO. 44 or an amino acid sequence encoded by said nucleic acid sequence.

149. The tolerance inducing construct according to any one of the preceding embodiments, wherein the dimerization unit comprises a hinge region.

150. The tolerance inducing construct according to any one of the preceding embodiments, wherein the dimerization unit comprises a hinge region and another domain that promotes dimerization.

151. The tolerance-inducing construct according to any one of the preceding embodiments, wherein 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 to the other dimerization promoting domain.

152. The tolerance-inducing construct according to any one of the preceding embodiments, wherein 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 to the other dimerization promoting domain.

153. The tolerance inducing construct according to any one of the preceding embodiments, wherein the dimerization unit linker is a glycine-serine rich linker, e.g. GGGSSGGGSG (SEQ ID NO: 139).

154. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the hinge region is Ig-derived, e.g. IgG1, igG2 or IgG 3-derived.

155. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the hinge region is derived from IgM and optionally comprises or consists of: the nucleotide sequence of SEQ ID NO. 47 or an amino acid sequence encoded by said nucleic acid sequence.

156. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the hinge region has the ability to form one or more covalent bonds, such as one or more disulfide bonds.

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

158. The tolerance-inducing construct according to any one of the preceding embodiments, wherein 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 with amino acid sequences 1-27 of SEQ ID No. 1, 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.

159. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the dimerization unit comprises or consists of: hinge exon h1 and hinge exon h4 having the amino acid sequences 1 to 27 of SEQ ID NO. 1 or a nucleotide sequence encoding said amino acid sequences.

160. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the dimerization unit comprises or consists of: hinge exon h1 and hinge exon h4 having the amino acid sequences 1-27 of SEQ ID NO. 1, with the difference that at most 10 amino acids have been substituted, deleted or inserted, for example at most 9 amino acids, for example at most 8 amino acids, for example at most 7 amino acids, for example at most 6 amino acids, for example at most five amino acids, for example at most four amino acids, for example at most 3 amino acids, for example at most 2 amino acids or for example at most 1 amino acid have been substituted, deleted or inserted.

161. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the dimerization unit comprises another domain that promotes dimerization, such as 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. CH3 domain), or a sequence substantially identical to such a C domain, or a variant thereof.

162. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the further dimerization promoting domain is a carboxy-terminal C domain derived from IgG, e.g. a carboxy-terminal C domain derived from IgG 3.

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

164. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the dimerization unit comprises or consists of: a carboxy-terminal C domain derived from IgG3 having an amino acid sequence having at least 85% sequence identity to amino acid sequences 39-144 of SEQ ID No. 1, 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.

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

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

167. The tolerance inducing construct according to any one of the preceding embodiments, wherein the immunoglobulin domain has the ability to form dimers by non-covalent interactions, such as hydrophobic interactions.

168. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the dimerization unit comprises a CH3 domain and does not comprise a CH2 domain, or wherein the dimerization unit comprises a CH2 domain and does not comprise a CH3 domain.

169. The tolerance inducing construct according to any one of the preceding embodiments, wherein the dimerization unit comprises hinge exon h1, hinge exon h4, dimerization unit linker and CH3 domain of human IgG 3.

170. The tolerance inducing construct according to any one of the preceding embodiments, wherein the dimerization unit comprises a polypeptide consisting of hinge exon h1, hinge exon h4, dimerization unit linker and CH3 domain of human IgG 3.

171. The tolerance inducing construct according to any one of the preceding embodiments, wherein the dimerization unit consists of a polypeptide consisting of hinge exon h1, hinge exon h4, dimerization unit linker and CH3 domain of human IgG 3.

172. The tolerance inducing construct according to any one of the preceding embodiments, wherein the dimerization unit comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID No. 1.

173. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the dimerization unit comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:1, such as at least 86%, 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.

174. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the dimerization unit consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:1, such as at least 85%, 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%.

175. The tolerance inducing construct according to any one of the preceding embodiments, wherein the dimerization unit consists of the amino acid sequence of SEQ ID No. 1 or a nucleotide sequence encoding the amino acid sequence.

176. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the dimerization unit comprises the amino acid sequence of SEQ ID No. 1 except that up to 22 amino acids have been substituted, deleted or inserted, e.g. up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid has been substituted, deleted or inserted.

177. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the dimerization unit consists of the amino acid sequence of SEQ ID No. 1, except that up to 22 amino acids have been substituted, deleted or inserted, e.g. up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid has been substituted, deleted or inserted.

178. The tolerance inducing construct according to any one of the preceding embodiments, wherein the dimerization unit linker is a glycine-serine rich linker, e.g. GGGSSGGGSG (SEQ ID NO: 139).

179. The tolerance inducing construct according to any one of the preceding embodiments, wherein the polynucleotide further comprises a nucleotide sequence encoding a signal peptide.

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

181. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the signal peptide is a human Ig VH signal peptide or a signal peptide naturally occurring at the N-terminus of any targeting unit described herein, e.g., a human signal peptide of human IL-10 or a human signal peptide of human tgfβ.

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

183. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the polynucleotide comprises a nucleotide sequence encoding a human Ig VH signal peptide, and preferably comprises a nucleotide sequence encoding a scFv such as human anti-DEC 205.

184. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the polynucleotide comprises a nucleotide sequence encoding a signal peptide comprising an amino acid sequence having at least 85%, such as at least 86%, 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. 6 or SEQ ID No. 48.

185. The tolerance inducing construct according to any one of the preceding embodiments, wherein the polynucleotide comprises a nucleotide sequence encoding a signal peptide comprising the amino acid sequence of SEQ ID No. 6 or SEQ ID No. 48.

186. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the polynucleotide comprises a nucleotide sequence encoding a signal peptide consisting of an amino acid sequence having at least 80%, preferably at least 85%, e.g. at least 86%, e.g. at least 87%, e.g. at least 88%, e.g. at least 89%, e.g. at least 90%, e.g. at least 91%, e.g. at least 92%, e.g. at least 93%, e.g. at least 94%, e.g. at least 95%, e.g. at least 96%, e.g. at least 97%, e.g. at least 98% or e.g. at least 99% sequence identity to the amino acid sequence of SEQ ID No. 6 or SEQ ID No. 48.

187. The tolerance inducing construct according to any one of the preceding embodiments, wherein the polynucleotide comprises a nucleotide sequence encoding a signal peptide having the amino acid sequence of SEQ ID No. 6 or SEQ ID No. 48.

188. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the polynucleotide comprises a nucleotide sequence encoding a signal peptide comprising or consisting of the amino acid sequence of SEQ ID No. 6 or SEQ ID No. 48, with the difference that at most 5 amino acids have been substituted, deleted or inserted, such as at most 4 amino acids, such as at most 3 amino acids, such as at most 2 amino acids or such as at most 1 amino acid have been substituted, deleted or inserted.

189. The tolerance inducing construct according to any one of the preceding embodiments, wherein the polynucleotide is a DNA sequence or an RNA sequence.

190. A polynucleotide as defined in any one of the preceding embodiments.

191. A vector comprising the polynucleotide of embodiment 190.

192. A host cell comprising the polynucleotide of embodiment 190 and/or the vector of embodiment 191.

193. A polypeptide encoded by a nucleotide sequence as defined in any one of embodiments 1 to 189.

194. A dimeric protein as defined in any one of embodiments 1 to 189, which consists of two polypeptides.

195. A multimeric protein defined in any one of embodiments 1 to 189, which consists of two or more polypeptides.

196. A pharmaceutical composition comprising the tolerance-inducing construct of any one of embodiments 1-189 and a pharmaceutically acceptable carrier.

197. A pharmaceutical composition comprising the polynucleotide of any one of embodiments 1 to 189, the vector of embodiment 191, the polypeptide of embodiment 193, the dimeric protein of embodiment 194, or the multimeric protein of embodiment 195, and a pharmaceutically acceptable carrier.

198. The pharmaceutical composition of any one of embodiments 196-197, further comprising one or more pharmaceutically acceptable excipients and/or diluents.

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

200. A method of preparing the pharmaceutical composition of any one of embodiments 196-199, comprising a multimeric protein, a dimeric protein, or a polypeptide, wherein the method comprises the steps of:

i) Transfecting a cell with the polynucleotide of embodiment 190;

ii) culturing the cells;

iii) Collecting and purifying multimeric, dimeric or polypeptides expressed from the cells; and

iv) mixing the dimeric protein or polypeptide obtained from step iii) with a pharmaceutically acceptable carrier.

201. A method of preparing the pharmaceutical composition of any one of embodiments 196-199, wherein the pharmaceutical composition comprises the polynucleotide of embodiment 190, the method comprising the steps of:

i) Preparing the polynucleotide;

ii) optionally cloning the polynucleotide into an expression vector; and

iii) Mixing the polynucleotide obtained from step i) or the vector obtained from step ii) with a pharmaceutically acceptable carrier.

