CN117377483A

CN117377483A - Tolerance-inducing constructs and compositions and their use in the treatment of immune disorders

Info

Publication number: CN117377483A
Application number: CN202280034552.7A
Authority: CN
Inventors: 阿涅特·布伦斯维克·弗雷德里克森; H·迈塞特; A·T·H·伯萨斯; 斯蒂因·格拉纳姆; P·迪拉德
Original assignee: Necord Therapeutics Ltd
Current assignee: Necord Therapeutics Ltd
Priority date: 2021-05-10
Filing date: 2022-05-10
Publication date: 2024-01-09

Abstract

The present disclosure relates to tolerance-inducing constructs for inducing tolerance, e.g., by targeting the tolerance-inducing constructs to Antigen Presenting Cells (APCs). Polynucleotides, vectors, host cells, pharmaceutical compositions and kits comprising the tolerance-inducing constructs are further disclosed. Tolerance-inducing constructs and compositions are also disclosed for use in the treatment of immune disorders, such as 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

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

Background

An immune response is necessary to protect against diseases, such as those caused by pathogens such as viruses, bacteria or parasites. However, undesired immune activation may lead to a process that causes damage or destruction of the own tissue. Undesired immune activation occurs, for example, in autoimmune diseases, wherein antibodies and/or T lymphocytes react with autoantigens, resulting in, for example, tissue damage and pathology. Undesired immune activation can also occur in allergic reactions, characterized by excessive immune responses to substances that are normally harmless to the environment, and possibly inflammatory responses that lead to tissue destruction. In addition, undesired immune activation also occurs in graft rejection, for example, rejection of transplanted organs or tissues significantly mediated by alloreactive T cells present in the host, which recognize donor alloantigens or xenogeneic antigens, which results in destruction of the transplanted organs or tissues.

Immune tolerance refers to the inability to obtain a specific immune response to a substance or tissue capable of eliciting an immune response in a given organism. In general, in order to induce tolerance to a specific antigen, the antigen must be presented to other immune cells by Antigen Presenting Cells (APCs) without an activation signal, which leads to death or functional inactivation of antigen-specific effector lymphocytes, or to the generation of antigen-specific cells that maintain tolerance. This process generally accounts for tolerance to self-antigens or self-tolerance. Immunosuppressant drugs are useful for preventing or reducing undesired immune responses, for example, for treating patients suffering from autoimmune diseases or receiving allografts. Conventional strategies for immunosuppression of unwanted immune responses are based on widely acting immunosuppressive drugs. Furthermore, immunosuppressive drug therapy is often a life-long recommendation in order to maintain immunosuppression. Unfortunately, the use of widely acting immunosuppressive drugs is associated with the risk of serious side effects, such as immunodeficiency, because most of these drugs act non-selectively, resulting in increased susceptibility to infection and decreased immune surveillance of cancer. Thus, novel compounds and compositions that induce antigen-specific tolerance are beneficial.

Antigen presenting cells, 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 either combat the presented antigen (putative pathogen), or silence the response against the presented antigen (putative non-pathogenic antigen) and induce peripheral tolerance. The challenge in developing tolerogenic immunotherapy is to deliver antigen to APC/dendritic cells efficiently in a manner that does not trigger an inflammatory immune response.

Disclosure of Invention

The present disclosure relates to tolerance-inducing constructs comprising an antigenic unit and first and second targeting units that interact with surface molecules on antigen presenting cells, such as dendritic cells, in a non-inflammatory manner or in a tolerogenic manner, resulting in antigen presentation in the absence of an inflammatory activation state.

The inventors have surprisingly found that the constructs of the present disclosure can deliver disease-associated antigens to optimal Antigen Presenting Cells (APCs) in an optimal manner for inducing a preferred antigen-specific tolerogenic response (anti-specific tolerogenic response) by binding to and signaling through selected surface receptors on APCs that internalize the construct and present the antigen in a tolerogenic manner, such as inducing regulatory T cells (tregs) and suppressing memory and effector T cell responses.

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

i) A polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the order specified:

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

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

ii) a polypeptide encoded by the nucleotide sequence defined in i); or alternatively

iii) A multimeric protein consisting of a plurality of polypeptides defined in ii), for example a dimeric protein consisting of two polypeptides defined in ii).

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

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

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

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

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

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

Also provided herein are multimeric proteins, e.g., dimeric proteins, as described herein, wherein the plurality of polypeptides are linked to each other via their respective first junction regions and via their respective second junction regions.

Also provided herein are multimeric proteins as described herein, wherein a plurality of polypeptides, e.g., two polypeptides, are linked to each other via their respective first junction regions and via their respective second junction regions.

Also provided herein are dimeric proteins as described herein, wherein the two polypeptides are linked to each other via their respective first junction regions and via their respective second junction regions.

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

a) Providing a polynucleotide, polypeptide, or multimeric protein, such as a dimeric protein, as described herein; and

b) The polynucleotide, polypeptide, or multimeric protein, e.g., a dimeric protein, is combined with a pharmaceutically acceptable carrier.

a) Providing a polynucleotide, polypeptide or multimeric protein described herein; and

b) The polynucleotide, polypeptide or multimeric protein is combined with a pharmaceutically acceptable carrier.

a) Providing a polynucleotide, polypeptide, or dimeric protein described herein; and

b) The polynucleotide, polypeptide, or dimeric protein is combined with a pharmaceutically acceptable carrier.

In another aspect, the present disclosure provides a pharmaceutical composition comprising a polynucleotide, polypeptide, or multimeric protein, such as a dimeric protein, as described herein, and a pharmaceutically acceptable carrier.

Also provided herein are pharmaceutical compositions comprising a polynucleotide, polypeptide, or multimeric protein described herein, and a pharmaceutically acceptable carrier.

Also provided herein are pharmaceutical compositions comprising a polynucleotide, polypeptide, or dimeric protein described herein, and a pharmaceutically acceptable carrier.

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

In another aspect, the present disclosure provides a host cell comprising a vector described herein.

In another aspect, the present disclosure provides a method of preparing a polypeptide or multimeric protein, such as a dimeric protein, comprising:

a) Transfecting a cell with a vector as described herein or a polynucleotide as described herein;

b) Culturing a cell, whereby the cell expresses a polypeptide encoded by the polynucleotide; and

c) Obtaining and purifying the multimeric protein, e.g., a dimeric protein, and/or the polypeptide expressed by the cell.

In another aspect, the present disclosure provides a method of preparing a polypeptide or multimeric protein, the method comprising:

c) Obtaining and purifying the multimeric protein and/or polypeptide expressed by the cells.

In another aspect, the present disclosure provides a method of preparing a polypeptide or a dimeric protein, the method comprising:

c) Obtaining and purifying the dimeric protein and/or polypeptide expressed by the cell.

In another aspect, the present disclosure provides a method for treating a disorder involving an undesired immune response, for example in the prophylactic or therapeutic treatment of autoimmune diseases, allergic diseases, and graft rejection, the method comprising administering to a subject in need thereof a polynucleotide, polypeptide, or multimeric protein as described herein, such as a dimeric protein, a vector as described herein, or a pharmaceutical composition as described herein.

In another aspect, the present disclosure provides a method of treating a disorder involving an undesired immune response, for example in the prophylactic or therapeutic treatment of autoimmune diseases, allergic diseases, and graft rejection, the method comprising administering to a subject in need thereof a polynucleotide, polypeptide, or multimeric protein as described herein, a vector as described herein, or a pharmaceutical composition as described herein.

In another aspect, the present disclosure provides a method of treating a disorder involving an undesired immune response, for example in the prophylactic or therapeutic treatment of autoimmune diseases, allergic diseases, and graft rejection, the method comprising administering to a subject in need thereof a polynucleotide, polypeptide, or dimeric protein as described herein, a vector as described herein, or a pharmaceutical composition as described herein.

Drawings

FIG. 1

A schematic of the immunotherapeutic constructs of the present disclosure is shown.

The above figures illustrate embodiments of the construct as a polypeptide. The lower panel shows an embodiment of a dimeric protein formed from two polypeptides joined via their respective first and second junction regions.

A shows a first targeting unit

B shows a second targeting unit

C shows an antigenic unit comprising at least one T cell epitope

D illustrates imparting flexibility to the targeting unit due to the presence of the flexible unit

A.A the first bond region

B.A shows the second land.

FIG. 2

The figure shows one embodiment of the landing zone.

A shows three covalent bonds formed between the covalent binding units contained in each of the two polypeptide chains.

B shows how the flexible unit is located between the binding unit and the targeting unit, providing flexibility to the targeting unit, as indicated by arrow D in fig. 1.

FIG. 3

The figure shows another embodiment of the landing zone.

A shows dimerization of two polypeptides by hydrophobic interactions between non-covalent binding units contained in each of the two polypeptide chains.

FIG. 4

The figure shows the expression and secretion levels of the tolerance-inducing constructs encoding MOG and IL-10 of the invention (capture antibody: mouse anti-MOG antibody, 0.25. Mu.g/ml, 100. Mu.l/well, sc-73330,Santa Cruz Biotechnology, detection antibody: goat anti-mouse IL-10 biotinylated antibody, 0.8. Mu.g/ml, 100. Mu.l/well, BAF417, R & D Systems) detected in the supernatant of transfected cells by sandwich ELISA.

A) Results of transient transfection of Expi293F cells with DNA vectors VB5042, VB5050, VB5072, VB5073, VB5074 and VB5075 are shown.

B) Results of transiently transfected HEK293 cells with DNA vector VB5038 are shown.

All MOG and IL-10 encoding constructs were highly expressed and secreted. (A) The negative control in (a) was the supernatant of the Expi293F cells treated with the transfection reagent expictamine alone, and the negative control in (B) was the supernatant of the HEK293 cells treated with the transfection reagent Lipofectamine alone.

FIG. 5

The figure shows the protein expression and secretion levels (capture antibody: mouse anti-MOG antibody, 0.25. Mu.g/ml, 100. Mu.l/well, sc-73330,Santa Cruz Biotechnology, detection antibody: goat anti-mouse CTLA-4 biotinylated antibody, 0.8. Mu.g/ml, 100. Mu.l/well, BAF476, R & D Systems) of MOG encoding construct (VB 5067) with CTLA-4 as the second targeting unit detected by sandwich ELISA in the supernatant of the Expi293F cells transiently transfected with DNA vector VB 5067. The negative control was the supernatant of the pi293F cells treated with the transfection reagent, epifectamine alone.

FIG. 6

The figure shows the protein expression and secretion levels (capture antibodies: mouse anti-MOG antibody, 0.25. Mu.g/ml, 100. Mu.l/well, sc-73330,Santa Cruz Biotechnology, detection antibodies: goat anti-SCGB 3A2 biotinylated antibody, 3.3. Mu.g/ml, 100. Mu.l/well, BAF3465, R & D Systems) of MOG-encoded tolerance-inducing constructs (VB 5072 and VB 5073) with MARCO ligand SCGB3A2 as the first targeting unit and IL-10 as the second targeting unit detected by sandwich ELISA in supernatants of Expi293F cells transiently transfected with DNA vectors VB5072 and VB 5073. The negative control was the supernatant of the pi293F cells treated with the transfection reagent, epifectamine alone.

FIG. 7

The figure shows that MOG-encoded tolerance-inducing constructs (VB 5072 and VB 5073) with MARCO ligand SCGB3A2 as the first targeting unit and IL-10 as the second targeting unit were secreted as full-length fusion proteins (capture antibodies: mouse anti-murine IL-10 antibody, 2. Mu.g/ml, 100. Mu.l/well, MAB417, R & D Systems, detection antibodies: goat anti-murine SCGB3A2, 3.3. Mu.g/ml, 100. Mu.l/well, BAF3465, R & D Systems) detected by sandwich ELISA in supernatants of Exp 293F cells transiently transfected with DNA vectors VB5072 and VB 5073. The negative control was the supernatant of the pi293F cells treated with the transfection reagent, epifectamine alone.

FIG. 8

The figure shows the binding of scFv anti-DEC 205 encoding constructs to DEC205 receptor and secretion of full length proteins detected by direct ELISA in supernatants of HEK293 cells transiently transfected with DNA vector VB 5038. ELISA wells were coated with recombinant DEC205 receptor (aa 216-503) and binding was detected by antibodies directed against MOG or murine IL-10. OD from negative control (i.e. supernatant of HEK293 cells treated with transfection reagent Lipofectamine) was subtracted prior to mapping _450nm A signal.

FIG. 9

The figure shows the binding of the construct containing IL-10 to the IL-10 receptor detected by direct ELISA in the supernatant of HEK293 cells transiently transfected with the DNA vector VB 5038. ELISA wells were coated with recombinant IL-10 receptor and binding was detected by antibodies directed against MOG. OD from negative control (i.e. supernatant of HEK293 cells treated with transfection reagent Lipofectamine) was subtracted prior to mapping _450nm Signal signal

FIG. 10

The figure shows the secretion of MOG (27-63) peptide (detection antibody: mouse anti-MOG antibody, 3.3. Mu.g/ml, 100. Mu.l/well, sc-73330,Santa Cruz Biotechnology) detected by direct ELISA in the supernatant of the Expi293F cells transiently transfected with the DNA vector VB 5051. The negative control was the supernatant of the pi293F cells treated with the transfection reagent, epifectamine alone.

FIG. 11

A. The expression and secretion levels of the pro-inflammatory control constructs encoded by DNA vector VB5052 (capture antibodies: mouse anti-MOG antibody, 0.25. Mu.g/ml, 100. Mu.l/well, sc-73330,Santa Cruz Biotechnology, detection antibodies: goat anti-human CCL3 biotin antibody, 0.2. Mu.g/ml, 100. Mu.l/well, BAF270, R & D Systems) detected by sandwich ELISA in supernatants of Expi293F cells transiently transfected with vector VB5052 containing CCL3L1 are shown. Detection of MOG and CCL3L1 portions of the fusion proteins showed full-length secretion of the fusion proteins. The negative control was the supernatant of the pi293F cells treated with the transfection reagent, epifectamine alone.

B. The expression and secretion levels of the pro-inflammatory control constructs encoded by DNA vector VB5002b (capture antibodies: mouse anti-human IgG (CH 3 domain), 1. Mu.g/ml, 100. Mu.l/well, 153272, biorad, detection antibodies: goat anti-human CCL3 biotin antibodies, 0.2. Mu.g/ml, 100. Mu.l/well, BAF270, R & D Systems) detected by sandwich ELISA in supernatants of HEK293 cells transiently transfected with vector VB5002b containing CCL3L1 are shown. The negative control was the supernatant of HEK293 cells treated with transfection reagent Lipofectamine alone.

FIG. 12

A. Western blot of full length secretion of tolerance-inducing protein encoded by VB5038 and pro-inflammatory control encoded by VB5002 b. Reduced supernatant samples (10 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). Chemidoc channel dyight 800.

B. Western blots of the protein encoded by VB5038 detected with anti-murine IL-10 antibodies under reducing and non-reducing conditions are shown. The left side shows a reduced supernatant sample from transfected Expi293F cells and the right side shows a non-reduced supernatant sample (loading 10 μl) from transfected Expi293F cells. An antibody: rat anti-murine IL10 (MAB 417). And (2) secondary antibody: donkey was resistant to rats, dylight 488 (SA 5-1006). Chemidoc channel Dylight 488. Specific bands of a size corresponding to homodimeric proteins were detected under non-reducing conditions (indicated by black arrows).

C. Western blots (black arrows) of full-length secretion of the proteins encoded by VB5041, VB5042 and VB5050 detected by anti-MOG antibodies are shown. Reduced supernatant samples (30 μl loaded) of 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.

D. Western blots (black arrows) of full-length secretion of the proteins encoded by VB5041, VB5042 and VB5050 detected by anti-murine IL-10 antibodies are shown. Reduced supernatant samples (30. Mu.L loaded) of transfected Expi293F cells. An antibody: rat anti-murine IL10 (MAB 417). And (2) secondary antibody: donkey is resistant to rat, dylight 650 (SA 5-10029). Chemidoc channel Dylight 650.

E. Western blots of the proteins encoded by VB5041, VB5042 and VB5050 are shown, which show that the proteins dimerize under non-reducing conditions (black arrows). A non-reduced supernatant sample (30 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). Chemidoc channel dlight 650 (for protein standards) and 800.

F. Shows a western blot of full-length secretion of the protein encoded by VB5074 and VB5075 with VSIG-3 as the first targeting unit detected by the anti-MOG antibody. VB5042 was included as a positive control. Supernatant samples (25. Mu.L loaded) from the supernatant of the upper reduction of 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). Chemidoc channel dyight 800.

G. Shows a western blot of full-length secretion of proteins encoded by VB5074 and VB5075, using VSIG-3 as the first targeting unit, detected by an anti-murine IL-10 antibody. VB5042 was included as a positive control. 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. 13

The figure shows a bicolor IL-10/IFNgamma FluoSpot. C57BL/6 mice were inoculated once (day 0) with 50. Mu.g of the indicated DNA vector and spleens were harvested on day 7 post-inoculation. Individual mice and mean ± SEM are shown, n=5 mice per group. * Double tail Mann-Whitney test (p < 0.01). Construct ID numbers are shown on the x-axis.

A. Shows IL-10 and IFN-gamma secretion (SFU/10) from mouse spleen cells detected with two-color FluoSpot for unstimulated spleen cells ⁶ Individual spleen cells).

B. Shows IL-10 and IFN-gamma secretion (SFU/10) from spleen cells of mice detected with two-color FluoSpot after an additional 44 hours of stimulation with MOG (35-55) peptide ⁶ Individual spleen cells).

C. A plot of IL-10/IFN-gamma ratio from data from MOG (35-55) -restimulated spleen cells in (B) is shown. Individual mice and mean ± range are shown. * Double tail Mann-Whitney test (p < 0.01).

FIG. 14

The figure shows the detection of MOG (38-49) -specific foxp3+ cells. C57BL/6 mice were inoculated once (day 0) with 50. Mu.g of the indicated DNA vector and spleens were harvested on day 7 post-inoculation. The percentage of splenic CD4+Foxp3+ cells was determined by the H-2Iab/MOG (38-49) tetramer. Tetramer staining was performed ex vivo and spleen cells were not re-stimulated with MOG (35-55) peptide. Data were generated from pools of 5 mice per group (pooled spleens into pools prior to analysis). Construct ID numbers are shown on the x-axis.

FIG. 15

The figure shows a bicolor IL-10/IFNgamma fluoroSpot. C57BL/6 mice were inoculated once (day 0) with 50. Mu.g of the indicated DNA vector and spleens were harvested on day 7 post-inoculation. Individual mice and mean ± SEM are shown, n=5 mice per group. * Double tail Mann-Whitney test (p < 0.01). Construct ID numbers are shown on the x-axis.

FIG. 16

FIG. 17

C. A plot of IL-10/IFN-gamma ratio from data from MOG (35-55) -restimulated spleen cells in (B) is shown. Individual mice and mean ± range are shown. * (p < 0.05), two-tailed Mann-Whitney test.

FIG. 18

The figure shows the detection of MOG (38-49) -specific foxp3+ cells. C57BL/6 mice were inoculated once (day 0) with 50. Mu.g of the indicated DNA vector and spleens were harvested on day 7 post-inoculation. The frequency of splenic CD4+Foxp3+ cells was detected by the H-2Iab/MOG (38-49) tetramer. Tetramer staining was performed ex vivo and spleen cells were not re-stimulated with MOG (35-55) peptide. Data were generated from pools of 5 mice per group (pooled spleens into pools prior to analysis). Construct ID numbers are shown on the x-axis.

FIG. 19

The figure shows the expression and secretion levels of Met e 1-containing tolerance-inducing constructs VB5077 and VB5078 with IL-10 as the second targeting unit. Sandwich ELISA: capture antibody: anti-murine IL-10 antibody (MAB 417, R & D Systems), detection antibody: anti-murine IL-10 biotinylated antibodies (BAF 417, R & D Systems) were used with supernatant from Expi293F cells transiently transfected with VB5077 and VB 5078. Both Met e 1 containing constructs were highly expressed and secreted. The negative control was the supernatant of the pi293F cells treated with the transfection reagent, epifectamine alone.

FIG. 20

The figure shows a western blot (black arrow) of full-length secretion of proteins encoded by the Met e 1-containing DNA vectors VB5077 and VB 5078. Reduced supernatant samples (35 μl loaded) from transfected Expi293F cells. An antibody: rat anti-IL 10 (MAB 417). And (2) secondary antibody: donkey is resistant to rat, dylight 650 (SA 5-10029). Chemidoc channel Dylight 650.

Detailed Description

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

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

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

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

Since the tolerance-inducing construct results in down-regulation of disease-specific cells in the immune system that cause 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 not result in increased susceptibility to infection and decreased immune surveillance of cancer. However, bystander suppression of immune cells specific for the associated disease antigen is expected to occur due to short-range inhibitory cytokines released 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 transplant rejection.

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

The term "tolerability" as used herein refers to a reduced level of an inflammatory immune response to a harmless antigen, such as an autoantigen, allergen or alloantigen, a delayed onset or progression of an inflammatory immune response and/or a reduced risk of onset or progression of an inflammatory immune response.

A "subject" is an animal, such as a mouse or a human, preferably a human. The subject may be a patient (i.e., a human) in need of therapeutic treatment having an immune disorder such as an autoimmune disorder, allergy or graft rejection, or may be a subject in need of prophylactic treatment or a subject suspected of having an immune disorder. The terms "subject" and "individual" are used interchangeably herein.

A "disease" is an abnormal medical condition, often associated with specific signs and symptoms in a subject affected by 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, e.g., rejection by a host of cells, tissues or organs from the same species (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 (removed from and/or present in) a cell or tissue that, when transferred from a donor to a recipient, can be recognized and bound by antibodies of the recipient's B or T cell recipient. Alloantigens are typically the products of polymorphic genes. An alloantigen is a protein or peptide that shows a slight structural difference when compared between a donor and a recipient (belonging to the same species). Such donor antigens, when present in a recipient, can elicit an inflammatory immune response in the recipient. This alloreactive immune response is specific for alloantigens.

The term "heterologous antigen" as used herein 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 that exhibits only early signs or symptoms of an immune disease, with the aim of preventing or at least reducing the risk of developing an immune disease. Prophylactic treatment acts as a prophylactic treatment against immune disease, or as a treatment to inhibit or reduce further development or enhancement of immune disease and/or its associated symptoms. The terms "prophylactic treatment", "prevention" and "prevention" are used interchangeably herein.

A "therapeutic treatment" is a treatment administered to a subject exhibiting symptoms or signs of an immune disease, wherein the treatment is administered to the subject for the purpose of reducing or eliminating those signs or symptoms, or to delay or stop disease progression.

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

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

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. Thus, the term minimal epitope may refer to a short subsequence of an epitope predicted to bind to MHC I or MHC II. Thus, a 27-mer epitope can encompass a plurality of 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, provided that it is predicted to bind to MHC I or MHC II, or consist of amino acids 9 to 18 of the epitope, or consist of amino acids 7 to 22, provided that these sequences are predicted to bind to MHC I or MHC II.

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

The terms "mouse" and "mouse" are used interchangeably herein.

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

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

Tolerance-inducing constructs

The structure of some embodiments of the construct is shown in fig. 1-3, which is based on a polypeptide and a dimeric protein formed from two polypeptides linked via their respective first and second junction regions. The polypeptide (fig. 1, top panel) comprises, in the order specified, a first targeting unit (a), a first junction region (A.A), an antigenic unit (C) as described herein, a second junction region (B.A) and a second targeting unit (B). The lower part of fig. 1 shows how the flexible unit comprised in the second engagement zone provides flexibility to the second targeting unit (arrow D).

In some embodiments, the polypeptide and multimeric protein, such as a dimeric protein, are formed from a plurality of polypeptides joined via their respective first and second junction regions. The polypeptide comprises, in the order specified, a first targeting unit, a first junction region, an antigenic unit as described herein, a second junction region, and a second targeting unit. The flexible unit included in the second engagement zone provides flexibility to the second targeting unit.

In some embodiments, the polypeptides and multimeric proteins are formed from a plurality of polypeptides linked via their respective first and second junction regions. The polypeptide comprises, in the order specified, a first targeting unit, a first junction region, an antigenic unit as described herein, a second junction region, and a second targeting unit. The flexible unit included in the second engagement zone provides flexibility to the second targeting unit.

In some embodiments, the polypeptide and the dimeric protein are formed from two polypeptides joined via their respective first and second junction regions. The polypeptide comprises, in the order specified, a first targeting unit, a first junction region, an antigenic unit as described herein, a second junction region, and a second targeting unit. The flexible unit included in the second engagement zone provides flexibility to the second targeting unit.

In the disclosure below, the individual units of the construct will be discussed in detail. They are contained in polynucleotides in the nucleic acid sequences encoding these units, while they are contained in polypeptides or multimeric/dimeric proteins in the amino acid sequences. For ease of reading, in the disclosure below, the units of the construct are mainly explained with respect to polypeptides or multimeric/dimeric proteins, i.e. based on their amino acid sequences.

Junction region

The polypeptides of the invention comprise a first junction region and a second junction region. The first bonding region and the second bonding region may be any one of the regions described below.

The first junction region is located between the first targeting unit described herein and the antigenic unit described herein.

In some embodiments, the second junction region is located between the antigenic unit described herein and the second targeting unit described herein.

In some embodiments, a multimeric protein of the present disclosure, such as a dimeric protein, is one in which multiple polypeptides, such as two polypeptides, are linked to each other by their junction regions. In other embodiments, a multimeric protein of the present disclosure, such as a dimeric protein, is one in which multiple polypeptides, such as two polypeptides, are linked to each other via their respective first junction regions and via their respective second junction regions.

In some embodiments, the multimeric proteins of the present disclosure are multimeric proteins in which multiple polypeptides are linked to each other through their junction regions. In other embodiments, the multimeric proteins of the present disclosure are multimeric proteins in which a plurality of polypeptides are linked to each other via their respective first junction regions and via their respective second junction regions.

In some embodiments, the dimeric proteins of the present disclosure are dimeric proteins in which two polypeptides are linked to each other via their junction regions. In other embodiments, the dimeric proteins of the present disclosure are dimeric proteins in which two polypeptides are linked to each other via their respective first junction regions and via their respective second junction regions. The term "junction" as used herein refers to the amino acid sequence between an antigenic unit and a targeting unit. Any amino acid sequence capable of joining multiple polypeptides (for embodiments involving multimeric proteins) such as can join two polypeptides (for embodiments involving dimeric proteins) but simultaneously providing flexibility and appropriate protein conformation to the multimeric or dimeric protein is a suitable junction region.

The junction region provides flexibility to multimeric proteins, such as dimeric proteins, so that the targeting unit can interact with surface molecules on an APC, for example, with surface molecules on the same APC, even though they are located at different distances. In addition, the junction region links multiple monomeric polypeptides into a multimeric protein, such as a dimeric protein. Any amino acid sequence that meets one or more of these requirements is a suitable junction region.

The junction region provides flexibility to the multimeric protein so that the targeting unit can interact with surface molecules on the APC, for example, with surface molecules on the same APC, even though they are located at different distances. In addition, the junction region joins multiple monomeric polypeptides into a multimeric protein. Any amino acid sequence that meets one or more of these requirements is a suitable junction region.

The junction region provides flexibility to the dimeric protein so that the targeting unit can interact with surface molecules on the APC, for example, with surface molecules on the same APC, even though they are located at different distances. In addition, the junction region joins two monomeric polypeptides into a dimeric protein. Any amino acid sequence that meets one or more of these requirements is a suitable junction region.

Preferably, the junction region comprises a flexible unit that provides flexibility and a binding unit that joins multiple polypeptides, such as two polypeptides, to form a multimer, such as a dimer. In a preferred embodiment, the flexible unit comprised in the junction is closest to the targeting unit and the binding unit is closest to the antigenic unit. In other embodiments, the junction region comprises a flexible unit that provides flexibility and a binding unit that joins the plurality of polypeptides to form a multimeric protein.

Preferably, the junction region comprises a flexible unit that provides flexibility and a binding unit that binds the plurality of polypeptides to form a multimer. In a preferred embodiment, the flexible unit comprised in the junction is closest to the targeting unit and the binding unit is closest to the antigenic unit. In other embodiments, the junction region comprises a flexible unit that provides flexibility and a binding unit that joins the plurality of polypeptides to form a multimeric protein. Preferably, the junction region comprises a flexible unit that provides flexibility and a binding unit that joins the two polypeptides to form a dimer. In a preferred embodiment, the flexible unit comprised in the junction is closest to the targeting unit and the binding unit is closest to the antigenic unit. In other embodiments, the junction region comprises a flexible unit that provides flexibility and a binding unit that joins the two polypeptides to form a dimeric protein. In some embodiments, the binding units of the first and second landing zones are different.

In some embodiments, the binding unit comprised in the first junction region of one polypeptide molecule is capable of binding to the binding unit comprised in the first junction region of another polypeptide molecule, whereby the plurality of molecules are linked via their respective first junction regions. Likewise, the binding units comprised in the second junction region of one polypeptide molecule are capable of binding to the binding units comprised in the second junction region of another polypeptide molecule, whereby the plurality of molecules are linked via their respective second junction regions. Thus, the plurality of polypeptide molecules form a multimeric protein, e.g., a dimeric protein, by linking to each other via their respective first junction regions and via their respective second junction regions.

In some embodiments, the binding unit comprised in the first junction region of one polypeptide molecule is capable of binding to the binding unit comprised in the first junction region of another polypeptide molecule, whereby the plurality of molecules are linked via their respective first junction regions. Likewise, the binding units comprised in the second junction region of one polypeptide molecule are capable of binding to the binding units comprised in the second junction region of another polypeptide molecule, whereby the plurality of molecules are linked via their respective second junction regions. Thus, the plurality of polypeptide molecules form a multimeric protein by linking to each other via their respective first junction regions and via their respective second junction regions.

Thus, in some embodiments, the binding unit comprised in the first junction region of one polypeptide molecule is capable of binding to the binding unit comprised in the first junction region of another polypeptide molecule, whereby the two molecules are linked via their respective first junction regions. Likewise, the binding unit comprised in the second junction region of one polypeptide molecule is capable of binding to the binding unit comprised in the second junction region of another polypeptide molecule, whereby the two molecules are linked via their respective second junction regions. Thus, the two polypeptide molecules form a dimeric protein by being linked to each other via their respective first junction regions and via their respective second junction regions.

In other embodiments, the first and second landing zones are the same.

Thus, in some embodiments, a binding unit contained in a first junction region of one polypeptide molecule is capable of binding to a binding unit contained in a first junction region or a second junction region of another polypeptide molecule. The same applies to the bonding units contained in the second bonding region.