202. A method of treating a subject suffering from or in need of prevention of a condition involving an undesired immune response, such as an autoimmune disease, allergic disease, or graft rejection, comprising administering to the subject the pharmaceutical composition of any one of embodiments 196-199.

203. The pharmaceutical composition of any one of embodiments 196-199 for use in treating a condition involving an undesired immune response, such as an autoimmune disease, allergic disease, or graft rejection.

204. The use of the pharmaceutical composition of any one of embodiments 196-199 for treating a condition involving an undesired immune response, such as an autoimmune disease, allergic disease, or graft rejection.

205. Use of the pharmaceutical composition of any one of embodiments 196-199 in the manufacture of a medicament for treating a condition involving an undesired immune response, such as an autoimmune disease, allergic disease, or graft rejection.

206. The use of the pharmaceutical composition of any one of embodiments 196-199 for treating a subject having a condition involving an undesired immune response, such as an autoimmune disease, allergic disease, or graft rejection.

207. A medicament comprising the tolerance-inducing construct of any one of embodiments 1 to 189 for use in the treatment of a condition involving an undesired immune response, such as an autoimmune disease, allergic disease or graft rejection.

208. Use of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the tolerance-inducing construct of any one of embodiments 1 to 189 in the manufacture of a medicament for treating a subject suffering from a condition involving an undesired immune response, such as an autoimmune disease, allergic disease or graft rejection.

209. Use of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the tolerance-inducing construct of any one of embodiments 1 to 189 for treating a subject suffering from a condition involving an undesired immune response, such as an autoimmune disease, allergic disease or graft rejection.

210. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the tolerance-inducing construct of any one of embodiments 1 to 189, when used to treat a condition involving an undesired immune response, such as an autoimmune disease, allergic disease or graft rejection.

211. A method of increasing tolerance to an autoantigen, allergen, alloantigen or xenogeneic antigen using the tolerance-inducing construct of any one of embodiments 1 to 189.

212. A method of increasing tolerance of a subject to an autoantigen, allergen, alloantigen or xenogeneic antigen, the method comprising administering to the subject the tolerance-inducing construct of any one of embodiments 1-189 or the pharmaceutical composition of any one of embodiments 196-199.

Sequence listing

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

<120> tolerance-inducing constructs and compositions and their use in the treatment of immune disorders

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465

<210> 3

<211> 484

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5004

<400> 3

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 Pro Ser Val Ile Phe Leu Thr

260 265 270

Lys Arg Gly Arg Gln Val Cys Ala Asp Pro Ser Glu Glu Trp Val Gln

275 280 285

Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys Thr Pro Leu

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Glu Ala Gln Pro

<210> 4

<211> 294

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5012

<400> 4

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 Pro Ser Val Ile Phe

85 90 95

Leu Thr Lys Arg Gly Arg Gln Val Cys Ala Asp Pro Ser Glu Glu Trp

100 105 110

Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys Thr

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro

195 200 205

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

210 215 220

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

225 230 235 240

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

245 250 255

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

260 265 270

Leu Met Glu Val Gly Trp Tyr Arg Ser Pro Phe Ser Arg Val Val His

275 280 285

Leu Tyr Arg Asn Gly Lys

290

<210> 5

<211> 631

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5013

<400> 5

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

Gly Val Ile Ala Gly Ala Val Gly Thr Gly Ala Val Leu Ile Val Ile

245 250 255

Cys Leu Ala Leu Ile Ser Gly Ala Phe Phe Tyr Trp Arg Ser Lys Asn

260 265 270

Lys Glu Glu Glu Glu Glu Glu Ile Pro Asn Glu Ile Arg Glu Asp Asp

275 280 285

Leu Pro Pro Lys Cys Ser Ser Ala Lys Ala Phe His Thr Glu Ile Ser

290 295 300

Ser Ser Glu Asn Asn Thr Leu Thr Ser Ser Asn Thr Tyr Asn Ser Arg

305 310 315 320

Tyr Trp Asn Asn Asn Pro Lys Pro His Arg Asn Thr Glu Ser Phe Asn

325 330 335

His Phe Ser Asp Leu Arg Gln Ser Phe Ser Gly Asn Ala Val Ile Pro

340 345 350

Ser Ile Tyr Ala Asn Gly Asn His Leu Val Leu Gly Pro His Lys Thr

355 360 365

Leu Val Val Thr Ala Asn Arg Gly Ser Ser Pro Gln Val Leu Pro Arg

370 375 380

Asn Asn Gly Ser Val Ser Arg Lys Pro Trp Pro Gln His Thr His Ser

385 390 395 400

Tyr Thr Val Ser Gln Met Thr Leu Glu Arg Ile Gly Ala Val Pro Val

405 410 415

Met Val Pro Ala Gln Ser Arg Ala Gly Ser Leu Val Pro Ser Val Ile

420 425 430

Phe Leu Thr Lys Arg Gly Arg Gln Val Cys Ala Asp Pro Ser Glu Glu

435 440 445

Trp Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln

530 535 540

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

545 550 555 560

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

565 570 575

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

580 585 590

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

595 600 605

Gly Leu Met Glu Val Gly Trp Tyr Arg Ser Pro Phe Ser Arg Val Val

610 615 620

His Leu Tyr Arg Asn Gly Lys

625 630

<210> 6

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> murine IgVH Signal peptide (1-19)

<400> 6

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

1 5 10 15

Val Gln Cys

<210> 7

<211> 467

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5016

<400> 7

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 Pro Ser Val Ile Phe Leu Thr

260 265 270

Lys Arg Gly Arg Gln Val Cys Ala Asp Pro Ser Glu Glu Trp Val Gln

275 280 285

Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys Thr Pro Leu

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly Gly Leu Thr

435 440 445

Asn Met Phe Thr Tyr Glu Ile Ala Pro Val Phe Val Leu Leu Glu Tyr

450 455 460

Val Thr Leu

465

<210> 8

<211> 381

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5005

<400> 8

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

1 5 10 15

Arg Ile Ser Arg Gly Gln Tyr Ser Arg Glu Asp Asn Asn Cys Thr His

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Glu Lys Leu Lys Thr Leu Arg Met 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 Ser

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro

290 295 300

Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

305 310 315 320

Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe Ser Cys Ser Val Met

325 330 335

His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser

340 345 350

Pro Gly Lys Gly Leu Gly Gly Leu Met Glu Val Gly Trp Tyr Arg Ser

355 360 365

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

370 375 380

<210> 9

<211> 397

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5006

<400> 9

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

1 5 10 15

Arg Ile Ser Arg Gly Gln Tyr Ser Arg Glu Asp Asn Asn Cys Thr His

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Glu Lys Leu Lys Thr Leu Arg Met 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 Ser

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro

290 295 300

Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

305 310 315 320

Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe Ser Cys Ser Val Met

325 330 335

His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser

340 345 350

Pro Gly Lys Gly Leu Gly Gly Leu Ser Pro Gly Lys Asn Ala Thr Gly

355 360 365

Met Glu Val Gly Trp Tyr Arg Ser Pro Phe Ser Arg Val Val His Leu

370 375 380

Tyr Arg Asn Gly Lys Asp Gln Asp Ala Glu Ala Gln Pro

385 390 395

<210> 10

<211> 593

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5009

<400> 10

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 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 Asp 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 Asp Arg Asn Asn Ala Ile Tyr Glu Lys Thr Lys Asp Ile Ser

115 120 125

His Ser Ile Tyr Met Phe Phe Asn Thr Ser Asp Ile Arg Glu Ala Val

130 135 140

Pro Glu Pro Pro Leu Leu Ser Arg Ala Glu Leu Arg Leu Gln Arg Leu

145 150 155 160

Lys Ser Ser Val Glu Gln His Val Glu Leu Tyr Gln Lys Tyr Ser Asn

165 170 175

Asn Ser Trp Arg Tyr Leu Gly Asn Arg Leu Leu Thr Pro Thr Asp Thr

180 185 190

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

195 200 205

Asn Gln Gly Asp Gly Ile Gln Gly Phe Arg Phe Ser Ala His Cys Ser

210 215 220

Cys Asp Ser Lys Asp Asn Lys Leu His Val Glu Ile Asn Gly Ile Ser

225 230 235 240

Pro Lys Arg Arg Gly Asp Leu Gly Thr Ile His Asp Met Asn Arg Pro

245 250 255

Phe Leu Leu Leu Met Ala Thr Pro Leu Glu Arg Ala Gln His Leu His

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

Ser 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 Pro Ser Val Ile Phe Leu Thr Lys Arg Gly