Preferably, if the first and second junction regions are the same, the first and second targeting units are different but interact with the same surface molecule on the APC; or the first and second targeting units are identical.

In some embodiments, the amino acid sequence of the first and/or second junction region comprises or consists of at least one naturally occurring sequence. In some embodiments, the amino acid sequence of the first and/or second junction region comprises or consists of at least one artificial sequence.

In some embodiments, the binding units are covalent binding units, in other embodiments, the binding units are non-covalent binding units.

In a preferred embodiment, the amino acid sequence of the junction region is a non-immunogenic sequence.

Embodiments of the junction region comprised in some embodiments of the dimeric protein are shown in figures 2 and 3.

The junction region shown in fig. 2 (junction region 1 or junction region 2) comprises a flexible unit (B) nearest the targeting unit and a covalent binding unit nearest the antigenic unit adjacent thereto. The covalent binding unit of fig. 2 shows three covalent bonds (a) formed between two polypeptide chains.

The junction region shown in fig. 3 (junction region 1 or junction region 2) comprises a flexible unit (B) nearest the targeting unit and a non-covalent binding unit nearest the antigenic unit adjacent thereto. The non-covalent binding unit of fig. 3 promotes dimerization of the two polypeptide chains by, for example, hydrophobic interactions (a).

Flexible unit

In some embodiments, the landing zones described herein comprise flexible units.

In a preferred embodiment, the amino acid sequence of the flexible unit is a non-immunogenic sequence.

In some embodiments, the amino acid sequence of the flexible unit is a naturally occurring peptide sequence. In some embodiments, the flexible unit is derived from an immunoglobulin. In some embodiments, the flexible unit is a hinge region of an immunoglobulin, wherein the hinge region does not comprise a cysteine residue.

In some embodiments, the amino acid sequence of the flexible unit is an artificial sequence.

In some embodiments, the flexible unit comprises a small nonpolar (e.g., glycine, alanine, or leucine) or polar (e.g., serine or threonine) amino acid. The small size of these amino acids provides flexibility and allows mobility of the attached amino acid sequences. The introduction of serine or threonine can reduce adverse interactions between the linker and the antigen by maintaining the stability of the linker in aqueous solution through hydrogen bonding with water molecules. In some embodiments, the flexible unit is an artificial sequence, such as a serine (S) and/or glycine (G) rich linker, i.e., a linker comprising multiple serine and/or multiple glycine residues. Preferred examples are GGGGSGGGSS (SEQ ID NO: 75), GGGSG (SEQ ID NO: 76), GGSGG (SEQ ID NO: 77), SGSSGS (SEQ ID NO: 78), GGGGS (SEQ ID NO: 79) or variants thereof such as GGGGSGGGGS (SEQ ID NO: 80), (GGGGS) m (SEQ ID NO: 81), (GGGSS) m (SEQ ID NO: 82), (GGGSG) m (SEQ ID NO: 83) or (SGSSGS) m (SEQ ID NO: 84), wherein m is an integer from 1 to 5, such as 1, 2, 3, 4 or 5. In a preferred embodiment, m is 2. In 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 flexible unit comprises or consists of LGGGS (SEQ ID NO: 85), GLGGS (SEQ ID NO: 86), GGLGS (SEQ ID NO: 87), GGGLS (SEQ ID NO: 88), or GGGGL (SEQ ID NO: 89). In other embodiments, the flexible unit comprises or consists of LGGSG (SEQ ID NO: 90), GLGSG (SEQ ID NO: 91), GGLSG (SEQ ID NO: 92), GGGLG (SEQ ID NO: 93) or GGGSL (SEQ ID NO: 94). In other embodiments, the flexible unit comprises or consists of LGGSS (SEQ ID NO: 95), GLGSS (SEQ ID NO: 96) or GGLSS (SEQ ID NO: 97).

In other embodiments, the flexible unit comprises or consists of LGLGS (SEQ ID NO: 98), GLGLS (SEQ ID NO: 99), GLLGS (SEQ ID NO: 100), LGGLS (SEQ ID NO: 101) or GLGGL (SEQ ID NO: 102). In other embodiments, the flexible unit comprises or consists of LGLSG (SEQ ID NO: 103), GLLSG (SEQ ID NO: 104), GGLSL (SEQ ID NO: 105), GGLLG (SEQ ID NO: 106) or GLGSL (SEQ ID NO: 107). In other embodiments, the flexible unit comprises or consists of LGLSS (SEQ ID NO: 108) or GGLLS (SEQ ID NO: 109).

In other embodiments, the flexible unit is a serine-glycine linker having a length of 10 amino acids and comprising 1 or 2 leucine residues.

In some embodiments, the flexible unit comprises or consists of LGGGSGGGGS (SEQ ID NO: 110), GLGGSGGGGS (SEQ ID NO: 111), GGLGSGGGGS (SEQ ID NO: 112), GGGLSGGGGS (SEQ ID NO: 113), or GGGGLGGGGS (SEQ ID NO: 114). In other embodiments, the flexible unit comprises or consists of LGGSGGGGSG (SEQ ID NO: 115), GLGSGGGGSG (SEQ ID NO: 116), GGLSGGGGSG (SEQ ID NO: 117), GGGLGGGGSG (SEQ ID NO: 118) or GGGSLGGGSG (SEQ ID NO: 119). In other embodiments, the flexible unit comprises or consists of LGGSSGGGSS (SEQ ID NO: 120), GLGSSGGGSS (SEQ ID NO: 121), GGLSSGGGSS (SEQ ID NO: 122), GGGLSGGGSS (SEQ ID NO: 123) or GGGSLGGGSS (SEQ ID NO: 124).

In further embodiments, the flexible unit comprises or consists of LGGGSLGGGS (SEQ ID NO: 125), GLGGSGLGGS (SEQ ID NO: 126), GGLGSGGLGS (SEQ ID NO: 127), GGGLSGGGLS (SEQ ID NO: 128) or GGGGLGGGGL (SEQ ID NO: 129). In other embodiments, the flexible unit comprises or consists of LGGSGLGGSG (SEQ ID NO: 130), GLGSGGLGSG (SEQ ID NO: 131), GGLSGGGLSG (SEQ ID NO: 132), GGGLGGGGLG (SEQ ID NO: 133) or GGGSLGGGSL (SEQ ID NO: 134). In other embodiments, the flexible unit comprises or consists of LGGSSLGGSS (SEQ ID NO: 135), GLGSSGLGSS (SEQ ID NO: 136) or GGLSSGGLSS (SEQ ID NO: 137).

In other embodiments, the flexible unit comprises or consists of GSGGGA (SEQ ID NO: 138), GSGGGAGSGGGA (SEQ ID NO: 139), GSGGGAGSGGGAGSGGGA (SEQ ID NO: 140), GSGGGAGSGGGAGSGGGAGSGGGA (SEQ ID NO: 141) or GENLYFQSGG (SEQ ID NO: 142). In other embodiments, the flexible unit comprises or consists of SGGGSSGGGS (SEQ ID NO: 143), SSGGGSSGGG (SEQ ID NO: 144), GGSGGGGSGG (SEQ ID NO: 145), GSGSGSGSGS (SEQ ID NO: 146), GGGSSGGGSG (SEQ ID NO:147, and amino acids 121-130 of SEQ ID NO: 1), GGGSSS (SEQ ID NO: 148), GGGSSGGGSSGGGSS (SEQ ID NO: 149), or GLGGLAAA (SEQ ID NO: 150).

In other embodiments, the flexible unit comprises or consists of sequence TQKSLSLSPGKGLGGL (SEQ ID NO: 151). In other embodiments, the flexible unit 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 flexible unit comprises or consists of a GSAT linker, i.e., a linker comprising one or more glycine, serine, alanine, and threonine residues, such as a linker comprising or consisting of sequence GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 154); or comprises or consists of a SEG linker, i.e.a linker comprising one or more serine, glutamic acid and glycine residues, e.g.a linker comprising or consisting of the sequences GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 155) or ELKTPLGDTTHT (SEQ ID NO: 19).

In some embodiments, the flexible unit is not a target for a protease.

In some embodiments, the flexible unit consists of up to 20 amino acids, for example, up to 15 amino acids, for example, 14 amino acids, for example, 13 amino acids, for example, 12 amino acids, for example, 11 amino acids, or 10 amino acids.

In some embodiments, the flexible unit comprises or consists of an amino acid sequence having at least 50% sequence identity with the amino acid sequence of 1-12 of SEQ ID No. 1, such as 60% or such as 70% or such as 80% or such as 90% sequence identity.

In a preferred embodiment, the flexible unit is hinge exon h1 of IgG 3.

In a preferred embodiment, the flexible unit comprises or consists of the amino acid sequence 1-12 of SEQ ID NO. 1.

In some embodiments, the flexible unit comprises or consists of an amino acid sequence having at least 50% sequence identity with the amino acid sequence of 16-23 of SEQ ID No. 2, such as 60% or such as 70% or such as 80% or such as 90% sequence identity.

In some embodiments, the flexible unit comprises or consists of the amino acid sequence of 16-23 of SEQ ID NO. 2, wherein any one amino acid of the flexible unit has been substituted, deleted or inserted with another amino acid, provided that NO more than 5 amino acids have been so substituted, deleted or inserted, such as NO more than 4 amino acids, such as NO more than 3 amino acids, such as NO more than 2 amino acids or NO more than 1 amino acid have been so substituted, deleted or inserted.

In other embodiments, the flexible unit is the lower hinge region of IgG 1.

In a preferred embodiment, the flexible unit comprises or consists of the amino acid sequence 16-23 of SEQ ID NO. 2.

Covalent binding units

In some embodiments, the junction regions described herein comprise a covalent binding unit.

In a preferred embodiment, the covalent binding unit comprises one or more cysteine residues, and the polypeptides described herein are linked by one or more disulfide bonds formed between the cysteine residues comprised in the covalent binding units of the respective first and second junction regions.

In some embodiments, the covalent binding unit consists of or comprises a cysteine-rich sequence.

In some embodiments, the covalent binding unit comprises at least 2 cysteine residues, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 cysteine residues.

In some embodiments, the covalent binding unit of the first junction region comprises a different number of cysteine residues than the covalent binding unit of the second junction region.

In some embodiments, the position of the cysteine residue of the covalent binding unit of the first junction region is different from the position of the cysteine residue of the covalent binding unit of the second junction region. For example, the number of amino acid residues between cysteine residues of the covalent binding unit of the first junction region is different from the number of amino acid residues between cysteine residues of the covalent binding unit of the second junction region.

In some embodiments, the number of cysteine residues is based on the length of the antigenic unit: the more amino acid residues contained in an antigenic unit, the higher the number of cysteine residues in the covalently bound unit.

In some embodiments, the covalent binding unit comprises the sequence EPKSCDTPPPCPRCP (SEQ ID NO:156; amino acids 13-27 corresponding to SEQ ID NO: 1).

In a preferred embodiment, the amino acid sequence of the covalent binding unit is a non-immunogenic sequence.

In some embodiments, the amino acid sequence of the covalent binding unit is an artificial sequence.

In some embodiments, the amino acid sequence of the covalent binding unit is a naturally occurring peptide sequence.

In some embodiments, the covalent binding unit consists of 2 to 100 amino acids, e.g., 3 to 70 amino acids, e.g., 4 to 50 amino acids, or 5 to 30 amino acids. In further embodiments, the covalent binding unit consists of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. In a preferred embodiment, the covalent binding unit consists of 15 amino acids. In a more preferred embodiment, the covalent binding unit consists of 15 amino acids, 3 of which are cysteine residues.

In some embodiments, the covalent binding unit is derived from an immunoglobulin.

In some embodiments, the covalent binding unit is a hinge region derived from an immunoglobulin, such as exon h4 of IgG3 or the mid-hinge region of IgG 1. The hinge region may be Ig derived, e.g. derived from IgG, e.g. IgG2 or IgG3. In some embodiments, the hinge region is derived from IgM, e.g., comprises SEQ ID NO:157 or an amino acid sequence encoded by said nucleotide sequence, or a combination thereof.

In some embodiments, the covalent binding unit comprises or consists of an amino acid sequence having at least 40% sequence identity to the amino acid sequence of 13-27 of SEQ ID NO. 1, e.g., at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity.

In some embodiments, the covalent binding unit comprises or consists of the amino acid sequence of 13-27 of SEQ ID NO. 1, wherein any one amino acid of the flexible unit has been substituted, deleted or inserted with another amino acid, provided that NO more than 6 amino acids are so substituted, deleted or inserted, e.g. NO more than 5 amino acids, e.g. NO more than 4 amino acids, e.g. NO more than 3 amino acids, e.g. NO more than 2 amino acids or NO more than 1 amino acid are so substituted, deleted or inserted.

In a preferred embodiment, the covalent binding unit is hinge exon h4 of IgG 3.

In other preferred embodiments, the covalent binding region consists of the amino acid sequence of 13-27 of SEQ ID NO. 1.

In some embodiments, the covalent binding unit comprises or consists of an amino acid sequence having at least 40% sequence identity to the amino acid sequence of 5-15 of SEQ ID NO. 2, e.g., at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity, provided that the number and position of cysteine residues are retained.

In some embodiments, the covalent binding unit comprises or consists of the amino acid sequence of 5-15 of SEQ ID NO. 2, wherein any one amino acid of the flexible unit has been substituted, deleted or inserted with another amino acid, provided that NO more than 5 amino acids have been so substituted, deleted or inserted, such as NO more than 4 amino acids, such as NO more than 3 amino acids, such as NO more than 2 amino acids or NO more than 1 amino acid have been so substituted, deleted or inserted.

In a preferred embodiment, the covalent binding unit is the mid-hinge region of IgG 1.

In other embodiments, the covalent binding unit consists of the amino acid sequence of 5-15 of SEQ ID NO. 2 or comprises the amino acid sequence of 5-15 of SEQ ID NO. 2.

Non-covalent binding units

In some embodiments, the junction regions described herein comprise non-covalent binding units.

In some embodiments, the non-covalent binding units promote multimerization, e.g., dimerization, by non-covalent interactions, e.g., hydrophobic interactions. In some embodiments, the non-covalent binding units have the ability to form multimers, e.g., dimers, by non-covalent interactions.

In some embodiments, the non-covalent binding units promote multimerization by non-covalent interactions such as hydrophobic interactions. In some embodiments, the non-covalent binding units have the ability to form multimers by non-covalent interactions.

Non-covalent binding units promote dimerization through non-covalent interactions such as hydrophobic interactions. In some embodiments, the non-covalent binding units have the ability to form dimers through non-covalent interactions.

In a preferred embodiment, the amino acid sequence of the non-covalent binding unit is a non-immunogenic sequence.

In some embodiments, the amino acid sequence of the non-covalent binding unit is an artificial sequence.

In some embodiments, the amino acid sequence of the non-covalent binding unit is a naturally occurring sequence.

In some embodiments, the non-covalent binding unit is or comprises an immunoglobulin domain, e.g., an immunoglobulin constant domain (C domain), e.g., a carboxy-terminal C domain (i.e., CH3 domain), CH1 domain, or CH2 domain, or a sequence substantially identical to the C domain, or a variant thereof. In some embodiments, the non-covalent binding unit is a carboxy-terminal C domain derived from IgG, e.g., derived from IgG3 or IgG1, preferably derived from IgG 1.

Preferably, if the non-covalent binding unit in one junction region comprises a CH3 domain, it does not additionally comprise a CH2 domain, and vice versa.

In some embodiments, the non-covalent binding unit comprises or consists of a carboxy-terminal C domain derived from IgG3 having an amino acid sequence with at least 80% sequence identity to the amino acid sequence of SEQ ID NO. 3.

In preferred embodiments, the non-covalent binding 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 the amino acid sequence of SEQ ID NO:3, 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 non-covalent binding unit comprises or consists of a carboxy-terminal C domain derived from IgG3 having the amino acid sequence of SEQ ID NO: 3.

In some embodiments, the non-covalent binding unit comprises or consists of a carboxy-terminal C domain derived from IgG3 having the amino acid sequence of SEQ ID NO 3, wherein any one amino acid of the flexible unit has been substituted, deleted, or inserted with another amino acid, provided that NO more than 21 amino acids have been so substituted, deleted, or inserted, such as NO more than 20 amino acids, such as NO more than 19 amino acids, such as NO more than 18 amino acids, such as NO more than 17 amino acids, such as NO more than 16 amino acids, such as NO more than 15 amino acids, such as NO more than 14 amino acids, such as NO more than 13 amino acids, such as NO more than 12 amino acids, such as NO more than 11 amino acids, such as NO more than 10 amino acids, such as NO more than 9 amino acids, such as NO more than 8 amino acids, such as NO more than 7 amino acids, such as NO more than 6 amino acids, such as NO more than 5 amino acids, such as NO more than 4 amino acids, such as NO more than 3 amino acids, such as NO more than 2 amino acids, such as NO more than 1 amino acid, such as substituted, or such.

In some embodiments, the non-covalent binding unit comprises or consists of a CH3 domain derived from IgGl having an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID No. 4.

In some preferred embodiments, the non-covalent binding unit comprises or consists of a CH3 domain from IgGl, has an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:4, 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 some embodiments, the non-covalent binding unit comprises or consists of a CH3 domain derived from IgGl having the amino acid sequence of SEQ ID NO 3, wherein any one amino acid of the flexible unit has been substituted, deleted or inserted by another amino acid, provided that NO more than 21 amino acids have been so substituted, deleted or inserted, such as NO more than 20 amino acids, such as NO more than 19 amino acids, such as NO more than 18 amino acids, such as NO more than 17 amino acids, such as NO more than 16 amino acids, such as NO more than 15 amino acids, such as NO more than 14 amino acids, such as NO more than 13 amino acids, such as NO more than 12 amino acids, such as NO more than 11 amino acids, such as NO more than 10 amino acids, such as NO more than 9 amino acids, such as NO more than 8 amino acids, such as NO more than 7 amino acids, such as NO more than 6 amino acids, such as NO more than 5 amino acids, such as NO more than 4 amino acids, such as NO more than 3 amino acids, such as NO more than 2 amino acids, such as NO more than 1 amino acid, such as substituted or such.

In some preferred embodiments, the non-covalent binding unit is or comprises CH3 of IgG 1.

In other embodiments, the non-covalent binding unit is or comprises a leucine zipper motif.

Leucine zippers are common three-dimensional structural motifs in proteins where leucine side chains from one alpha helix cross leucine side chains from another alpha helix, thus promoting dimerization.

Leucine zipper is a dimerization motif of the bZIP (basic region leucine zipper) class of eukaryotic transcription factors. bZIP domains are 60 to 80 amino acids in length, with highly conserved DNA binding basic regions and more diverse leucine zipper dimerization regions. In some embodiments, the non-covalent binding unit is or comprises a leucine zipper motif derived from the bZIP class of eukaryotic transcription factors.

In some embodiments, the non-covalent binding unit is or comprises a Jun/Fos-based leucine zipper. In some embodiments, the non-covalent binding unit is or comprises an ATF6 based leucine zipper. In some embodiments, the non-covalent binding unit is or comprises a PAR-based leucine zipper. In some embodiments, the non-covalent binding unit is or comprises a C/ebpα based leucine zipper. In some embodiments, the non-covalent binding unit is or comprises an OASIS-based leucine zipper.

In a further preferred embodiment, the non-covalent binding unit is or comprises a leucine zipper motif (SEQ ID NO: 5) from a CREB transcription factor (amino acids 308-336).

In a further preferred embodiment, the non-covalent binding unit comprises or consists of an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO. 5, e.g. at least 81% or at least 81%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity.

In a further preferred embodiment, the non-covalent binding unit comprises or consists of the amino acid sequence of SEQ ID NO. 5, wherein any one amino acid of the flexible unit has been substituted, deleted or inserted with another amino acid, provided that NO more than 12, such as NO more than 11, such as NO more than 10, such as NO more than 9, such as NO more than 8, such as NO more than 7, such as NO more than 6, such as NO more than 5, such as NO more than 4 amino acids, such as NO more than 3 amino acids, such as NO more than 2 amino acids, such as NO more than 1 amino acid.

In some embodiments, the junction region comprises a flexible unit and a binding unit that is a covalent or non-covalent binding unit. In some embodiments, the junction region comprises a binding unit comprising both a covalent binding unit and a non-covalent binding unit.

In other embodiments, the junction region comprises a flexible unit, a covalent binding unit, and a non-covalent binding unit. In some embodiments, the non-covalent binding unit is located between the antigenic unit and the covalent binding unit. In other embodiments, the covalent binding unit is located between the antigenic unit and the non-covalent binding unit.

In other embodiments, the junction region comprises a flexible unit and a non-covalent binding unit. In other embodiments, the junction region comprises a flexible unit and a covalent binding unit. In a preferred embodiment, the flexible unit is located closest to the targeting unit, i.e. between the targeting unit and the covalent and/or non-covalent binding unit.

In some embodiments, the junction region further comprises a linker. In a further embodiment, the linker is located between the covalent binding unit and the non-covalent binding unit.

Non-covalent binding units that promote multimerization/conjugation of more than two polypeptides

In addition to linking the antigenic unit and the targeting unit, the non-covalent binding unit also facilitates multimerization/linking of multiple polypeptides (e.g., two, three, four or more polypeptides) into multimeric proteins, such as dimeric, trimeric or tetrameric proteins.

In some embodiments, the non-covalent binding unit is or comprises 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 non-covalent binding unit is a trimerisation unit comprising the amino acid sequence of SEQ ID NO:158 or an amino acid sequence encoded by said nucleic acid sequence, or a combination thereof. In other embodiments, the trimerization unit is the C-terminal domain of T4 fibritin. Thus, in some embodiments, the non-covalent binding unit is a trimerisation unit comprising or consisting of the amino acid sequence of SEQ ID NO 159 or a nucleic acid sequence encoding said nucleic acid sequence.

In other embodiments, the non-covalent binding unit is or comprises a tetramerization unit, e.g. a domain derived from p53, optionally further comprising a hinge region as described below. Thus, in some embodiments, the non-covalent binding unit is a tetramerisation unit comprising or consisting of the nucleic acid sequence of SEQ ID NO. 160 or an amino acid sequence encoded by said nucleic acid sequence, optionally further comprising a hinge region as described below.

Specific embodiments of the junction region

In a preferred embodiment, the junction region comprises hinge exon h1 and hinge exon h4 of IgG 3. In a further preferred embodiment, the junction region comprises or consists of an amino acid sequence having at least 40% sequence identity with the amino acid sequence of SEQ ID NO. 1, e.g. at least 50% sequence identity, at least 60%, at least 70%, at least 80% or at least 90% sequence identity, provided that the number and position of cysteine residues in the sequence are preserved.

In a further preferred embodiment, the junction region comprises or consists of the amino acid sequence of SEQ ID NO. 1, provided that the number and position of cysteine residues in the sequence are preserved, wherein any one amino acid of the flexible unit has been substituted, deleted or inserted by another amino acid, provided that NO more than 16 amino acids have been so substituted, deleted or inserted, such as NO more than 15 amino acids, such as NO more than 14 amino acids, such as NO more than 13 amino acids, such as NO more than 12 amino acids, such as NO more than 11 amino acids, such as NO more than 10 amino acids, such as NO more than 9 amino acids, such as NO more than 8 amino acids, such as NO more than 7 amino acids, such as NO more than 6 amino acids, such as NO more than 5 amino acids, such as NO more than 4 amino acids, such as NO more than 3 amino acids, such as NO more than 2 amino acids, such as NO more than 1 amino acid has been so substituted, deleted or inserted.

In some embodiments, the junction region is hinge exon h1 and hinge exon h4 of IgG 3. In other embodiments, the junction region consists of the amino acid sequence of SEQ ID NO. 1 or comprises the amino acid sequence of SEQ ID NO. 1.

If the above-mentioned junction region is the second junction region, the junction region comprises hinge exons in the order h4 to h1, i.e. the above-mentioned sequence is "flipped" so that the flexible unit h1 is closest to the second targeting unit.

In other preferred embodiments, the junction region comprises a middle hinge region and a lower hinge region of IgG 1. In a further preferred embodiment, the junction region comprises or consists of an amino acid sequence having at least 40% sequence identity with the amino acid sequence of 5-23 of SEQ ID NO. 2, e.g. at least 50% sequence identity, at least 60%, at least 70%, at least 80% or at least 90% sequence identity, provided that the number and position of cysteine residues in the sequence are preserved.

In a further preferred embodiment, the junction region comprises or consists of the amino acid sequence 5-23 of SEQ ID NO. 2, provided that the number and position of cysteine residues in the sequence are preserved, wherein any one amino acid of the flexible unit has been substituted, deleted or inserted by another amino acid, provided that NO more than 11 amino acids have been so substituted, deleted or inserted, such as NO more than 10 amino acids, such as NO more than 9 amino acids, such as NO more than 8 amino acids, such as NO more than 7 amino acids, such as NO more than 6 amino acids, such as NO more than 5 amino acids, such as NO more than 4 amino acids, such as NO more than 3 amino acids, such as NO more than 2 amino acids, such as NO more than 1 amino acid have been so substituted, deleted or inserted.

In some embodiments, the junction region is the middle and lower hinge regions of IgG 1. In other preferred embodiments, the junction region consists of the amino acid sequence of 5-23 of SEQ ID NO. 2 or comprises the amino acid sequence of 5-23 of SEQ ID NO. 2.

If the above-mentioned junction region is the first junction region, the junction region comprises hinge regions in the order of lower hinge region to middle hinge region, i.e. the above-mentioned sequence is "flipped" so that the flexible unit (lower hinge region) is closest to the first targeting unit.

In some embodiments, the junction region comprises hinge exon h1 and hinge exon h4 of IgG3 and/or the junction region comprising the middle and lower hinge regions of IgGl may further comprise a non-covalent binding region, such as the non-covalent binding regions described above, preferably an immunoglobulin constant domain.

Targeting unit

The tolerance-inducing constructs of the present disclosure comprise a first targeting unit and a second targeting unit that target Antigen Presenting Cells (APCs).

The first and second targeting units are attached to the first and second junction regions, respectively, as described herein.

The term "targeting unit" as used herein refers to a unit that delivers a construct of the present disclosure to an antigen presenting cell and interacts with a surface molecule on an APC, e.g., binds to a surface receptor on the APC, without inducing maturation of the cell. APC internalizes constructs and presents T cell epitopes contained in antigenic units on the MHC on its surface in an anti-inflammatory, tolerogenic manner, e.g., by not upregulating costimulatory signals and/or by upregulating inhibitory surface receptors and/or by promoting secretion of inhibitory cytokines. In some embodiments, the targeting unit comprises or consists of a moiety that binds to a surface molecule on an APC selected from the group consisting of tgfβ receptors (including tgfβr1, tgfβr2, and tgfβr3), IL10R (e.g., IL-10RA and IL 10-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, clec B, DCIR, langerin, MR, DC-Sign, treml4, dectin-1, PDL1, 2, HVEM, CD163, and CD141.

In a preferred embodiment, the targeting unit comprises or consists of a moiety that binds to a surface molecule on human (h) APC selected from hTGF beta receptors (including hTGF beta R1, hTGF beta R2 and hTGF beta R3), hIL-10R (e.g. hIL-10RA and hIL-10 RB), hIL-2R, hIL-4R, hIL-6R, hIL-11R, hIL-13R, hIL-27R, hIL-35R, hIL-37R, hGM-CSFR, hFLT3, hCR 7, hCD11b, hCD11c, hCD103, hCD14, hCD36, hCD205, hCD109, hVISTA, hMARCO, hMHCII, hCD83, hSIGLEC, hClec A (hMGL), hASGR (hASGR 1/hASGR 2), hCD80, hCD86, hCD 9A, hClec12A, hClec B, hDCIR, hLangerin, hMR, hDC-Sign, hTreml4, hDectin-1, hDL 1, hPDL2, hCD163, and hCD141.

The moiety may be a natural ligand, an antibody or a portion thereof, such as an 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 the presentation of a T cell epitope contained in an antigenic unit 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 the presentation of the T cell epitope contained in the antigenic unit in an anti-inflammatory, tolerogenic manner. Protein modeling can be used to design such synthetic ligands.

In other embodiments, the moiety is a natural ligand.

In some embodiments, the natural ligand is selected from the group consisting of TGF-beta (e.g., 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 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 IL-10 or TGF-beta, preferably human IL-10 or human TGF-beta.

In other embodiments, the targeting unit comprises or consists of an amino acid sequence that has at least 80% sequence identity to human tgfβ.

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., 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.

In other embodiments, the targeting unit comprises or consists of the amino acid sequence of human tgfβ, 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 an amino acid sequence having at least 80% sequence identity to the amino acid sequence of human IL-10 (SEQ ID NO: 66).

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. 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.

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

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 or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of human SCGB3A2.

In other embodiments, the targeting unit comprises or consists of an amino acid sequence having at least 85% sequence identity with the amino acid sequence of human SCGB3A2, 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%, e.g. at least 99% or e.g. 100% sequence identity.

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

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 VSIG-3.

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., 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.

In other embodiments, the targeting unit comprises or consists of the amino acid sequence of human VSIG-3, 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 is or comprises an antibody or portion thereof, e.g., scFv, specific for CD 205.

Antigenic unit

The antigenic units of the tolerance-inducing constructs of the present disclosure comprise one or more T cell epitopes of self-antigens, including but not limited to T reg epitopes, or inhibitory neoantigens, allergens, alloantigens or xenogeneic antigens.

The antigenic unit is located between the first and second junction regions described herein.

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 one embodiment, the plurality of T cell epitopes are from, i.e. comprised in, the same autoantigen. In another embodiment, the plurality of T cell epitopes are from, i.e., contained in, a plurality of different autoantigens.

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 a plurality of antigens, such as 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 autoantigens, allergens, alloantigens or xenogeneic antigens, wherein each T cell epitope is separated from the other antigens by a linker. Another way of describing the separation of each T cell epitope of a self, allergen, alloantigen or xenogeneic antigen from other T cell epitopes by a linker is that all epitopes are arranged in subunits, with each subunit comprising or consisting of an antigen as described herein and a linker, except for the terminal T cell epitope, i.e. except for the antigen located at the beginning of the N-terminus of the polypeptide or the antigen located at the C-terminus of the polypeptide (i.e. located at the end of an antigenic unit that is 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 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 arranged in subunits separated from each other by linkers.

In some embodiments, the linker is designed to be non-immunogenic. It may be a rigid linker, which means that it does not substantially allow the two amino acid sequences to which it is attached to move 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.