385 390 395 400

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

405 410 415

Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys Thr Pro Leu Gly Asp Thr

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

Lys

<210> 11

<211> 364

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5017

<400> 11

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

35 40 45

Ser Ser His Gly Val Ala Ser Phe Pro Cys Glu Tyr Ser Pro Ser His

50 55 60

Asn Thr Asp Glu Val Arg Val Thr Val Leu Arg Gln Thr Asn Asp Gln

65 70 75 80

Met Thr Glu Val Cys Ala Thr Thr Phe Thr Glu Lys Asn Thr Val Gly

85 90 95

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

100 105 110

Asn Leu Thr Ile Gln Gly Leu Arg Ala Val Asp Thr Gly Leu Tyr Leu

115 120 125

Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Phe Val Gly Met 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 Pro Ser Val Ile Phe Leu Thr Lys Arg Gly Arg Gln Val Cys Ala

165 170 175

Asp Pro Ser Glu Glu Trp Val Gln Lys Tyr Val Ser Asp Leu Glu Leu

180 185 190

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

195 200 205

Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly Gly

210 215 220

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

225 230 235 240

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

245 250 255

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

260 265 270

Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met

275 280 285

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

290 295 300

Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe Ser Cys Ser Val Met His

305 310 315 320

Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro

325 330 335

Gly Lys Gly Leu Gly Gly Leu Met Glu Val Gly Trp Tyr Arg Ser Pro

340 345 350

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

355 360

<210> 12

<211> 468

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5022

<400> 12

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 Pro Ser Val Ile Phe Leu Thr

260 265 270

Lys Arg Gly Arg Gln Val Cys Ala Asp Pro Ser Glu Glu Trp Val Gln

275 280 285

Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys Thr Pro Leu

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly Gly Leu Ala

435 440 445

Tyr Lys Glu Gln Ile Lys Thr Leu Thr Asn Lys Leu Lys Ala Ala Glu

450 455 460

Ala Arg Ala Glu

465

<210> 13

<211> 435

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5003b

<400> 13

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 Glu

405 410 415

Val Gly Trp Tyr Arg Ser Pro Phe Ser Arg Val Val His Leu Tyr Arg

420 425 430

Asn Gly Lys

435

<210> 14

<211> 348

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5005b

<400> 14

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

1 5 10 15

Arg Ile Ser Arg Gly Gln Tyr Ser Arg Glu Asp Asn Asn Cys Thr His

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Glu Lys Leu Lys Thr Leu Arg Met 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 Ser

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

245 250 255

Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met

260 265 270

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

275 280 285

Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe Ser Cys Ser Val Met His

290 295 300

Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro

305 310 315 320

Gly Lys Gly Leu Gly Gly Leu Met Glu Val Gly Trp Tyr Arg Ser Pro

325 330 335

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

340 345

<210> 15

<211> 331

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5017a

<400> 15

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

35 40 45

Ser Ser His Gly Val Ala Ser Phe Pro Cys Glu Tyr Ser Pro Ser His

50 55 60

Asn Thr Asp Glu Val Arg Val Thr Val Leu Arg Gln Thr Asn Asp Gln

65 70 75 80

Met Thr Glu Val Cys Ala Thr Thr Phe Thr Glu Lys Asn Thr Val Gly

85 90 95

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

100 105 110

Asn Leu Thr Ile Gln Gly Leu Arg Ala Val Asp Thr Gly Leu Tyr Leu

115 120 125

Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Phe Val Gly Met 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 Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Glu Pro Lys

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

225 230 235 240

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

245 250 255

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

260 265 270

Ser Arg Trp Gln Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu

275 280 285

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

290 295 300

Lys Gly Leu Gly Gly Leu Met Glu Val Gly Trp Tyr Arg Ser Pro Phe

305 310 315 320

Ser Arg Val Val His Leu Tyr Arg Asn Gly Lys

325 330

<210> 16

<211> 426

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5046

<400> 16

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

420 425

<210> 17

<211> 451

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB50048

<400> 17

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> 18

<211> 279

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5052

<400> 18

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 Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln

165 170 175

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

180 185 190

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Gln Asp Ala Glu Gln Ala Pro

275

<210> 19

<211> 364

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5058

<400> 19

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

1 5 10 15

Arg Ile Ser Arg Gly Gln Tyr Ser Arg Glu Asp Asn Asn Cys Thr His

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Gly Glu Lys Leu Lys Thr Leu Arg Met 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 Ser

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Gly Gly Gly

195 200 205

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

210 215 220

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

225 230 235 240

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

245 250 255

Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro Met

260 265 270

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

275 280 285

Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe Ser Cys Ser Val Met His

290 295 300

Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro

305 310 315 320

Gly Lys Gly Leu Gly Gly Leu Ser Pro Gly Lys Asn Ala Thr Gly Met

325 330 335

Glu Val Gly Trp Tyr Arg Ser Pro Phe Ser Arg Val Val His Leu Tyr

340 345 350

Arg Asn Gly Lys Asp Gln Asp Ala Glu Gln Ala Pro

355 360

<210> 20

<211> 576

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5059

<400> 20

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 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 Asp 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 Asp Arg Asn Asn Ala Ile Tyr Glu Lys Thr Lys Asp Ile Ser

115 120 125

His Ser Ile Tyr Met Phe Phe Asn Thr Ser Asp Ile Arg Glu Ala Val

130 135 140

Pro Glu Pro Pro Leu Leu Ser Arg Ala Glu Leu Arg Leu Gln Arg Leu

145 150 155 160

Lys Ser Ser Val Glu Gln His Val Glu Leu Tyr Gln Lys Tyr Ser Asn

165 170 175

Asn Ser Trp Arg Tyr Leu Gly Asn Arg Leu Leu Thr Pro Thr Asp Thr

180 185 190

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

195 200 205

Asn Gln Gly Asp Gly Ile Gln Gly Phe Arg Phe Ser Ala His Cys Ser

210 215 220

Cys Asp Ser Lys Asp Asn Lys Leu His Val Glu Ile Asn Gly Ile Ser

225 230 235 240

Pro Lys Arg Arg Gly Asp Leu Gly Thr Ile His Asp Met Asn Arg Pro

245 250 255

Phe Leu Leu Leu Met Ala Thr Pro Leu Glu Arg Ala Gln His Leu His

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

Ser 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 Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr

385 390 395 400

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

405 410 415

Pro Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly Gly Gln Pro Arg Glu

420 425 430

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

435 440 445

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

450 455 460

Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr Asn Thr

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

<210> 21

<211> 277

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5060

<400> 21

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

275

<210> 22

<211> 347

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5061

<400> 22

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

35 40 45

Ser Ser His Gly Val Ala Ser Phe Pro Cys Glu Tyr Ser Pro Ser His

50 55 60

Asn Thr Asp Glu Val Arg Val Thr Val Leu Arg Gln Thr Asn Asp Gln

65 70 75 80

Met Thr Glu Val Cys Ala Thr Thr Phe Thr Glu Lys Asn Thr Val Gly

85 90 95

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

100 105 110

Asn Leu Thr Ile Gln Gly Leu Arg Ala Val Asp Thr Gly Leu Tyr Leu

115 120 125

Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Phe Val Gly Met 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 Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Glu Pro Lys

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

225 230 235 240

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

245 250 255

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

260 265 270

Ser Arg Trp Gln Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu

275 280 285

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

290 295 300

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

305 310 315 320

Val Gly Trp Tyr Arg Ser Pro Phe Ser Arg Val Val His Leu Tyr Arg

325 330 335

Asn Gly Lys Asp Gln Asp Ala Glu Gln Ala Pro

340 345

<210> 23

<211> 355

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5071

<400> 23

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

355

<210> 24

<211> 56

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5051

<400> 24

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> 25

<211> 39

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5014b

<400> 25

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

1 5 10 15

Val Gln Cys Thr Asn Met Phe Thr Tyr Glu Ile Ala Pro Val Phe Val

20 25 30

Leu Leu Glu Tyr Val Thr Leu

35

<210> 26

<211> 262

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5015b

<400> 26

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 Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln

165 170 175

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

180 185 190

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

195 200 205

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

210 215 220

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

225 230 235 240

Gly Leu Thr Asn Met Phe Thr Tyr Glu Ile Ala Pro Val Phe Val Leu

245 250 255

Leu Glu Tyr Val Thr Leu

260

<210> 27

<211> 434

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5016b

<400> 27

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 Thr Asn

405 410 415

Met Phe Thr Tyr Glu Ile Ala Pro Val Phe Val Leu Leu Glu Tyr Val

420 425 430

Thr Leu

<210> 28

<211> 45

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<223> dimerization unit

<400> 28

gagcccaaat cttgtgacac acctcccccg tgcccaaggt gccca 45

<210> 29

<211> 171

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> Met e 1 antigenic Unit

<400> 29

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> 30

<211> 274

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> Met e 1 antigenic Unit

<400> 30

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

1 5 10 15

Gln Gln Asn Lys Glu Ala Asn Asn Arg Ala Glu Lys Ser Glu Glu Glu

20 25 30

Val Phe Gly Leu Gln Lys Lys Leu Gln Gln Leu Glu Asn Asp Leu Asp

35 40 45

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

50 55 60

Asp Lys Ala Leu Ser Asn Ala Glu Gly Glu Val Ala Ala Leu Asn Arg

65 70 75 80

Arg Ile Gln Leu Leu Glu Glu Asp Leu Glu Arg Ser Glu Glu Arg Leu

85 90 95

Asn Thr Ala Thr Thr Lys Leu Ala Glu Ala Ser Gln Ala Ala Asp Glu

100 105 110

Ser Glu Arg Met Arg Lys Val Leu Glu Asn Arg Ser Leu Ser Asp Glu

115 120 125

Glu Arg Met Asp Ala Leu Glu Asn Gln Leu Lys Glu Ala Arg Phe Leu

130 135 140

Ala Glu Glu Ala Asp Arg Lys Tyr Asp Glu Val Ala Arg Lys Leu Ala

145 150 155 160

Met Val Glu Ala Asp Leu Glu Arg Ala Glu Glu Arg Ala Glu Thr Gly

165 170 175

Glu Ser Lys Ile Val Glu Leu Glu Glu Glu Leu Arg Val Val Gly Asn

180 185 190

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

195 200 205

Glu Ala Tyr Lys Glu Gln Ile Lys Thr Leu Thr Asn Lys Leu Lys Ala

210 215 220

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

225 230 235 240

Lys Glu Val Asp Arg Leu Glu Asp Glu Leu Val Asn Glu Lys Glu Lys

245 250 255

Tyr Lys Ser Ile Thr Asp Glu Leu Asp Gln Thr Phe Ser Glu Leu Ser

260 265 270

Gly Tyr

<210> 31

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

The sequence shown in <223> may be repeated m times, where m is an integer greater than or equal to 1

<400> 31

Glu Ala Ala Lys Gly Ser

1 5

<210> 32

<211> 434

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5024?

<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 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

<210> 33

<211> 153

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> human IL2

<400> 33

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> 34

<211> 413

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5030

<400> 34

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 Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln

165 170 175

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

180 185 190

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

195 200 205

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

210 215 220

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

225 230 235 240

Gly Leu Lys Glu Val Asp Arg Leu Glu Asp Glu Leu Val Asn Glu Lys

245 250 255

Glu Lys Tyr Lys Ser Ile Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

260 265 270

Ala Tyr Lys Glu Gln Ile Lys Thr Leu Thr Asn Lys Leu Lys Ala Ala

275 280 285

Glu Ala Arg Ala Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn

290 295 300

Gln Leu Lys Glu Ala Arg Phe Leu Ala Glu Glu Ala Asp Arg Lys Tyr

305 310 315 320

Asp Glu Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ala Leu

325 330 335

Asn Arg Arg Ile Gln Leu Leu Glu Glu Asp Leu Glu Arg Ser Glu Glu

340 345 350

Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Leu Asp Gln Val

355 360 365

Gln Glu Ser Leu Leu Lys Ala Asn Asn Gln Leu Val Glu Lys Asp Gly

370 375 380

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Gln Gln Asn Lys Glu Ala

385 390 395 400

Asn Asn Arg Ala Glu Lys Ser Glu Glu Glu Val His Asn

405 410

<210> 35

<211> 516

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5079?

<400> 35

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 Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln

165 170 175

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

180 185 190

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

195 200 205

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

210 215 220

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

225 230 235 240

Gly Leu Met Lys Leu Glu Lys Asp Asn Ala Met Asp Arg Ala Asp Thr

245 250 255

Leu Glu Gln Gln Asn Lys Glu Ala Asn Asn Arg Ala Glu Lys Ser Glu

260 265 270

Glu Glu Val Phe Gly Leu Gln Lys Lys Leu Gln Gln Leu Glu Asn Asp

275 280 285

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

290 295 300

Glu Lys Asp Lys Ala Leu Ser Asn Ala Glu Gly Glu Val Ala Ala Leu

305 310 315 320

Asn Arg Arg Ile Gln Leu Leu Glu Glu Asp Leu Glu Arg Ser Glu Glu

325 330 335

Arg Leu Asn Thr Ala Thr Thr Lys Leu Ala Glu Ala Ser Gln Ala Ala

340 345 350

Asp Glu Ser Glu Arg Met Arg Lys Val Leu Glu Asn Arg Ser Leu Ser

355 360 365

Asp Glu Glu Arg Met Asp Ala Leu Glu Asn Gln Leu Lys Glu Ala Arg

370 375 380

Phe Leu Ala Glu Glu Ala Asp Arg Lys Tyr Asp Glu Val Ala Arg Lys

385 390 395 400

Leu Ala Met Val Glu Ala Asp Leu Glu Arg Ala Glu Glu Arg Ala Glu

405 410 415

Thr Gly Glu Ser Lys Ile Val Glu Leu Glu Glu Glu Leu Arg Val Val

420 425 430

Gly Asn Asn Leu Lys Ser Leu Glu Val Ser Glu Glu Lys Ala Asn Gln

435 440 445

Arg Glu Glu Ala Tyr Lys Glu Gln Ile Lys Thr Leu Thr Asn Lys Leu

450 455 460

Lys Ala Ala Glu Ala Arg Ala Glu Phe Ala Glu Arg Ser Val Gln Lys

465 470 475 480

Leu Gln Lys Glu Val Asp Arg Leu Glu Asp Glu Leu Val Asn Glu Lys

485 490 495

Glu Lys Tyr Lys Ser Ile Thr Asp Glu Leu Asp Gln Thr Phe Ser Glu

500 505 510

Leu Ser Gly Tyr

515

<210> 36

<211> 462

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<223> human IL2

<400> 36

atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacaaacagt 60

gcacctactt caagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 120

ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 180

acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 240

gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 300

agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 360

acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 420

tggattacct tttgtcaaag catcatctca acactgactt ga 462

<210> 37

<211> 56

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5001b

<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 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 Ala Gln Pro

50 55

<210> 38

<211> 279

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5002b

<400> 38

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 Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala Glu Leu Lys

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln

165 170 175

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

180 185 190

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

Gln Asp Ala Glu Ala Gln Pro

275

<210> 39

<211> 451

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5004b

<400> 39

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

Ala Gln Pro

450

<210> 40

<211> 363

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5006b

<400> 40

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

245 250 255

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

260 265 270

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

275 280 285

Ser Arg Trp Gln Gln Gly Asn Ile Phe Ser Cys Ser Val Met His Glu

290 295 300

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

305 310 315 320

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

325 330 335

Val Gly Trp Tyr Arg Ser Pro Phe Ser Arg Val Val His Leu Tyr Arg

340 345 350

Asn Gly Lys Asp Gln Asp Ala Glu Ala Gln Pro

355 360

<210> 41

<211> 277

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> VB5012b

<400> 41

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 Ala Gln Pro

275

<210> 42

<211> 180

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<223> trimerization unit

<400> 42

gctgggcagg tgaggatctg ggccacatac cagaccatgc tggacaagat ccgggaggtg 60

ccggagggct ggctcatctt tgtggccgag agggaagagc tctatgtacg cgttagaaat 120

ggcttccgga aggtgctgct ggaggcccgg acagccctcc cgagaggcac gggcaatgag 180

<210> 43

<211> 27

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> trimerization unit

<400> 43

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> 44

<211> 117

<212> DNA

<213> Homo sapiens (Homo sapiens)

<220>

<221> misc_feature

<223> tetramerization Unit

<400> 44

aagcctctgg acggagagta tttcactctc cagatccggg gccccgaaag gttcgaaatg 60

ttccgggagc ttaacgaggc cttggagctg aaagacgcac aggccggaaa ggaaccg 117

<210> 45

<211> 8

<212> PRT

<213> Homo sapiens (Homo sapiens)

<220>

<221> MISC_FEATURE

<223> OVA (257-264)

<400> 45

Ser Ile Ile Asn Phe Glu Lys Leu

1 5

<210> 46

<211> 15

<212> PRT

<213> Homo sapiens (Homo sapiens)

<220>

<221> MISC_FEATURE

<223> dimerization unit

<400> 46

Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

1 5 10 15

<210> 47

<211> 333

<212> DNA

<213> Homo sapiens (Homo sapiens)

<220>

<221> misc_feature

<223> IgM hinge

<400> 47

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> 48

<211> 18

<212> PRT

<213> Homo sapiens (Homo sapiens)

<220>

<221> MISC_FEATURE

<223> murine Ig VH Signal peptide

<400> 48

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

1 5 10 15

Val Gln

<210> 49

<211> 246

<212> PRT

<213> Homo sapiens (Homo sapiens)

<220>

<221> MISC_FEATURE

<223> murine scFv anti-DEC 205

<400> 49

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> 50

<211> 18

<212> PRT

<213> Homo sapiens (Homo sapiens)

<220>

<221> MISC_FEATURE

<223> native leader murine IL-10?