Since the antigens 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 for participation in Multiple Sclerosis (MS), and an antigenic unit 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 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 one embodiment, 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 allergies to cats, and an antigenic unit may comprise, for example, one or more T cell epitopes of Fel d1, i.e. one cell epitope of T 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 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 a patient population or patient, i.e., in end-product therapy.

Personalized tolerance-inducible constructs

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

Taking the patient with MS 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 contained in one or more autoantigens (e.g., autoantigens that have been studied and proposed as autoantigens involved in MS)

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

4) One or more tolerance-inducing test constructs are designed and produced, 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. selected on computer using predictive HLA binding algorithms. After identifying relevant epitopes, the epitopes are ranked according to their ability to bind to the patient's HLA class I/II allele, 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, for example 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

tolerance-inducing constructs for end products

The antigenic units of the finished tolerance-inducing construct preferably comprise hot spots of minimal T cell epitopes, i.e. one or more regions (e.g. having a length of 8-15 amino acids) of the antigen containing multiple minimal T cell epitopes, which regions are intended to be presented by different HLA alleles so as to cover a broad range of subjects, such as ethnic groups or even worldwide groups.

By including such hot spots, the chance of the construct inducing tolerance in a broad range of subjects is maximized.

Further description of antigenic units

The T cell epitopes comprised in the antigenic units of the constructs of the invention have a length of 7 to about 200 amino acids, longer T cell epitopes possibly comprising the hot spot of the smallest 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., about 10 to about 100 amino acids or about 15 to about 100 amino acids or about 20 to about 75 amino acids or about 25 to about 50 amino acids, 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 in length.

T cell epitopes of about 60 to 200 amino acids in length can be divided into shorter sequences and included in antigenic units separated by linkers described herein. For example, a T cell epitope of 150 amino acids in length may be separated into 3 sequences of 50 amino acids each and contained in an antigenic unit, with a linker separating the 3 sequences from each other.

In some embodiments, the length of one T cell epitope is such that the protein does not fold properly. For example, the most well known cat allergen Fel d 1 is a protein formed from two heterodimers, each consisting of two chains, chain 1 comprising 70 amino acid residues and chain 2 comprising 90 or 92 residues. The inclusion of long T cell epitopes of both chains into the antigenic unit can lead to correct folding of the protein and may trigger allergic reactions if more than one IgE on the subject's mast cells and basophils bind to the antigenic unit of the construct.

If longer T cell epitopes are included in the antigenic unit, protein folding can be detected in vitro by, for example, ELISA using antibodies to the protein (e.g., cat allergen) and determining whether the antibodies bind to the T cell epitopes.

In some embodiments, the T cell epitope has a length suitable for presentation by MHC (major histocompatibility complex). MHC molecules are mainly of two classes: MHC class I and MHC class II. The terms MHC class I and MHC class II are used interchangeably herein with HLA class I and HLA class II. HLA (human leukocyte antigen) is the major histocompatibility complex in humans. Thus, in a preferred embodiment, the antigenic unit comprises a T cell epitope having a length suitable for specific presentation on MHC class I or MHC class II. In 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, such as 9 to 30 amino acids, such as 15 to 60 amino acids, such as 15 to 30 amino acids, for MHC class II presentation. In a preferred embodiment, 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, such as the T cell epitope linkers described herein.

In some embodiments, 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.

In some embodiments, the antigenic unit comprises 1 to 10T cell epitopes, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, or 10T cell epitopes, or 11 to 20T cell epitopes, e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20T cell epitopes, or 21 to 30T cell epitopes, e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30T cell epitopes, or 31 to 40T cell epitopes, e.g., 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40T cell epitopes, or 41 to 50T cell epitopes, e.g., 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, 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.

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

In some embodiments, the T cell epitopes are arranged in a sequence from more antigenic to less antigenic along the direction from the multimerization/dimerization unit to the end of the antigenic unit (see figure 1). Alternatively, particularly if the hydrophilicity/hydrophobicity varies greatly between T cell epitopes, the most hydrophobic T cell epitope may be located substantially in the middle of the antigenic unit and the most hydrophilic T cell epitope located closest to the multimerization/dimerization unit or the end of the antigenic unit.

In some embodiments, the T cell epitopes are arranged in order of higher antigenicity to lower antigenicity along 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 in the middle of the antigenic unit and the most hydrophilic T cell epitope located closest to the end of the multimerization unit or antigenic unit.

In some embodiments, the T cell epitopes are arranged in order of higher antigenicity to lower antigenicity along the direction from the dimerization unit to the end of the antigenic unit (see figure 1). Alternatively, especially if the hydrophilicity/hydrophobicity varies greatly between T cell epitopes, the most hydrophobic T cell epitope may be located substantially in the middle of the antigenic unit and the most hydrophilic T cell epitope located closest to the dimerization unit or the end of the antigenic unit.

Since it is only possible that an antigenic unit comprises an odd number of T cell epitopes, it is true that the term "substantially" in this context means that the most hydrophobic T cell epitope is as close to the middle as possible when an antigenic unit comprises an even number of T cell epitopes.

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

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

Alternatively, T cell epitopes may alternate 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 such that there is at least one non-GC-rich T cell epitope between them. In some embodiments, the GC-rich sequences encoding the T cell epitopes are arranged such that there is at least one non-GC-rich T cell sequence between them. 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, the 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 separating each T cell epitope from one or two other T cell epitopes.

T cell epitope linkers are designed to be non-immunogenic and preferably also flexible linkers, which allow presentation of T cell epitopes to the immune system in an optimal way even though the antigenic unit contains 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, 10 to 15 amino acids or 8 to 12 amino acids, e.g. 8, 9, 10, 11 or 12 amino acids. In a specific embodiment, the T cell epitope linker consists of 10 amino acids.

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

Preferably, the T cell epitope linker is a serine (S) and/or glycine (G) rich linker, i.e. a linker comprising a plurality of serine and/or glycine residues. Preferred examples are GGGGSGGGSS (SEQ ID NO: 75), GGGSG (SEQ ID NO: 76), GGGGS (SEQ ID NO: 77), SGSSGS (SEQ ID NO: 78), GGSGG (SEQ ID NO: 79) or variants thereof such as GGGGSGGGGS (SEQ ID NO: 80), (GGGGS) m (SEQ ID NO: 81), (GGGSS) m (SEQ ID NO: 82), (GGSGG) m (SEQ ID NO: 161), (GGGSG) m (SEQ ID NO: 83) or (SGSSGS) m (SEQ ID NO: 84), wherein m is an integer from 1 to 5, such as 1, 2, 3, 4 or 5. In a preferred embodiment, m is 2. In 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: 85), GLGGS (SEQ ID NO: 86), GGLGS (SEQ ID NO: 87), GGGLS (SEQ ID NO: 88), or GGGGL (SEQ ID NO: 89). In other embodiments, the T cell epitope linker comprises or consists of LGGSG (SEQ ID NO: 90), GLGSG (SEQ ID NO: 91), GGLSG (SEQ ID NO: 92), GGGLG (SEQ ID NO: 93) or GGGSL (SEQ ID NO: 94). In other embodiments, the T cell epitope linker comprises or consists of LGGSS (SEQ ID NO: 95), GLGSS (SEQ ID NO: 96) or GGLSS (SEQ ID NO: 97).

In other embodiments, the T cell epitope linker comprises or consists of LGLGS (SEQ ID NO: 98), GLGLS (SEQ ID NO: 99), GLLGS (SEQ ID NO: 100), LGGLS (SEQ ID NO: 101), GLGGL (SEQ ID NO: 102), or (GLGGL) m (SEQ ID NO: 162). In other embodiments, the T cell epitope linker comprises or consists of LGLSG (SEQ ID NO: 103), GLLSG (SEQ ID NO: 104), GGLSL (SEQ ID NO: 105), GGLLG (SEQ ID NO: 106), or GLGSL (SEQ ID NO: 107). In other embodiments, the T cell epitope linker comprises or consists of LGLSS (SEQ ID NO: 108) or GGLLS (SEQ ID NO: 109).

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: 110), GLGGSGGGGS (SEQ ID NO: 111), GGLGSGGGGS (SEQ ID NO: 112), GGGLSGGGGS (SEQ ID NO: 113) or GGGGLGGGGS (SEQ ID NO: 114). In other embodiments, the T cell epitope linker comprises or consists of LGGSGGGGSG (SEQ ID NO: 115), GLGSGGGGSG (SEQ ID NO: 116), GGLSGGGGSG (SEQ ID NO: 117), GGGLGGGGSG (SEQ ID NO: 118) or GGGSLGGGSG (SEQ ID NO: 119). In other embodiments, the T cell epitope linker comprises or consists of LGGSSGGGSS (SEQ ID NO: 120), GLGSSGGGSS (SEQ ID NO: 121), GGLSSGGGSS (SEQ ID NO: 122), GGGLSGGGSS (SEQ ID NO: 123) or GGGSLGGGSS (SEQ ID NO: 124).

In further embodiments, the T cell epitope linker comprises or consists of LGGGSLGGGS (SEQ ID NO: 125), GLGGSGLGGS (SEQ ID NO: 126), GGLGSGGLGS (SEQ ID NO: 127), GGGLSGGGLS (SEQ ID NO: 128) or GGGGLGGGGL (SEQ ID NO: 129). In other embodiments, the T cell epitope linker comprises or consists of LGGSGLGGSG (SEQ ID NO: 130), GLGSGGLGSG (SEQ ID NO: 131), GGLSGGGLSG (SEQ ID NO: 132), GGGLGGGGLG (SEQ ID NO: 133) or GGGSLGGGSL (SEQ ID NO: 134). In other embodiments, the T cell epitope linker comprises or consists of LGGSSLGGSS (SEQ ID NO: 135), GLGSSGLGSS (SEQ ID NO: 136), GGLSSGGLSS (SEQ ID NO: 137).

In other embodiments, the T cell epitope linker comprises or consists of GSGGGA (SEQ ID NO: 138), GSGGGAGSGGGA (SEQ ID NO: 139), GSGGGAGSGGGAGSGGGA (SEQ ID NO: 140), GSGGGAGSGGGAGSGGGAGSGGGA (SEQ ID NO: 141) or GENLYFQSGG (SEQ ID NO: 142). In other embodiments, the T cell epitope linker comprises or consists of SGGGSSGGGS (SEQ ID NO: 143), GGGGSGGGGS (SEQ ID NO: 80), SSGGGSSGGG (SEQ ID NO: 144), GGSGGGGSGG (SEQ ID NO: 145), GSGSGSGSGS (SEQ ID NO: 146), GGGSSGGGSG (SEQ ID NO: 147), GGGSSS (SEQ ID NO: 148), GGGSSGGGSSGGGSS (SEQ ID NO: 149), or GLGGLAAA (SEQ ID NO: 150).

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 subunit linker comprises or consists of KPEPKPAPAPKP (SEQ ID NO: 163), AEAAAKEAAAKA (SEQ ID NO: 164), (EAAAK) mGS (SEQ ID NO: 165), (EAAK) mGS (SEQ ID NO: 39), PSRLEEELRRRLTEP (SEQ ID NO: 166), or SACGELS (SEQ ID NO: 167). 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: 168). 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 one or more glycine, serine, alanine, and threonine residues, such as a linker comprising or consisting of sequence GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 154); or a SEG linker, i.e.a linker comprising one or more serine, glutamic acid and glycine residues, e.g.a linker comprising or consisting of the sequences GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 155) or ELKTPLGDTTHT (SEQ ID NO: 19).

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 and method for producing the same

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 milk allergen. In some embodiments, the 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: 168).

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, an inhibitory protein (profilin), a nsLTP, a sweet protein-like protein (thaumatin-like protein), a gibberellin-regulating protein, an isoflavone reductase-related protein, a class 1 chitinase, a beta 1,3 glucanase, a germinatin-like protein (germin like protein), an alkaline serine protease, a pathogenic related protein 1, actinidin, a plant cystatin (phytostatin), kiwellin, a major 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 type 10, profilin, nsLTP type 1, nsLTP type protein 2, an osmolyte-like protein, an 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 Gly m lectin, gly m Bd28K, gly m 30kD, gly m CPI or 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 h8, ara h 9, ara h 10, ara h 11, ara h 12, ara h 13, ara h 14, ara h 15, ara h 16, or Ara h 17. In some embodiments, the allergen is a woody nut or seed allergen. In some embodiments, the allergen is 11S globulin, 7S globulin, 2S globulin, PR10, PR-14NSLTP, oleosin (oleosin), or an inhibitor protein.

In other embodiments, the food allergen is 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, or Hev b 15.

In some embodiments, the allergen is a dust mite allergen. In some embodiments, the allergen is a house dust mite allergen. In some embodiments, the allergen is a stored dust allergen (storage dust allergen). In some embodiments, the house dust mite allergen is Der p 1, der p 2, der p 3, der p 4, der p 5, der p 7, der p 8, der p 10, der p 11, der p 21, or Der p 23. In some embodiments, the antigenic unit 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 f 2, asp f 3, asp f 4, asp f 5, asp f 6, asp f 7, asp f 8, asp f 9, asp f 10, asp f 11, asp f 12, asp f 13, asp f 14, asp f 15, asp f 16, asp f 17, asp f 18, asp f 22, asp f 23, asp f 27, asp f 28, asp f 29, or Asp f 34.

In some embodiments, the allergen is a fungal allergen. In some embodiments, the fungal allergen is a malassezia allergen. In some embodiments, the malassezia allergen is Mala f 1, mala f 2, mala f 3, mala f 4, mala f 5, mala f 6, mala f 7, mala f 8, mala f 9, mala f 10, mala f 11, mala f 12, or Mala f 13, or MGL_1204.

In some embodiments, the allergen is a furred animal allergen. In some embodiments, the allergen is a 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 horse 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 timothy grass (timothy grass), fescue (orchard grass), kentucky grass (Kentucky bluegrass), perennial rye (perennial rye), sweet spring grass (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, smut (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, cor a 2, cor a 6, cor a 8, cor a 9, cor a 10, cor a 11, cor a 12, cor a 13, cor a 14, ost c 1, cas 15, cas 18, or Cas 1 9, fag s 1, que a 1, fre 1, lig v 1, ole e 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, chamomile, wallflower (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 2, 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 Amar 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 embodiment we provide a construct for multiple sclerosis comprising a short (35-55 amino acids) or longer (27-63 amino acids) T cell epitope 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 as well as in MOG (35-55) -induced C57/BL6 mice and Lewis rats Experimental Autoimmune Encephalomyelitis (EAE).

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 autoantigens are citrullinated proteins, homocysteine proteins, and the 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.

Signal peptides

In some embodiments, the constructs of the present disclosure are polynucleotides 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.

In a preferred embodiment, the polynucleotide comprises a nucleotide sequence encoding a signal peptide comprising the amino acid sequence of SEQ ID NO. 6. In other embodiments, the polynucleotide comprises a nucleotide sequence encoding a signal peptide consisting of an amino acid sequence having at least 80%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or such as at least 99% sequence identity to the amino acid sequence of SEQ ID NO. 6.

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

In some embodiments, the signal peptide comprises or consists of the amino acid sequence of SEQ ID NO. 6, wherein any one amino acid of the signal peptide has been substituted, deleted or inserted with another amino acid, provided that NO more than 5 amino acids have been so substituted, deleted or inserted, such as NO more than 4 amino acids, such as NO more than 3 amino acids, such as NO more than 2 amino acids, or NO more than 1 amino acid have been so substituted, deleted or inserted.

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 that there be amino acid sequence identity 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 acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), aliphatic amino acids (alanine, valine, leucine, isoleucine), polar amino acids (glutamine, asparagine, serine, threonine), aromatic amino acids (phenylalanine, tryptophan, tyrosine), hydroxy amino acids (serine, threonine), large amino acids (phenylalanine, tryptophan) and small amino acids (glycine, alanine).

Substitutions may also be made 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 alkyl groups such as methyl, ethyl or propyl in addition to amino acid spacer groups such as glycine or β -alanine residues. Another form of variation involves the presence of one or more amino acid residues in the peptidomimetic 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 polypeptide comprising, in the order specified:

a. a first targeting unit comprising a first group of targeting units,

b. a first land;

c. an antigenic unit;

d. a second land; and

e. a second targeting unit; wherein the antigenic unit comprises one or more T cell epitopes of autoantigens, allergens, alloantigens or xenogeneic antigens.

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 tolerance-inducing constructs of the present disclosure may be in the form of a polypeptide encoded by a polynucleotide as described above.

Another aspect of the disclosure is a polypeptide comprising, in the order specified:

a. a first targeting unit, a first junction region;

b. An antigenic unit;

c. a second land; and

d. a second targeting unit; wherein the antigenic unit comprises one or more T cell epitopes of autoantigens, allergens, alloantigens or xenogeneic antigens.

The polypeptide may be expressed in vitro to produce a tolerance-inducing construct, e.g., for the production of a pharmaceutical composition comprising the construct, or the polypeptide may be expressed in vivo as a result of administration of the polynucleotide to a subject, as described above. Due to the presence of multimerization/dimerization units, multimerization/dimerization proteins are formed when polypeptides are expressed, i.e., by conjugating multiple polypeptides via their respective multimerization/dimerization units.

Multimeric proteins

Another aspect of the disclosure is a multimeric protein consisting of a plurality of polypeptides, wherein each polypeptide comprises, in the indicated order,

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

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

and wherein the plurality of polypeptides are linked to each other via their respective first junction regions and via their respective second junction regions.

Multimeric proteins may be prepared by in vitro expression of polypeptides.

Accordingly, another aspect of the present disclosure is a method for preparing a multimeric protein consisting of a plurality of polypeptides, wherein each polypeptide comprises, in the indicated order,

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

and wherein the plurality of polypeptides are linked to each other via their respective first junction regions and via their respective second junction regions, wherein the method comprises:

a. transfecting a cell with a polynucleotide comprising a nucleotide sequence encoding the polypeptide;

b. culturing the cells;

c. collecting multimeric proteins from the cells; and

d. isolating and purifying the fraction of multimeric proteins, wherein the plurality of polypeptides are linked to each other via their respective first junction regions and via their respective second junction regions.

Isolation and optional purification of the multimeric protein in step d may be carried out by methods known in the art, including precipitation, differential solubilization and chromatography.

The multimeric proteins of the present disclosure may be used as active ingredients in protein vaccines for prophylactic or therapeutic treatment of autoimmune diseases, allergic diseases and graft rejection.

The multimeric/dimeric protein may be a homomultimer or a heteromultimer, e.g., if the protein is a dimeric protein, the dimeric protein may be a homodimer, i.e., a dimeric protein in which the two polypeptide chains are identical and 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 in its antigenic unit a different antigen sequence than polypeptide 2. The latter may be of interest if the number of antigens incorporated into the antigenic unit exceeds the upper size limit of the antigenic unit. Preferably, the dimeric protein is a homodimeric protein.

Dimeric proteins

Another aspect of the disclosure is a dimeric protein consisting of two polypeptides, wherein each polypeptide comprises, in the order indicated,

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

And wherein the two polypeptides are linked to each other via their respective first junction regions and via their respective second junction regions.

Dimeric proteins may be prepared by in vitro expression of the polypeptides.

Another aspect of the present disclosure is a method for preparing a dimeric protein consisting of two polypeptides, wherein each polypeptide comprises, in the order indicated,

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

and wherein the two polypeptides are linked to each other via their respective first junction regions and via their respective second junction regions, wherein the method comprises:

b. culturing the cells;

c. collecting the dimeric protein from the cells; and

d. a fraction of the dimeric protein is isolated and purified, wherein the two polypeptides are linked to each other via their respective first junction regions and via their respective second junction regions.

Isolation and purification of the dimeric protein in step d) may be carried out by methods known in the art, including precipitation, differential solubilization and chromatography.

The dimeric proteins of the present disclosure may be used as active ingredients in protein vaccines for prophylactic or therapeutic treatment of autoimmune diseases, allergic diseases and graft rejection.

Carrier body

The polynucleotide sequence of the tolerance-inducing construct may be a DNA polynucleotide comprised in a vector suitable for transfection of host cells and expression of the polypeptide or multimeric/dimeric protein encoded by said polynucleotide, i.e. an expression vector, e.g. a DNA plasmid or a viral vector, preferably a DNA plasmid. In another embodiment, the vector is suitable for transfecting a host cell and expressing mRNA encoding the polypeptide or multimeric/dimeric protein.

The vector of the present invention 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 one embodiment, the vector is a DNA vector, such as a DNA plasmid, or a DNA viral vector, such as a DNA viral vector selected from the group consisting of adenovirus, vaccinia virus, adeno-associated virus, cytomegalovirus, and sendai virus.

In another embodiment, the vector is an RNA vector, such as an RNA plasmid, or an RNA viral vector, such as a retroviral vector 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.

Preferably, the vector allows easy exchange of the various units described above, in particular antigenic units in the case of an individualized tolerance-inducing construct.

Accordingly, the present disclosure provides a vector comprising a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the order specified:

a. a first targeting unit, a first junction region;

b. an antigenic unit comprising at least one T cell epitope;

c. a second land; and

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

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: 102) and/or GLSGL (SEQ ID NO: 40) unit linker and the 3' site is comprised after the stop codon in the vector.

In a preferred embodiment, the vector is a DNA plasmid and the polynucleotide is DNA.

DNA plasmid

A plasmid is a small extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. 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 near the transcription initiation site of the gene upstream of 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 the 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 exports the cell and performs its function elsewhere, sometimes after a considerable amount of post-translational modification.

When the protein is finally 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 invention 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 by methods known in the art to comprise the elements and units of the present disclosure.

Host cells

In some embodiments, the present disclosure provides host cells comprising the vectors described herein.

In some embodiments, the present disclosure provides a host cell comprising:

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

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

ii) a vector comprising said polynucleotide.

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.

Polycistronic vector

In some embodiments, the above-described vectors are polycistronic vectors, allowing for expression of the polypeptides of the present disclosure, and further allowing for expression of one or more immunosuppressive compounds as separate molecules.

Another aspect of the disclosure is a vector comprising:

(A) A polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising in the indicated order

a. A first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

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

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

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

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

The polycistronic vector of the present disclosure may be any suitable vector, such as a DNA plasmid or a viral vector, such as a retroviral vector. In a preferred embodiment, the vector is a polycistronic DNA plasmid. The polycistronic vectors of the present disclosure will be described by discussing DNA plasmids (i.e., polycistronic DNA plasmids of the present disclosure), but it should be understood that the discussion is also applicable to other vectors, such as viral vectors.

Polycistronic plasmids are known in the art, and thus, the skilled artisan is able to design and construct the polycistronic plasmids of the present disclosure.

In a preferred embodiment, the polycistronic plasmid of the present disclosure comprises one or more coexpression elements, i.e., nucleic acid sequences that allow for co-expression of the polypeptide and the one or more immunosuppressive compounds from the plasmid as separate molecules.

In some embodiments of the invention, the polycistronic plasmid comprises a co-expression element that results in transcription of the polypeptide and the one or more immunosuppressive compounds on a single transcript, but independent translation into the polypeptide and the one or more immunosuppressive compounds. Thus, the presence of the coexpression element results in the final production of an individual translation product.

In some embodiments, such co-expression elements are IRES elements (internal ribosome entry sites). In other embodiments, such co-expression elements are 2A self-cleaving peptides (2A peptides). Both of these co-expression elements are known in the art.

If the polycistronic plasmids of the present disclosure express more than one immunosuppressive compound, then the IRES element and/or the 2A peptide need to be present in the plasmid, e.g., upstream of each nucleic acid sequence encoding the immunosuppressive compound.

In other embodiments, the polycistronic plasmid comprises a co-expression element that results in transcription of the polypeptide and the one or more immunosuppressive compounds into separate transcripts, which produce separate transcripts and thus separate proteins.

In some embodiments, such a co-expression element is a bi-directional promoter.

In other embodiments, such coexpression elements are the various promoters, i.e., the polycistronic plasmid comprises a promoter encoding each nucleotide sequence of the polypeptide or the one or more immunosuppressive compounds. Both of these co-expression elements are known in the art.

The above-described co-expression elements may be combined in any manner, i.e., the polycistronic plasmid of the present disclosure may comprise one or more such identical or different co-expression elements.

Immunosuppressive compounds

The polycistronic plasmids of the present disclosure comprise one or more nucleic acid sequences encoding one or more immunosuppressive compounds.

In some embodiments of the present disclosure, an immunosuppressive compound is a compound known to induce, increase, or maintain immune tolerance.

In some embodiments of the present disclosure, the immunosuppressive compound is the extracellular portion of an inhibitory checkpoint molecule. In some embodiments, the inhibitory checkpoint molecule is selected from the group consisting of CLTA-4 (SEQ ID NO: 72), PD-1 (SEQ ID NO: 74), BTLA and TIM-3. In some embodiments, the inhibitory checkpoint molecule is CLTA-4 (SEQ ID NO: 72). In some embodiments, the inhibitory checkpoint molecule is PD-1 (SEQ ID NO: 74). In some embodiments, the inhibitory checkpoint molecule is BTLA. In some embodiments, the inhibitory checkpoint molecule is TIM-3. In some embodiments of the present disclosure, the immunosuppressive compound is a cytokine selected from the group consisting of IL-10 (SEQ ID NO: 66), TGF-beta 1 (SEQ ID NO: 60), TGF-beta 2 (SEQ ID NO: 62), TGF-beta 3 (SEQ ID NO: 64), IL-27, IL-2, IL-37, and IL-35. In some embodiments, the cytokine is IL-10 (SEQ ID NO: 66). In some embodiments, the cytokine is TGF-beta 1 (SEQ ID NO: 60). In some embodiments, the cytokine is TGF-beta 2 (SEQ ID NO: 62). In some embodiments, the cytokine is TGF-beta 3 (SEQ ID NO: 64). In some embodiments, the cytokine is IL-27. In some embodiments, the cytokine is IL-2. In some embodiments, the cytokine is IL-37. In some embodiments, the cytokine is IL-35.

In some embodiments of the disclosure, the DNA plasmid comprises a nucleic acid sequence encoding 2, 3, 4, 5, 6, 7, or 8 immunosuppressive compounds. In a preferred embodiment, the DNA plasmid comprises a nucleic acid sequence encoding 2 to 6 immunosuppressive compounds, e.g. 2 or 3 or 4 or 5 or 6 different immunosuppressive compounds. The immunosuppressive compounds may be the same or different, preferably different.

In preferred embodiments, different immunosuppressive compounds produce or promote tolerance-inducing environments at a number of different levels. For example, a plasmid of the present disclosure may comprise nucleic acid sequences encoding 3 different immunosuppressive compounds, wherein the first induces tolerance, the second increases tolerance and the third maintains tolerance.

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 protein (e.g., a dimeric protein)) and a pharmaceutically acceptable carrier.

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 multimeric protein) and a pharmaceutically acceptable carrier.

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 dimeric protein) and a pharmaceutically acceptable carrier.

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

i) A polypeptide comprising, in the order indicated:

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

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

i) A polypeptide comprising, in the order indicated:

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

i) A polypeptide comprising, in the order indicated:

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

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 pharmaceutically acceptable carrier or diluent is an aqueous buffer. In other embodiments, the aqueous buffer is a Tyrode buffer, e.g., a Tyrode buffer comprising 140mM NaCl, 6mM KCl, 3mM CaCl2, 2mM MgCl2, 10mM 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid (Hepes) pH7.4, and 10mM glucose.

Suitable adjuvants may include, but are not limited to, dexamethasone, the B subunit of the enterotoxin Cholera Toxin (CTB), TLR2 ligands, worm-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 poly (ethylene oxide) ("PEO") blocks and poly (propylene oxide) ("PPO") blocks. 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 ethylenediamido.

"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 (poloxamer 188 according to the manufacturer's data sheet) exhibit large molecular weight variations (between 7,680 and 9,510), and the values of a and b provided for these specific products are about 79 and 28, respectively. This reflects the heterogeneity of the block copolymer, indicating that the values of a and b are the average values in the most formulated recipe.

"Poloxamine" or "sequential Poloxamine" (under the trade nameCommercially available) is an X-block copolymer having four PEO-PPO arms that are linked to a central ethylenediamine moiety through a bond between a free OH group contained in the PEO-PPO-arm and a primary amine group in the ethylenediamine moiety. Reverse poloxamine is also an X-shaped blockA segmented copolymer having four PPO-PEO arms that are linked to a central ethylenediamine moiety through a bond between a free OH group contained in the PPO-PEO arm and a primary amine group in ethylenediamine.

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 respectively904. 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 also 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 intradermal or intramuscular injection.

In some embodiments, the pharmaceutical composition comprising a polynucleotide described herein (e.g., contained in a vector such as a polycistronic vector) may be administered in any manner suitable for administration to a subject, such as by intradermal, intramuscular, or subcutaneous injection, or by mucosal or epithelial administration, such as intranasal or oral administration.

In a preferred embodiment, the pharmaceutical composition comprises a polynucleotide as described herein, e.g., in a vector such as a polycistronic vector, and is administered by intramuscular or intradermal injection.

The pharmaceutical compositions of the present disclosure typically comprise from 0.1 μg to 10mg polynucleotide, e.g., 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. The pharmaceutical compositions of the present disclosure typically comprise 5 μg to 5mg of the polypeptide/dimer protein.

The amount of the polynucleotide/polypeptide/multimeric or dimeric protein may be dependent 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 medical history.

Process for preparing pharmaceutical compositions

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 of preparing a pharmaceutical composition comprising a multimeric protein, such as a dimeric protein, or a 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.

Accordingly, another aspect of the invention is a method of preparing a pharmaceutical composition comprising a multimeric protein, such as a dimeric protein, or polypeptide, consisting of a plurality of polypeptides, wherein the method comprises:

a) Transfecting a cell with a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising in the indicated order:

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit; 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 the multimeric protein, e.g., a dimeric protein, or the polypeptide expressed from the cell; and

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

Accordingly, another aspect of the present disclosure is a method of preparing a pharmaceutical composition comprising a multimeric protein or polypeptide consisting of a plurality of polypeptides, wherein the method comprises:

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

b) Culturing the cells;

c) Collecting and purifying the multimeric protein or polypeptide 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 present disclosure is a method of preparing a pharmaceutical composition comprising a dimeric protein or polypeptide consisting of two polypeptides, wherein the method comprises:

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

b) Culturing the cells;

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

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

In some embodiments, the polynucleotide is comprised in a vector described herein.

In a preferred embodiment, the multimeric protein, e.g. a 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 dissolution.

In another aspect, the present disclosure relates to a method of preparing a pharmaceutical composition comprising a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the indicated order,

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting 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

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

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

The expression vector may be any vector described herein.