<400> 50

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

1 5 10 15

Arg Ile

<210> 51

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 51

Gly Gly Gly Gly Ser Gly Gly Gly Ser Ser

1 5 10

<210> 52

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 52

Gly Gly Gly Ser Gly

1 5

<210> 53

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 53

Gly Gly Gly Gly Ser

1 5

<210> 54

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 54

Ser Gly Ser Ser Gly Ser

1 5

<210> 55

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 55

Gly Gly Ser Gly Gly

1 5

<210> 56

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 56

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 57

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 57

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 58

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 58

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 59

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 59

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25

<210> 60

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 60

Gly Gly Gly Ser Ser

1 5

<210> 61

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 61

Gly Gly Gly Ser Ser Gly Gly Gly Ser Ser

1 5 10

<210> 62

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 62

Gly Gly Gly Ser Ser Gly Gly Gly Ser Ser Gly Gly Gly Ser Ser

1 5 10 15

<210> 63

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 63

Gly Gly Gly Ser Ser Gly Gly Gly Ser Ser Gly Gly Gly Ser Ser Gly

1 5 10 15

Gly Gly Ser Ser

20

<210> 64

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 64

Gly Gly Gly Ser Ser Gly Gly Gly Ser Ser Gly Gly Gly Ser Ser Gly

1 5 10 15

Gly Gly Ser Ser Gly Gly Gly Ser Ser

20 25

<210> 65

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 65

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10

<210> 66

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 66

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

<210> 67

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 67

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Gly Ser Gly

20

<210> 68

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 68

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Gly Ser Gly Gly Gly Gly Ser Gly

20 25

<210> 69

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 69

Ser Gly Ser Ser Gly Ser Ser Gly Ser Ser Gly Ser

1 5 10

<210> 70

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 70

Ser Gly Ser Ser Gly Ser Ser Gly Ser Ser Gly Ser Ser Gly Ser Ser

1 5 10 15

Gly Ser

<210> 71

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 71

Ser Gly Ser Ser Gly Ser Ser Gly Ser Ser Gly Ser Ser Gly Ser Ser

1 5 10 15

Gly Ser Ser Gly Ser Ser Gly Ser

20

<210> 72

<211> 30

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 72

Ser Gly Ser Ser Gly Ser Ser Gly Ser Ser Gly Ser Ser Gly Ser Ser

1 5 10 15

Gly Ser Ser Gly Ser Ser Gly Ser Ser Gly Ser Ser Gly Ser

20 25 30

<210> 73

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 73

Leu Gly Gly Gly Ser

1 5

<210> 74

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 74

Gly Leu Gly Gly Ser

1 5

<210> 75

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 75

Gly Gly Leu Gly Ser

1 5

<210> 76

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 76

Gly Gly Gly Leu Ser

1 5

<210> 77

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 77

Gly Gly Gly Gly Leu

1 5

<210> 78

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 78

Leu Gly Gly Ser Gly

1 5

<210> 79

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 79

Gly Leu Gly Ser Gly

1 5

<210> 80

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 80

Gly Gly Leu Ser Gly

1 5

<210> 81

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 81

Gly Gly Gly Leu Gly

1 5

<210> 82

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 82

Gly Gly Gly Ser Leu

1 5

<210> 83

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 83

Leu Gly Gly Ser Ser

1 5

<210> 84

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 84

Gly Leu Gly Ser Ser

1 5

<210> 85

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 85

Gly Gly Leu Ser Ser

1 5

<210> 86

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 86

Leu Gly Leu Gly Ser

1 5

<210> 87

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 87

Gly Leu Gly Leu Ser

1 5

<210> 88

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 88

Gly Leu Leu Gly Ser

1 5

<210> 89

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 89

Leu Gly Gly Leu Ser

1 5

<210> 90

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 90

Gly Leu Gly Gly Leu

1 5

<210> 91

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 91

Gly Leu Gly Gly Leu Gly Leu Gly Gly Leu

1 5 10

<210> 92

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 92

Gly Leu Gly Gly Leu Gly Leu Gly Gly Leu Gly Leu Gly Gly Leu

1 5 10 15

<210> 93

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 93

Gly Leu Gly Gly Leu Gly Leu Gly Gly Leu Gly Leu Gly Gly Leu Gly

1 5 10 15

Leu Gly Gly Leu

20

<210> 94

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 94

Gly Leu Gly Gly Leu Gly Leu Gly Gly Leu Gly Leu Gly Gly Leu Gly

1 5 10 15

Leu Gly Gly Leu Gly Leu Gly Gly Leu

20 25

<210> 95

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 95

Leu Gly Leu Ser Gly

1 5

<210> 96

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 96

Gly Leu Leu Ser Gly

1 5

<210> 97

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 97

Gly Gly Leu Ser Leu

1 5

<210> 98

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 98

Gly Gly Leu Leu Gly

1 5

<210> 99

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 99

Gly Leu Gly Ser Leu

1 5

<210> 100

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 100

Leu Gly Leu Ser Ser

1 5

<210> 101

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 101

Gly Gly Leu Leu Ser

1 5

<210> 102

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 102

Leu Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 103

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 103

Gly Leu Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 104

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 104

Gly Gly Leu Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 105

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 105

Gly Gly Gly Leu Ser Gly Gly Gly Gly Ser

1 5 10

<210> 106

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 106

Gly Gly Gly Gly Leu Gly Gly Gly Gly Ser

1 5 10

<210> 107

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 107

Leu Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10

<210> 108

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 108

Gly Leu Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10

<210> 109

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 109

Gly Gly Leu Ser Gly Gly Gly Gly Ser Gly

1 5 10

<210> 110

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 110

Gly Gly Gly Leu Gly Gly Gly Gly Ser Gly

1 5 10

<210> 111

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 111

Gly Gly Gly Ser Leu Gly Gly Gly Ser Gly

1 5 10

<210> 112

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 112

Leu Gly Gly Ser Ser Gly Gly Gly Ser Ser

1 5 10

<210> 113

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 113

Gly Leu Gly Ser Ser Gly Gly Gly Ser Ser

1 5 10

<210> 114

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 114

Gly Gly Leu Ser Ser Gly Gly Gly Ser Ser

1 5 10

<210> 115

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 115

Gly Gly Gly Leu Ser Gly Gly Gly Ser Ser

1 5 10

<210> 116

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 116

Gly Gly Gly Ser Leu Gly Gly Gly Ser Ser

1 5 10

<210> 117

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 117

Leu Gly Gly Gly Ser Leu Gly Gly Gly Ser

1 5 10

<210> 118

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 118

Gly Leu Gly Gly Ser Gly Leu Gly Gly Ser

1 5 10

<210> 119

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 119

Gly Gly Leu Gly Ser Gly Gly Leu Gly Ser

1 5 10

<210> 120

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 120

Gly Gly Gly Leu Ser Gly Gly Gly Leu Ser

1 5 10

<210> 121

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 121

Gly Gly Gly Gly Leu Gly Gly Gly Gly Leu

1 5 10

<210> 122

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 122

Leu Gly Gly Ser Gly Leu Gly Gly Ser Gly

1 5 10

<210> 123

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 123

Gly Leu Gly Ser Gly Gly Leu Gly Ser Gly

1 5 10

<210> 124

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 124

Gly Gly Leu Ser Gly Gly Gly Leu Ser Gly

1 5 10

<210> 125

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 125

Gly Gly Gly Leu Gly Gly Gly Gly Leu Gly

1 5 10

<210> 126

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 126

Gly Gly Gly Ser Leu Gly Gly Gly Ser Leu

1 5 10

<210> 127

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 127

Leu Gly Gly Ser Ser Leu Gly Gly Ser Ser

1 5 10

<210> 128

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 128

Gly Leu Gly Ser Ser Gly Leu Gly Ser Ser

1 5 10

<210> 129

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 129

Gly Gly Leu Ser Ser Gly Gly Leu Ser Ser

1 5 10

<210> 130

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 130

Gly Ser Gly Gly Gly Ala

1 5

<210> 131

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 131

Gly Ser Gly Gly Gly Ala Gly Ser Gly Gly Gly Ala

1 5 10

<210> 132

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 132

Gly Ser Gly Gly Gly Ala Gly Ser Gly Gly Gly Ala Gly Ser Gly Gly

1 5 10 15

Gly Ala

<210> 133

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 133

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> 134

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 134

Gly Glu Asn Leu Tyr Phe Gln Ser Gly Gly

1 5 10

<210> 135

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 135

Ser Gly Gly Gly Ser Ser Gly Gly Gly Ser

1 5 10

<210> 136

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 136

Ser Ser Gly Gly Gly Ser Ser Gly Gly Gly

1 5 10

<210> 137

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 137

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

1 5 10

<210> 138

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 138

Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser

1 5 10

<210> 139

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 139

Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly

1 5 10

<210> 140

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 140

Gly Gly Gly Ser Ser Ser

1 5

<210> 141

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 141

Gly Leu Gly Gly Leu Ala Ala Ala

1 5

<210> 142

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 142

Lys Pro Glu Pro Lys Pro Ala Pro Ala Pro Lys Pro

1 5 10

<210> 143

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 143

Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala

1 5 10

<210> 144

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 144

Glu Ala Ala Ala Lys

1 5

<210> 145

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 145

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

1 5 10

<210> 146

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 146

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

1 5 10 15

<210> 147

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 147

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu

1 5 10 15

Ala Ala Ala Lys

20

<210> 148

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 148

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu

1 5 10 15

Ala Ala Ala Lys Glu Ala Ala Ala Lys

20 25

<210> 149

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 149

Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu Pro

1 5 10 15

<210> 150

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 150

Ser Ala Cys Tyr Cys Glu Leu Ser

1 5

<210> 151

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 151

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu Gly Gly Leu

1 5 10 15

<210> 152

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 152

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

1 5 10

<210> 153

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 153

Gly Pro Gly Pro Gly

1 5

<210> 154

<211> 36

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 154

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> 155

<211> 36

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 155

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> 156

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> dimerization unit

<400> 156

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

1 5 10

<210> 157

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 157

His Cys Leu Gly Lys Trp Leu Gly His Pro Asp Lys Phe

1 5 10

<210> 158

<211> 29

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 158

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> 159

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 159

Asn Thr Trp Thr Thr Cys Gln Ser Ile Ala Phe Pro Ser Lys

1 5 10

<210> 160

<211> 30

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 160

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> 161

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<400> 161

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> 162

<211> 36

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 162

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> 163

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 163

Gly Leu Ser Gly Leu

1 5

<210> 164

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> linker, repeat: m is 1 or greater

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<400> 164

Gly Gly Gly Gly Ser

1 5

<210> 165

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> linker, repeat: m is 1 or greater

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<400> 165

Glu Ala Ala Ala Lys

1 5

<210> 166

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> linker, repeat: m is 1 or greater

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<400> 166

Glu Ala Ala Ala Lys Gly Ser

1 5

<210> 167

<211> 17

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 167

Gly Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu Pro

1 5 10 15

Gly

<210> 168

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> joint

<400> 168

His Glu Tyr Gly Ala Glu Ala Leu Glu Arg Ala Gly

1 5 10

<210> 169

<211> 160

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> murine IL-10

<400> 169

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> 170

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> Natural leader murine SG3A2

<400> 170

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> 171

<211> 70

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> murine SG3A2?

<400> 171

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> 172

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> natural leader murine VSIG-3?

<400> 172

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> 173

<211> 218

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> murine VSIG-3 extracellular Domain

<400> 173

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> 174

<211> 35

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> native leader murine CTLA4?

<400> 174

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> 175

<211> 126

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> murine CTLA-4 extracellular domain

<400> 175

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> 176

<211> 29

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> Natural leader murine TGF (beta) 1

<400> 176

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> 177

<211> 361

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> murine TGF (beta) 1 proprotein

<400> 177

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> 178

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> native leader murine PD-1?

<400> 178

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> 179

<211> 145

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> murine PD-1 extracellular Domain

<400> 179

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> 180

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> MOG(35-55)

<400> 180

Met Glu Val Gly Trp Tyr Arg Ser Pro Phe Ser Arg Val Val His Leu

1 5 10 15

Tyr Arg Asn Gly Lys

20

<210> 181

<211> 37

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> MOG(27-63)

<400> 181

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> 182

<211> 37

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> MOG with A/Q exchange (27-63)

<400> 182

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> 183

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> GAD65(202-221)

<400> 183

Thr Asn Met Phe Thr Tyr Glu Ile Ala Pro Val Phe Val Leu Leu Glu

1 5 10 15

Tyr Val Thr Leu

20

<210> 184

<211> 274

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> Met e 1

<400> 184

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

1 5 10 15

Gln Gln Asn Lys Glu Ala Asn Asn Arg Ala Glu Lys Ser Glu Glu Glu

20 25 30

Val His Asn Leu Gln Lys Arg Met Gln Gln Leu Glu Asn Asp Leu Asp

35 40 45

Gln Val Gln Glu Ser Leu Leu Lys Ala Asn Asn Gln Leu Val Glu Lys

50 55 60

Asp Lys Ala Leu Ser Asn Ala Glu Gly Glu Val Ala Ala Leu Asn Arg

65 70 75 80

Arg Ile Gln Leu Leu Glu Glu Asp Leu Glu Arg Ser Glu Glu Arg Leu

85 90 95

Asn Thr Ala Thr Thr Lys Leu Ala Glu Ala Ser Gln Ala Ala Asp Glu

100 105 110

Ser Glu Arg Met Arg Lys Val Leu Glu Asn Arg Ser Leu Ser Asp Glu

115 120 125

Glu Arg Met Asp Ala Leu Glu Asn Gln Leu Lys Glu Ala Arg Phe Leu

130 135 140

Ala Glu Glu Ala Asp Arg Lys Tyr Asp Glu Val Ala Arg Lys Leu Ala

145 150 155 160

Met Val Glu Ala Asp Leu Glu Arg Ala Glu Glu Arg Ala Glu Thr Gly

165 170 175

Glu Ser Lys Ile Val Glu Leu Glu Glu Glu Leu Arg Val Val Gly Asn

180 185 190

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

195 200 205

Glu Ala Tyr Lys Glu Gln Ile Lys Thr Leu Thr Asn Lys Leu Lys Ala

210 215 220

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

225 230 235 240

Lys Glu Val Asp Arg Leu Glu Asp Glu Leu Val Asn Glu Lys Glu Lys

245 250 255

Tyr Lys Ser Ile Thr Asp Glu Leu Asp Gln Thr Phe Ser Glu Leu Ser

260 265 270

Gly Tyr

<210> 185

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> Met e 1 (16-35)

<400> 185

Glu Gln Gln Asn Lys Glu Ala Asn Asn Arg Ala Glu Lys Ser Glu Glu

1 5 10 15

Glu Val His Asn

20

<210> 186

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> Met e 1 (46-65)

<400> 186

Asp Leu Asp Gln Val Gln Glu Ser Leu Leu Lys Ala Asn Asn Gln Leu

1 5 10 15

Val Glu Lys Asp

20

<210> 187

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> Met e 1 (76-95)

<400> 187

Ala Ala Leu Asn Arg Arg Ile Gln Leu Leu Glu Glu Asp Leu Glu Arg

1 5 10 15

Ser Glu Glu Arg

20

<210> 188

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> Met e 1 (136-155)

<400> 188

Asn Gln Leu Lys Glu Ala Arg Phe Leu Ala Glu Glu Ala Asp Arg Lys

1 5 10 15

Tyr Asp Glu Val

20

<210> 189

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> Met e 1 (210-230)

<400> 189

Ala Tyr Lys Glu Gln Ile Lys Thr Leu Thr Asn Lys Leu Lys Ala Ala

1 5 10 15

Glu Ala Arg Ala Glu

20

<210> 190

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> Met e 1 (241-260)

<400> 190

Lys Glu Val Asp Arg Leu Glu Asp Glu Leu Val Asn Glu Lys Glu Lys

1 5 10 15

Tyr Lys Ser Ile

20

<210> 191

<211> 284

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> Pan b 1

<400> 191

Met Asp Ala Ile Lys Lys Lys Met Gln Ala Met Lys Leu Glu Lys Asp

1 5 10 15

Asn Ala Met Asp Arg Ala Asp Thr Leu Glu Gln Gln Asn Lys Glu Ala

20 25 30

Asn Asn Arg Ala Glu Lys Ser Glu Glu Glu Val Phe Gly Leu Gln Lys

35 40 45

Lys Leu Gln Gln Leu Glu Asn Asp Leu Asp Ser Val Gln Glu Ala Leu

50 55 60

Leu Lys Ala Asn Gln His Leu Glu Glu Lys Asp Lys Ala Leu Ser Asn

65 70 75 80

Ala Glu Gly Glu Val Ala Ala Leu Asn Arg Arg Ile Gln Leu Leu Glu

85 90 95

Glu Asp Leu Glu Arg Ser Glu Glu Arg Leu Asn Thr Ala Thr Thr Lys

100 105 110

Leu Ala Glu Ala Ser Gln Ala Ala Asp Glu Ser Glu Arg Met Arg Lys

115 120 125

Val Leu Glu Asn Arg Ser Leu Ser Asp Glu Glu Arg Met Asp Ala Leu

130 135 140

Glu Asn Gln Leu Lys Glu Ala Arg Phe Leu Ala Glu Glu Ala Asp Arg

145 150 155 160

Lys Tyr Asp Glu Val Ala Arg Lys Leu Ala Met Val Glu Ala Asp Leu

165 170 175

Glu Arg Ala Glu Glu Arg Ala Glu Thr Gly Glu Ser Lys Ile Val Glu

180 185 190

Leu Glu Glu Glu Leu Arg Val Val Gly Asn Asn Leu Lys Ser Leu Glu

195 200 205

Val Ser Glu Glu Lys Ala Asn Gln Arg Glu Glu Ala Tyr Lys Glu Gln

210 215 220

Ile Lys Thr Leu Thr Asn Lys Leu Lys Ala Ala Glu Ala Arg Ala Glu

225 230 235 240

Phe Ala Glu Arg Ser Val Gln Lys Leu Gln Lys Glu Val Asp Arg Leu

245 250 255

Glu Asp Glu Leu Val Asn Glu Lys Glu Lys Tyr Lys Ser Ile Thr Asp

260 265 270

Glu Leu Asp Gln Thr Phe Ser Glu Leu Ser Gly Tyr

275 280

<210> 192

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> Pan b 1 (26-45)