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 described herein as a medicament.

Medicament

In one aspect, the disclosure relates to the use of a construct, polynucleotide, polypeptide, multimeric protein, or dimeric protein 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 it induces tolerance in the individual to whom such pharmaceutical composition is administered. Tolerance is induced by a single administration and preferably by multiple administrations at sufficiently spaced intervals over time.

In a further aspect, the present disclosure provides a method of treating a subject suffering from or in need of prophylaxis of an immune disorder selected from the group consisting of autoimmune disorders, allergic disorders, and graft rejection, the method comprising administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable carrier and

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

In another aspect, the present disclosure provides a method of treating a subject suffering from or in need of prophylaxis of an immune disorder selected from the group consisting of autoimmune disorders, allergic disorders, and graft rejection, the method comprising administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable carrier and i) a polynucleotide comprising a nucleotide sequence encoding a polypeptide comprising, in the order named:

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

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

In yet 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 a pharmaceutically acceptable carrier and

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

iii) A multimeric protein consisting of a plurality of polypeptides encoded by the nucleotides defined in i), for example a dimeric protein consisting of two polypeptides encoded by the nucleotides defined in i).

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

iii) A multimeric protein consisting of a plurality of polypeptides encoded by the nucleotides defined in i).

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

iii) A dimeric protein consisting of two polypeptides encoded by the nucleotides defined in i).

The first and second targeting units, the first and second junction regions, and the antigenic unit are described in detail above.

In yet another aspect, the present disclosure provides a pharmaceutical composition for the prophylactic or therapeutic treatment of a subject suffering from or suspected of suffering from an immune disorder selected from the group consisting of autoimmune disorders, allergic disorders, and transplant rejection, the pharmaceutical composition comprising a pharmaceutically acceptable carrier and

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

iii) A multimeric protein consisting of a polypeptide encoded by a plurality of nucleotides as defined in i), for example a dimeric protein consisting of polypeptides encoded by two nucleotides as defined in i),

wherein the pharmaceutical composition is administered to the subject.

a. a first targeting unit, a first junction region;

b. An antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

iii) A multimeric protein consisting of a plurality of polypeptides encoded by the nucleotides defined in i),

wherein the pharmaceutical composition is administered to the subject.

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

iii) A dimeric protein consisting of two polypeptides encoded by the nucleotides defined in i),

wherein the pharmaceutical composition is administered to the subject.

In yet another aspect, the present disclosure provides the use of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

iii) A multimeric protein consisting of a polypeptide encoded by a plurality of nucleotides as defined in i), for example a dimeric protein consisting of polypeptides encoded by two nucleotides as defined in i).

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

a. A first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. A second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

iii) A multimeric protein, e.g. a dimeric protein, consisting of a plurality of polypeptides encoded by the nucleotides defined in i).

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

Furthermore, disclosed herein is a use of a pharmaceutical composition for the preparation of a medicament for the prophylactic or therapeutic treatment of a subject suffering from an immune disorder selected from the group consisting of autoimmune disorders, allergic disorders and graft rejection, wherein the medicament is administered to the subject, the pharmaceutical composition comprising a pharmaceutically acceptable carrier and

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

Furthermore, disclosed herein is the use of a pharmaceutical composition for the prophylactic or therapeutic treatment of a subject suffering from an immune disorder selected from the group consisting of autoimmune disorders, allergic disorders and graft rejection, wherein the medicament is administered to the subject, the pharmaceutical composition comprising a pharmaceutically acceptable carrier and

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. A second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

Further, disclosed herein are pharmaceutical compositions comprising a pharmaceutically acceptable carrier and

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

for use in the prophylactic or therapeutic treatment of an immune disorder selected from autoimmune disorders, allergic disorders and graft rejection.

a. A first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

iii) A dimeric protein consisting of a plurality of polypeptides encoded by the nucleotides defined in i),

Further, disclosed herein is a medicament for prophylactic or therapeutic treatment of a subject suffering from an immune disorder selected from the group consisting of autoimmune disorders, allergic disorders, and graft rejection by administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable carrier and

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

Also disclosed herein is a medicament for the prophylactic or therapeutic treatment of a subject suffering from an immune disorder selected from the group consisting of autoimmune disorders, allergic disorders, and transplant rejection by administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable carrier and

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

a. A first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

iii) A dimeric protein consisting of a plurality of polypeptides encoded by the nucleotides defined in i).

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

Examples

Example 1: design, production and in vitro expression of tolerance-inducing constructs of the invention for the treatment of multiple sclerosis Sign of sign

Myelin Oligodendrocyte Glycoprotein (MOG) is a protein expressed in the central nervous system. Immunodominant 35-55 epitope of MOG, MOG (35-55), is the primary target of cellular and humoral immune responses during multiple sclerosis. MOG (35-55) -induced Experimental Autoimmune Encephalomyelitis (EAE) is the most commonly used animal model of multiple sclerosis (Hunterman, H.et al 2022).

Design of DNA vector

All the gene sequences described were ordered from GenScript (Genscript Biotech BV, netherlands) and cloned into the expression vector pALD-CV 77. A DNA vector was designed comprising a nucleotide sequence encoding the following units/parts:

1. signal peptides

2. First targeting unit

3. A first junction region: hinge region 1 from human IgG3 (amino acids 1-12 of SEQ ID NO: 1), hinge region 4 from human IgG3 (amino acids 13-27 of SEQ ID NO: 1), glycine-leucine linker (SEQ ID NO: 102)

4. Antigenic unit: MOG (27-63) (SEQ ID NO: 12)

5. Second junction region

6. A second targeting unit

Table 1 depicts the differences between the vectors, including insertion of hinge region 1 from human IgG3 in the targeting unit and the second dimerization unit.

TABLE 1

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* Antigenic unit: murine Myelin Oligodendrocyte Glycoprotein (MOG) 27-63 sequence, obtained from Krienke et al (Science 371,145-153,2021) (SEQ ID NO: 13), patent application US2020061166A1.

* Antigenic unit: MOG (35-55) (SEQ ID NO: 14)

* Extracellular domain

Plasmid DNA vectors VB5038, VB5041, VB5042, VB5043, VB5050, VB5066, VB5067, VB5072, VB5073, VB5074 and VB5075 are vectors of the disclosure and encode tolerance-inducing constructs comprising targeting units, dimerization units and antigenic units as shown in table 1.

The vectors used as controls are shown in table 2.

TABLE 2

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* MOG (27-63) sequences were obtained from Krienke et al 2021, patent application US2020061166A1.

The DNA vectors VB5052 (SEQ ID NO: 33) and VB5002b (SEQ ID NO: 34) encode fusion proteins comprising a human CCL3L1 targeting unit known to target APCs in a pro-inflammatory manner, i.e. an antigen specific construct comprising such a targeting unit will induce an inflammatory immune response in the subject to which it is administered, and which compound is expected to induce IFN- γ production (see e.g. WO2011161244 A1).

The DNA vectors VB5051 (SEQ ID NO: 35) and VB5001b (SEQ ID NO: 36) encode only the antigenic unit MOG (27-63), i.e.a single protein/peptide.

The murine MOG (27-63) antigenic unit comprises the T cell epitope MOG (35-55).

Protein expression and secretion of MOG-containing constructs

The aim of this experiment was to characterize protein expression and secretion in mammalian cell supernatants transiently transfected with a MOG-containing DNA vector.

Expi293F cells were obtained from Thermo Fisher Sci and transiently transfected with DNA vectors (VB 5042, VB5050, VB5067, VB5072, VB4073, VB5074 and VB 5075) containing MOG (27-63). Briefly, expi293F cells (1.7x10 ⁶ Individual cells/ml, 1 ml) were inoculated into 96-well culture plates. Cells were transfected with 0.64 μg/ml plasmid DNA using the ExpiFectamine 293 reagent (Thermo Fisher Sci.) and the plates were humidified in CO ₂ Cell incubator (8% CO) ₂ Incubate on an orbital shaker (diameter 3mm,900 rpm) at 37 ℃. Supernatants were harvested 72 hours post transfection.

HEK293 cells were obtained from ATCC and transiently transfected with DNA vector VB5038 containing MOG (27-63). Briefly, 2x10 ⁵ Individual cells/wells were plated in 24-well tissue culture plates containing 10% fbs growth medium and used under conditions recommended by the manufacturer (Thermo Fischer Scientific)2000 reagents were transfected with 1. Mu.g of the corresponding DNA vector. The transfected cells were incubated at 37℃with 5% CO ₂ For 5 days, and cell supernatants were collected.

Secreted proteins encoded by the MOG-containing vectors were characterized by sandwich ELISA of supernatants of transiently transfected Expi293F cells or HEK293 cells using antibodies against one of the MOG and targeting units. The results are shown in FIGS. 4-6.

FIGS. 4A and 4B show that all IL-10 encoding tolerance-inducing constructs were expressed and secreted in vitro using ELISA, mouse anti-MOG antibody as capture antibody (0.25. Mu.g/ml, 100. Mu.l/well, sc-73330,Santa Cruz Biotechnology) and goat anti-mouse IL-10 biotinylated antibody as detection antibody (0.8. Mu.g/ml, 100. Mu.l/well, BAF417, R & D Systems).

FIG. 5 shows expression and secretion of tolerance-inducing constructs encoding CTLA-4 in vitro using ELISA, mouse anti-MOG antibody as capture antibody (0.25. Mu.g/ml, 100. Mu.l/well, sc-73330,Santa Cruz Biotechnology) and goat anti-mouse CTLA-4 biotinylated antibody as detection antibody (0.8. Mu.g/ml, 100. Mu.l/well, BAF476, R & D Systems).

FIG. 6 shows expression and secretion of tolerance-inducing constructs encoding SCGB3A2 in vitro using ELISA, mouse anti-MOG antibody as capture antibody (0.25. Mu.g/ml, 100. Mu.l/well, sc-73330,Santa Cruz Biotechnology) and goat anti-mouse SCGB3A2 as detection antibody (3.3. Mu.g/ml, 100. Mu.l/well, BAF3465, R & D Systems).

Secretion of full-length tolerance-inducing constructs with SCGB3A2 and IL-10 as the first and second targeting units, respectively, was verified by sandwich ELISA on supernatants using antibodies against murine IL-10 (capture antibodies: rat anti-murine IL-10 antibodies, 2 μg/ml,100 μl/well, MAB417, R & D Systems) and murine SCGB3A2 (detection antibodies: goat anti-murine SCGB3A2,3.3 μg/ml,100 μl/well, BAF3465, R & D Systems). The results are shown in figure 7, which demonstrates that the vaccine encoded by DNA vectors VB5073 and VB5072 with IL-10 and SCGB3A2 as targeting units (with and without additional copies of hinge region 1 from human IgG3 in the second dimerization unit) is expressed and secreted as a full length fusion protein.

In vitro characterization of tolerance-inducible constructs binding to the DEC205 receptor

The aim of this experiment was to characterize the functional binding of scFv anti-DEC 205 targeting units to the recombinant DEC205 receptor. Functional binding of the targeting units was assessed in supernatants of HEK293 cells transiently transfected with DNA vector VB5038 encoding scFv anti-DEC 205 as the first targeting unit in ELISA by coating ELISA plates with recombinant DEC205 receptor and using antibodies against the antigenic unit or the second targeting unit as detection antibodies.

HEK293 cells were obtained from ATCC and transiently transfected with VB 5038. Briefly, 2x10 ⁵ Individual cells/wells were plated in 24-well tissue culture plates containing 10% fbs growth medium and used under conditions recommended by the manufacturer (Invitrogen, thermo Fischer Scientific)2000 reagents were transfected with 1. Mu.g of the corresponding DNA vector. The transfected cells were incubated at 37℃with 5% CO ₂ For 5 days, and cell supernatants were collected. Secreted proteins encoded by VB5038 were assessed by direct ELISA in the supernatant of transiently transfected cells. ELISA plates were coated with 100. Mu.l/well of 5. Mu.g/ml recombinant DEC205 receptor (aa 216-503,OPCD05072,Aviva Systems Biology) and blocked prior to addition of supernatant. By administering a therapeutic agent against MOG (mouse anti-MOG antibody, 1. Mu.g/ml, 100. Mu.l/well, sc-73330,Santa Cruz Biotechnology) or murine IL-10 (goat anti-murine IL-10 biotinylated antibody, 1. Mu.g/ml, 100. Mu.l/well, BAF417, R)&D Systems), detecting binding of vaccine proteins to recombinant receptors.

The results shown in figure 8 demonstrate binding of VB5038 containing scFv against DEC205 to DEC205 receptor and secretion of full length fusion protein.

In vitro characterization of tolerance-inducible constructs binding to IL-10 receptor

The aim of this experiment was to characterize the functional binding of IL-10 targeting units to recombinant IL-10 receptors. Functional binding of the targeting units was assessed in supernatants of HEK293 cells transiently transfected with DNA vaccine encoding IL-10 as the second targeting unit in ELISA by coating ELISA plates with recombinant IL-10 receptor (IL-10R) and using antibodies against the antigenic units of the detection antibodies.

HEK293 cells were obtained from ATCC and transiently transfected with VB 5038. Briefly, 2x10 ⁵ Individual cells/wells were plated in 24-well tissue culture plates containing 10% fbs growth medium and used under conditions recommended by the manufacturer (Invitrogen, thermo Fischer Scientific)2000 reagents were transfected with 1. Mu.g of the corresponding DNA vector. The transfected cells were incubated at 37℃with 5% CO ₂ For 5 days, and cell supernatants were collected. Secreted proteins encoded by VB5038 were assessed by direct ELISA in the supernatant of transiently transfected cells. ELISA plates were coated with 100. Mu.l/well of 2.5. Mu.g/ml recombinant IL-10 receptor and blocked prior to addition of supernatant. Binding of the vaccine protein to the recombinant receptor was detected by antibodies against MOG (100. Mu.l/well, 1. Mu.g/ml mouse anti-MOG antibody, sc-73330,Santa Cruz Biotechnology).

The results shown in FIG. 9 demonstrate that tolerance-inducing construct proteins with IL-10 as the second targeting unit are capable of binding to IL-10 receptor.

Protein expression and secretion after transient transfection of MOG (27-63) peptide encoded in vector VB5051 into mammalian cells In vitro characterization

The purpose of this experiment was to evaluate protein expression and secretion in transiently transfected mammalian cells in vitro of a MOG (27-63) antigen encoded by vector VB5051 alone control. Briefly, expi293F cells (2 x10 ⁶ Cells/ml, 1 ml) were inoculated in 96-well culture plates. Cells were transfected with 0.64 μg/ml plasmid DNA using the ExpiFectamine293 reagent (Thermo Fisher Sci.) and the plates were humidified in CO ₂ Cell incubator (8% CO) ₂ Incubate on an orbital shaker (diameter 3mm,900 rpm) at 37 ℃). Supernatants were harvested 72 hours post transfection.

Secretion of MOG (27-63) peptides was characterized by direct ELISA, coated with supernatant and detected using antibodies against MOG (capture antibody, 100 μl/well, 3.3 μg/ml mouse anti-MOG antibody, sc-73330,Santa Cruz Biotechnology). FIG. 10 shows that MOG (27-63) peptide is expressed by VB5051 and secreted by mammalian cells transfected with the vector.