<400> 192

Glu Gln Gln Asn Lys Glu Ala Asn Asn Arg Ala Glu Lys Ser Glu Glu

1 5 10 15

Glu Val Phe Gly

20

<210> 193

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> Pan b 1 (56-75)

<400> 193

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

1 5 10 15

Glu Glu Lys Asp

20

<210> 194

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> Pan b 1 (86-105)

<400> 194

Ala Ala Leu Asn Arg Arg Ile Gln Leu Leu Glu Glu Asp Leu Glu Arg

1 5 10 15

Ser Glu Glu Arg

20

<210> 195

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> Pan b 1 (146-165)

<400> 195

Asn Gln Leu Lys Glu Ala Arg Phe Leu Ala Glu Glu Ala Asp Arg Lys

1 5 10 15

Tyr Asp Glu Val

20

<210> 196

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> Pan b 1 (220-240)

<400> 196

Ala Tyr Lys Glu Gln Ile Lys Thr Leu Thr Asn Lys Leu Lys Ala Ala

1 5 10 15

Glu Ala Arg Ala Glu

20

<210> 197

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> Pan b 1 (251-270)

<400> 197

Lys Glu Val Asp Arg Leu Glu Asp Glu Leu Val Asn Glu Lys Glu Lys

1 5 10 15

Tyr Lys Ser Ile

20

<210> 198

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> Natural leader sequence human CCL3L1

<400> 198

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> 199

<211> 70

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> human CCL3L1

<400> 199

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> 200

<211> 36

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<223> hinge region 1 from human IgG3

<400> 200

gagctcaaaa ccccacttgg tgacacaact cacaca 36

<210> 201

<211> 30

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<223> hinge region 4 from human IgG3

<400> 201

ggcggtggaa gcagcggagg tggaagtgga 30

<210> 202

<211> 107

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> human IgG3 CH3 Domain

<400> 202

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

1 5 10 15

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

20 25 30

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

35 40 45

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

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

65 70 75 80

Asn Ile Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

100 105

<210> 203

<211> 321

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<223> human IgG3 CH3 Domain

<400> 203

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> 204

<211> 15

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<223> joint

<400> 204

ggcctcggtg gcctg 15

<210> 205

<211> 390

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> human transforming growth factor beta-1

<400> 205

Met Pro Pro Ser Gly Leu Arg Leu Leu Pro 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> 206

<211> 442

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> human transforming growth factor beta-2

<400> 206

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> 207

<211> 309

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> human transforming growth factor beta-3

<400> 207

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

305

<210> 208

<211> 1173

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<223> human transforming growth factor beta-1

<400> 208

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> 209

<211> 1329

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<223> human transforming growth factor beta 2

<400> 209

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> 210

<211> 930

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<223> human transforming growth factor beta 3

<400> 210

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> 211

<211> 178

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> human interleukin-10 isoform 1

<400> 211

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> 212

<211> 537

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<223> human interleukin 10 (IL 10)

<400> 212

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> 213

<211> 93

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> human secretoglobin family 3A member 2 precursor (SCGB 3A 2)

<400> 213

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> 214

<211> 282

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<223> human secretoglobin family 3A member 2 (SCGB 3A 2)

<400> 214

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> 215

<211> 241

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> IGSF11 (VSIG3) (ECD)

<400> 215

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> 216

<211> 723

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<223> IGSF11 (VSIG3) (ECD)

<400> 216

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> 217

<211> 161

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> CTLA4_human cytotoxic T lymphocyte protein 4 (ECD)

<400> 217

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> 218

<211> 483

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<223> human cytotoxic T lymphocyte-associated protein 4 (CTLA 4) (ECD)

<400> 218

atggcttgcc ttggatttca gcggcacaag gctcagctga acctggctac caggacctgg 60

ccctgcactc tcctgttttt tcttctcttc atccctgtct tctgcaaagc aatgcacgtg 120

gcccagcctg ctgtggtact ggccagcagc cgaggcatcg ccagctttgt gtgtgagtat 180

gcatctccag gcaaagccac tgaggtccgg gtgacagtgc ttcggcaggc tgacagccag 240

gtgactgaag tctgtgcggc aacctacatg atggggaatg agttgacctt cctagatgat 300

tccatctgca cgggcacctc cagtggaaat caagtgaacc tcactatcca aggactgagg 360

gccatggaca cgggactcta catctgcaag gtggagctca tgtacccacc gccatactac 420

ctgggcatag gcaacggaac ccagatttat gtaattgatc cagaaccgtg cccagattct 480

gac 483

<210> 219

<211> 170

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<223> human apoptosis protein 1 PDCD1 (PD-1) (ECD)

<400> 219

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> 220

<211> 510

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<223> human programmed cell death 1 PDCD1 (PD-1) (ECD)

<400> 220

atgcagatcc cacaggcgcc ctggccagtc gtctgggcgg tgctacaact gggctggcgg 60

ccaggatggt tcttagactc cccagacagg ccctggaacc cccccacctt ctccccagcc 120

ctgctcgtgg tgaccgaagg ggacaacgcc accttcacct gcagcttctc caacacatcg 180

gagagcttcg tgctaaactg gtaccgcatg agccccagca accagacgga caagctggcc 240

gccttccccg aggaccgcag ccagcccggc caggactgcc gcttccgtgt cacacaactg 300

cccaacgggc gtgacttcca catgagcgtg gtcagggccc ggcgcaatga cagcggcacc 360

tacctctgtg gggccatctc cctggccccc aaggcgcaga tcaaagagag cctgcgggca 420

gagctcaggg tgacagagag aagggcagaa gtgcccacag cccaccccag cccctcaccc 480

aggccagccg gccagttcca aaccctggtg 510

Claims

1. A tolerance-inducing construct comprising:

ii) a polypeptide encoded by a nucleic acid sequence as defined in (i); or (b)

2. The tolerance-inducing construct of claim 1, wherein the targeting unit interacts with a surface molecule on the antigen presenting cell without activating the cell.

3. The tolerance-inducing construct of any one of the preceding claims, wherein the construct comprises multimerization units.

4. The tolerance-inducing construct of any one of the preceding claims, wherein the construct comprises dimerization units and/or wherein the multimeric protein is a dimeric protein.

5. The tolerance-inducing construct of any one of the preceding claims, wherein the targeting unit comprises or consists of a moiety that binds to a receptor selected from tgfβr1, tgfβr2, tgfβr3, IL10R, IL-10RA and IL10-RB, IL2R, IL4R, IL6R, IL11R and IL13R, IL27R, IL35R, IL37R, CCR7, CD11b, CD11c, CD103, CD14, CD36, CD205, CD109, VISTA, MARCO, MHCII, MHCII, CD83, SIGLEC, MGL, CD80, CD86, clec9A, clec12A, clec12B, DCIR2, langerin, MR, DC-Sign, treml4, dectin-1, PDL2 and HVEM.

6. The tolerance-inducing construct of claim 5, wherein the moiety is an antibody or a portion thereof, e.g., an scFv.

7. The tolerance-inducing construct of any one of claims 5 to 6, wherein the moiety is an antibody specific for CD205, or a portion of an antibody specific for CD205, such as a scFv.

8. The tolerance-inducing construct of claim 5, wherein the moiety is a synthetic ligand.

9. The tolerance inducing construct of any one of claim 5, wherein the moiety is a natural ligand.

10. The tolerance-inducing construct of claim 9, wherein the natural ligand is selected from the group consisting of tgfβ, IL-10, IL2, IL4, IL6, IL11, IL13, IL27, IL35, IL37, CCL19, CCL21, ICAM-1, keratin, VSIG-3, SCGB3A2, CTLA-4, PD-1, and BTLA.

11. The tolerance-inducing construct of claim 9, wherein the natural ligand is selected from the group consisting of an extracellular domain of CTLA-4, an extracellular domain of PD-1, and an extracellular domain of BTLA.

12. The tolerance-inducing construct of claim 9, wherein the natural ligand is selected from IL-10, tgfβ, SCGB3A2 and VSIG-3.

13. The tolerance-inducing construct of any one of the preceding claims, wherein the antigenic unit comprises a T cell epitope of a self antigen or an allergen or an alloantigen or a xenogeneic antigen.

14. The tolerance-inducing construct of any one of the preceding claims, wherein the antigenic unit comprises a plurality of T cell epitopes of a self antigen or an allergen or an alloantigen or a xenogeneic antigen.

15. The tolerance-inducing construct of any one of the preceding claims, wherein the antigenic unit comprises a plurality of T cell epitopes of a plurality of different autoantigens or a plurality of different allergens or a plurality of different alloantigens.

16. The tolerance-inducing construct of any one of the preceding claims, wherein the T cell epitope is 7 to about 200 amino acids in length, 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 amino acids or 15 to 30 amino acids or 20 to 75 amino acids or 25 to 50 amino acids.

17. Tolerance-inducing construct according to any one of the preceding claims, wherein the T cell epitope has a length suitable for specific presentation on MHC class I or MHC class II, for example a length of 7 to 11 amino acids for MHC class I presentation, or for example a length of 15 amino acids for MHC class II presentation.

18. The tolerance-inducing construct of any one of the preceding claims, wherein the antigenic unit comprises up to 3500 amino acids, e.g. 60 to 3500 amino acids, e.g. about 80 or about 100 or about 150 amino acids to about 3000 amino acids, e.g. about 200 to about 2500 amino acids, e.g. about 300 to about 2000 amino acids or about 400 to about 1500 amino acids or about 500 to about 1000 amino acids.

19. The tolerance-inducing construct of any one of the preceding claims, 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.

20. The tolerance-inducing construct of any one of the preceding claims, wherein the antigenic unit comprises a plurality of T cell epitopes separated by T cell epitope linkers.

21. The tolerance-inducing construct of claim 20, wherein the T cell epitope linker is non-immunogenic and preferably also flexible.

22. The tolerance-inducing construct of any one of claims 20 to 21, wherein the T cell epitope linker consists of 4 to 20 amino acids, for example, of 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.

23. The tolerance-inducing construct of any one of the preceding claims, wherein the construct is a personalized tolerance-inducing construct.

24. The tolerance-inducing construct of any one of the preceding claims, wherein the construct is an off-the-shelf tolerance-inducing construct.

25. The tolerance-inducing construct of any one of the preceding claims, wherein the antigenic unit comprises one or more T cell epitopes of an allergen.

26. The tolerance-inducing construct of claim 25, wherein the allergen is selected from the group consisting of food allergens, bee venom allergens, latex allergens, dust mite allergens, cockroach allergens, mold allergens, fungus allergens, trichogenous animal allergens, pollen allergens, and allergens contained in a medicament.

27. The tolerance-inducing construct of any one of claims 1 to 24, wherein the antigenic unit comprises one or more T cell epitopes of a self antigen.

28. The tolerance-inducing construct of claim 27, wherein the self-antigen is selected from the group consisting of multiple sclerosis self-antigen, type 1 diabetes self-antigen, celiac disease self-antigen, rheumatoid arthritis self-antigen, chronic inflammatory demyelinating multiple radiculonephropathy self-antigen, hashimoto's thyroiditis self-antigen, pemphigus vulgaris self-antigen, thyroidemia self-antigen, grave's disease self-antigen, primary biliary cirrhosis self-antigen, myasthenia gravis self-antigen, insulin resistance diabetes self-antigen, and hemolytic anemia self-antigen.

29. The tolerance-inducing construct of any one of claims 1 to 24, wherein the antigenic unit comprises one or more T cell epitopes of an alloantigen or a xenogeneic antigen.

30. The tolerance inducing construct of any one of the preceding claims, wherein the dimerization unit comprises a hinge region.

31. The tolerance-inducing construct of claim 30, wherein the hinge region has the ability to form one or more covalent bonds.

32. The tolerance inducing construct of any one of claims 30 to 31, wherein the hinge region is Ig-derived.

33. The tolerance inducing construct of any one of claims 30 to 32, wherein the dimerization unit further comprises another domain that promotes dimerization.

34. The tolerance-inducing construct of claim 33, wherein the further domain is an immunoglobulin domain, preferably an immunoglobulin constant domain.

35. The tolerance-inducing construct of any one of claims 33 to 34, wherein the further domain is a carboxy-terminal C domain derived from IgG, preferably from IgG 3.

36. The tolerance inducing construct of any one of the preceding claims, wherein the dimerization unit further comprises a dimerization unit linker.

37. The tolerance-inducing construct of claim 36, wherein the dimerization unit linker connects the hinge region and the another dimerization promoting domain.

38. The tolerance inducing construct according to any one of the preceding claims, wherein the dimerization unit comprises hinge exon h1 and hinge exon h4, a dimerization unit linker and a CH3 domain of human IgG 3.

39. The tolerance-inducing construct of any one of the preceding claims, wherein the multimerization unit is a trimerization unit, e.g. a collagen-derived trimerization unit, e.g. a human collagen-derived trimerization domain, e.g. a human collagen-derived XVIII trimerization domain or a human collagen XV trimerization domain.

40. The tolerance inducing construct according to any one of the preceding claims, wherein the multimerization unit is a trimerization unit, is the C-terminal domain of T4 fibritin.

41. The tolerance-inducing construct according to any one of the preceding claims, wherein the multimerization unit is a tetramerization unit, such as a domain derived from P53.

42. The tolerance-inducing construct of any one of the preceding claims, wherein the construct is the polynucleotide (i).

43. The tolerance inducing construct of claim 42 wherein the polynucleotide is RNA or DNA, preferably DNA.

44. The tolerance inducing construct of any one of claims 42 to 43, wherein the polynucleotide further comprises a nucleotide sequence encoding a signal peptide.

45. A polynucleotide as defined in any one of claims 1 to 44.

46. A vector comprising the polynucleotide of claim 45.

47. A host cell comprising the polynucleotide of claim 45 or the vector of claim 46.

48. A polypeptide encoded by a nucleic acid as defined in any one of claims 1 to 44.

49. A dimeric protein consisting of two polypeptides according to claim 48.

50. The dimeric protein of claim 49, wherein the dimeric protein is a homodimeric protein.

51. A multimeric protein consisting of two or more polypeptides according to claim 48.

52. The polypeptide, dimeric protein or multimeric protein of any one of claims 48-51, wherein the targeting unit, dimerization unit and antigenic unit in the polypeptide or dimeric protein are in an N-terminal to C-terminal order.

53. A tolerance-inducing construct according to any one of claims 1 to 44, a polynucleotide according to claim 45, a vector according to claim 46, a polypeptide according to claim 48, a dimeric protein according to claim 49 or 50 or a multimeric protein according to claim 51 for use as a medicament.

54. A pharmaceutical composition comprising the tolerance-inducing construct of any one of claims 1 to 44 and a pharmaceutically acceptable carrier.

55. The pharmaceutical composition of claim 54, wherein the pharmaceutically acceptable carrier is selected from the group consisting of saline, buffered saline, PBS, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffer, and combinations thereof.

56. The tolerance-inducing construct of any one of claims 1 to 44 or the pharmaceutical composition of any one of claims 54 to 55 for use in the treatment of a condition involving an undesired immune response, for example for the treatment of autoimmune diseases, allergic diseases and graft rejection.

57. The tolerance inducing construct or pharmaceutical composition of claim 56 wherein the treatment is prophylactic or therapeutic.

58. A method of preparing a pharmaceutical composition of any one of claims 54 to 55, wherein the pharmaceutical composition comprises the polypeptide of claim 48 or the dimeric protein of any one of claims 49 or 50, wherein the method comprises the steps of:

a) Transfecting a cell with the polynucleotide of claim 45;

b) Culturing the cells;

c) Collecting and purifying the dimeric protein or polypeptide expressed from the cells; and

59. A method of preparing the pharmaceutical composition of any one of claims 54 to 55, wherein the pharmaceutical composition comprises the polynucleotide of claim 45, wherein the method comprises the steps of:

a) Preparing the polynucleotide;

b) Optionally cloning the polynucleotide into an expression vector; and

c) Mixing the polynucleotide obtained in step a) or the vector obtained in step b) with a pharmaceutically acceptable carrier.

60. A method of treating a subject suffering from a condition involving an undesired immune response, such as an autoimmune disease, allergic disease or graft rejection, or in need of prophylaxis of said condition, the method comprising administering to the subject a pharmaceutical composition as defined in any one of claims 54 to 55.

61. The pharmaceutical composition of any one of claims 54 to 55 for use in the treatment of a condition involving an undesired immune response, such as an autoimmune disease, allergic disease or graft rejection.

62. A method of improving tolerance to an autoantigen, allergen, alloantigen or xenogeneic antigen using a tolerance inducing construct according to any one of claims 1 to 44.

63. A method of improving tolerance of a subject to an autoantigen, allergen, alloantigen or xenogeneic antigen, the method comprising administering to the subject the tolerance-inducing construct of any one of claims 1 to 44 or the pharmaceutical composition of any one of claims 54 to 55.