After transient transfection of mammalian cells with the pro-inflammatory control constructs encoded by the DNA vectors VB5052 and VB5002b, eggs In vitro characterization of white expression and secretion

The DNA vectors VB5052 (SEQ ID NO: 33) and VB5002b (SEQ ID NO: 34) encode fusion proteins comprising a human CCL3L1 targeting unit known to target APCs in a pro-inflammatory manner, i.e. antigen specific vaccines comprising such targeting units will induce an inflammatory immune response in the subject to which they are administered, and the compounds are expected to induce IFN- γ production (see e.g. WO2011161244 A1).

The purpose of these experiments was to characterize the protein expression and secretion of the proteins encoded by the DNA vectors VB5052 and VB5002b in transiently transfected mammalian cells.

The Expi293F cells were obtained from Thermo Fisher Sci and transiently transfected with the DNA vector VB 5052. Briefly, expi293F cells (1.7x10 ⁶ Individual cells/ml, 1 ml) were inoculated into 96-well culture plates. Cells were transfected with 0.64 μg/ml plasmid DNA using the ExpiFectamine 293 reagent (Thermo Fisher Sci.) and the plates were humidified in CO ₂ Cell incubator (8% CO) ₂ Incubate on an orbital shaker (diameter 3mm,900 rpm) at 37 ℃. Supernatants were harvested 72 hours post transfection.

HEK293 cells were obtained from ATCC and transiently transfected with DNA vector VB5002 b. Briefly, 2x10 ⁵ Individual cells/well were inoculated into 24-well tissue culture plates containing 10% fbs growth medium and used under conditions recommended by the manufacturer (Thermo Fischer Scientific)2000 reagents were transfected with 1. Mu.g of the corresponding DNA vector. The transfected cells were incubated at 37℃with 5% CO ₂ Incubation under. Supernatants were harvested 5 days after transfection.

Secreted proteins encoded by DNA vector VB5052 were evaluated by sandwich ELISA in supernatants of transiently transfected cells using antibodies against MOG (mouse anti-MOG, 0.25 μg/ml,100 μl/well, sc-73330,Santa Cruz Biotechnology) and antibodies to human CCL3L1 (goat anti-human CCL3, 0.2 μg/ml,100 μl/well, BAF270, R & D Systems). The results are shown in fig. 11A, which shows that the pro-inflammatory control vaccine encoded by vector VB5052 is highly expressed and secreted as a full-length fusion protein.

The secreted protein encoded by DNA vector VB5002b was evaluated by sandwich ELISA in the supernatant of transiently transfected cells using antibodies against human IgG3 (CH 3) (mouse anti-human IgG (CH 3 domain), 1 μg/ml,100 μl/well, 153272, biorad) and antibodies to human CCL3L1 (goat anti-human CCL3,0.2 μg/ml,100 μl/well, BAF270, R & D Systems). The results are shown in fig. 11B, which shows that the immunogenic control vaccine encoded by vector VB5002B is expressed and secreted in protein form.

The DNA vectors VB5038, VB5041, VB5042, VB5050, VB5074 and VB5075 are subjected to Western blotting Characterization of the protein of interest

The supernatants of transfected Expi293F cells were subjected to western blot analysis to further characterize the proteins encoded by DNA vectors VB5038, VB5041, VB5042, VB5050, VB5074, VB5075 and VB5002 b.

The Expi293F cells were obtained from Thermo Fisher Sci and transiently transfected with DNA vectors VB5038 and VB5002 b. Briefly, expi293F cells (3 x10 ⁶ Individual cells/ml, 1.6 ml) was inoculated into 6-well culture plates. Cells were transfected with 1. Mu.g/ml plasmid DNA using the ExpiFectamine 293 reagent (Thermo Fisher Sci.) and plates were incubated on an orbital shaker (diameter 19mm,125 rpm) in a humidified CO2 cell incubator (8% CO2, 37 ℃). After 18 hours of incubation, an expictamine 293 transfection enhancer (Thermo Fisher sci.) was added to each well. Plates were incubated for an additional 78 hours and then supernatants were harvested. By mixing 105. Mu.l of supernatant from transfected Expi293F cells with 37.5. Mu.l of 4 XLaemmli sample buffer (Bio-Rad) and 7.5. Mu.l of DTT (Thermo Fisher Sci.) or 7.5 Mu.l of ultrapure water (for reducing and non-reducing conditions, respectively) was mixed to prepare a sample. The sample (reduced or non-reduced) was heated at 70℃for 10 minutes and then added (the added sample volume is shown in the drawing heading) to a 4% -20%Criterion TGX Stain-Free pre-gel (Bio-Rad). SDS-PAGE was performed in 1 XTris/glycine/SDS running buffer (Bio-Rad) using Precision Plus Protein All Blue pre-stained protein standards (Bio-Rad). Proteins were transferred from the gel onto EtOH-activated Low Fluorescence (LF) 0.45 μm PVDF membrane (Bio-Rad) using a Tran-Blot Turbo semi-dry transfer membrane system (Bio-Rad). PVDF membrane was blocked in EveryBlot buffer (Bio-Rad) for 5 min and used with mouse anti-MOG (sc-73330,Santa Cruz Biotechnology) and rat anti-mouse IL-10 antibodies (MAB 417, R)&D systems) detection to detect MOG and IL-10, respectively. The membrane was incubated with the fluorochrome conjugated secondary antibody for 1 hour at room temperature, then washed and dried. Using ChemiDoc ^TM MP imaging system (set Dyight 488 and 800, auto Optimal) acquires images. The results are shown in FIGS. 12A and 12B.

The Expi293F cells were obtained from Thermo Fisher Sci and transiently transfected with DNA vectors VB5041, VB5042, VB5050, VB5074 and VB 5075. Briefly, expi293F cells (2 or 1.7x10 ⁶ 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 the plates were humidified in CO ₂ Cell incubator (8% CO) ₂ Incubate on an orbital shaker (diameter 3mm,900 rpm) at 37 ℃. Plates were incubated for 72 hours and then supernatants were harvested. 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) (scale up of total sample volume according to the given ratio). The sample (reduced or non-reduced) was heated at 70℃for 10 minutes and then added (the added sample volume is shown in the drawing heading) to a 4% -20%Criterion TGX Stain-Free pre-gel (Bio-Rad). SDS-PAGE was performed in 1 XTris/glycine/SDS running buffer (Bio-Rad) using Precision Plus Protein All Blue pre-stained protein standards (Bio-Rad). Tran-Blot Turbo semi-dry transfer filmSystem (Bio-Rad) proteins were transferred from gels onto EtOH activated Low Fluorescence (LF) 0.45 μm PVDF membrane (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) or rat anti-murine IL-10 (MAB 417, R) &D Systems) to detect MOG or IL-10, 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 IL10 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. Using ChemiDoc ^TM The MP imaging system acquires an image. The results are shown in FIGS. 12C, 12D, 12E, 12F and 12G.

Western blot analysis (FIGS. 12A and 12C) of vaccines encoding scFv anti-DEC 205 as the first targeting unit, MOG (27-63) as the antigenic unit, IL-10 as the second targeting unit using anti-MOG antibodies (VB 5038, VB5041, VB5042 and VB 5050) showed that these vaccines were secreted as full-length fusion proteins. The detection with anti-murine IL-10 antibody (FIGS. 12B and D) showed a single band at the same molecular weight as the previously described anti-murine MOG antibody, which demonstrated that both antibodies detected the same protein band, confirming that MOG and IL-10 are part of the same fusion protein. The non-reduced samples in fig. 12B and 12E show dimerization of these proteins.

Western blotting (FIG. 12F) with anti-murine MOG antibodies on vaccines encoding VSIG-3 as the first targeting unit, MOG (27-63) as the antigenic unit, IL-10 as the second targeting unit (VB 5074 and VB 5075) showed that these vaccines were secreted as full-length fusion proteins. The migration rate of the protein was slower than expected from its calculated molecular weight, which can be explained by the known post-translational glycosylation (fig. 12F). Detection of supernatants of cells transfected with VB5074 and VB5075 with anti-murine IL-10 antibody (FIG. 12G) showed that both antibodies detected the same protein band, confirming that MOG and IL-10 are part of the same fusion protein.

Example 2: evaluation of tolerance-inducing ability of VB5067

The tolerance-inducing capacity of VB5067 was evaluated in the spleens of mice vaccinated once with 50 μg VB5067 (as shown in Table 1) and was determined by calculating the induced IL-10/IFN-gamma ratio. IL-10 (an anti-inflammatory cytokine known to function as immunosuppression) and IFN-gamma (a marker for inducing an inflammatory immune response) signals were determined in a two-color FluoSpot assay after re-stimulation of spleen cells harvested from vaccinated mice with MOG (35-55). The IL-10/IFN-gamma ratio indicates the degree to which the DNA vector-induced immune response is biased towards a tolerogenic response. Tolerogenic characteristics were further assessed by frequency of MOG (38-49) -specific foxp3+ T cells induced in response to vaccination and detected ex vivo. Foxp3 acts as a major regulator of the immunosuppressive pathway in the development and function of regulatory T cells (tregs) and suggests that Treg cells can suppress and control MOG-specific inflammatory immune responses, thereby maintaining self-tolerance. The results obtained were compared with the response elicited by the pro-inflammatory control vaccine VB5052 or the tolerance induction capacity of the VB5051 vaccination (all described in table 2).

Pestilence inoculation and Fluorospot

The following study design was used:

female C57BL/6 mice of 6 weeks of age were obtained from Janvier Labs (France). All animals were kept in an animal facility from Radium Hospital, norway Olympic. All animal protocols were approved by the norwegian food safety agency (Norwegian Food Safety Authority, norwegian oslo). Each group of 5 mice was used to test VB5067 (as described in table 1), VB5052 and VB5051 (as described in table 2). VB5052 serves as a pro-inflammatory version of the vaccine encoding MOG (27-63). VB5052 comprises a human CCL3L1 targeting unit known to target APC in a pro-inflammatory manner, i.e. a vaccine comprising such targeting unit will induce an inflammatory immune response in the subject to which it is administered, and the compound is expected to induce IFN-gamma production. The DNA vector VB5051 encoding MOG (27-63) peptide alone served as a comparison with VB 5067.

A dose of 50 μg of DNA vector VB5067 or control vector VB5051 or VB5052 dissolved in sterile PBS was administered to each tibialis anterior (2 x 25 μl,1000 μg/ml) and then electroporated using the agile in vivo electroporation system (BTX, usa). Spleens were harvested 7 days after vaccination and triturated in a cell filter to obtain a single cell suspension. Erythrocytes were lysed using potassium Ammonium Chloride (ACK) lysis buffer. After washing, spleen cells were counted using a NucleoCounter NC-202 (ChemoMetec, denmark) and resuspended to a final concentration of 6x10 ⁶ Individual cells/ml, at 6X10 ⁵ Individual cells/well were seeded in 96-well IFN- γ/IL-10 bicolor Fluorospot plates. Spleen cells were then re-stimulated with 16.67 μg/ml MOG (35-55) peptide for 44 hours, and IFN- γ and IL-10 cytokine production was then detected 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 are shown as triplicates of IL-10+ or IFN-gamma+ spots/10 ⁶ Average number of individual splenocytes.

As can be seen from FIG. 13A, IL-10 production was detected in unstimulated spleen cells harvested from mice vaccinated with all three constructs VB5067, VB5051 and VB5052, while only low background levels of IFN-gamma were observed. When MOG (35-55) re-stimulated splenocytes, an increase in IFN- γ levels was detected following inoculation with VB5052, which was significantly higher than the levels caused by VB5067 and VB5051, as shown in fig. 13B. In order to avoid excessive inflammation and ensure the final resolution of inflammation, it is important that the production of pro-inflammatory cytokines such as IFN-gamma is subject to a negative feedback mechanism (including the production of anti-inflammatory cytokines such as IL-10 ¹ ) Is provided. Thus, the observed increased IL-10 levels in response to VB5052 can be explained by this feedback mechanism controlling the induced inflammatory response. As shown in fig. 13C, VB5067 was detected to have a significantly higher IL-10/IFN- γ ratio compared to VB5052, indicating that VB5067 has a higher immunosuppressive potential compared to VB 5052.

MOG (38-49) -specific T-fines in the spleen of vaccinated mice using tetramer (H-2 IAb/GWYRSPFSRVVH) Flow cytometry analysis of cells

The production of MOG-specific foxp3+ cells was identified in mice by MOG-specific tetramer staining and flow cytometry, i.e. indicating that T cells were able to suppress and control MOG-specific inflammatory immune responses and thereby maintain self-tolerance (cd4+ MOG (38-49) -tet+ foxp3+ cells).

Briefly, 2x10 will be pooled from each group ⁶ Individual spleen cells were transferred to a 96-well V-bottom plate. Tetramers and antibodies were diluted in PBS containing 5% fbs and protected from light prior to use. Unless otherwise indicated, all steps requiring cell washing were performed using PBS containing 5% fbs. First, by a method specific to (MOG 38-49)MHC class II tetramer (1. Mu.g/ml, H-2IAb-GWYRSPFSRVVH->Tetramer PE,2958, proimmune) stained cells and plates were incubated in a humidified CO2 cell incubator (5% CO2, 37 ℃) for 2 hours. Without washing the cells, the FC receptor was blocked on ice for 5 minutes to prevent flow cytometry antibodies from binding to the Fc receptor (0.25. Mu.g/ml, truStain FcX ^TM PLUS (anti-mouse CD 16/32) antibodies, 156604, biolegend) bind non-specifically. Cells were stained on ice for 30 min with a surface antibody 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. Next, cells were stained on ice for 10 minutes using a fixable vital dye (150 μl per well, 1:8000 dilutions in PBS, fixable Viability Stain 780,565388,BD biosciences). Cells were washed twice with PBS only and fixed and permeabilized using Foxp 3/transcription factor staining buffer set (200. Mu.l per well, 00-5523-00, thermoficcher/eBioscience) according to the manufacturer's instructions. Cells were washed and pooled with a cell containing anti-mouse FOXP3 eFluor 660 (0.25. Mu.g/ml, clone: FJK-16s,50-5773-82, thermoficcher/eBioscience), anti-mouse Ki-67 Alexa Fluor 488 (0.25. Mu.g/ml, clone: 11F6, 15) 1204, biolegend) was stained on ice for 30 minutes. Cells were washed and resuspended in 150 μl of 5% FBS-containing PBS and BD FACSymphosy was used ^TM A3 cell analyzer was used for analysis. The following controls were used as a control using FlowJo ^TM v10.8 guidance of software (BD Life Sciences) gating on the required cell population: undyed control (=cells did not receive any antibody) and Fluorescence Minus One (FMO) control (=samples stained with all fluorophore labelled antibodies), minus one to accurately distinguish positive from negative signals).

As shown in FIG. 14, a higher percentage of MOG (38-49) -specific Foxp3+ cells was detected in response to VB5067 compared to VB 5051.

Thus, example 2 demonstrates that vaccination with VB5067 encoding a construct with scFv-anti-DEC 205 and CTLA-4 as targeting units and MOG (27-63) as antigenic units results in a higher ratio of anti-inflammatory cytokine to inflammatory cytokine (IL-10/IFN- γ) and a lack of inflammatory IFN- γ production compared to the pro-inflammatory vaccine VB 5052. Furthermore, scFv anti-DEC 205 and CTLA-4 targeting proteins induced a higher proportion of MOG (38-49) -specific foxp3+ cells than VB 5051. Taken together, these results demonstrate that vaccination with scFv anti-DEC 205 and CTLA-4 bispecific constructs exhibited a lack of pro-inflammatory cytokine production (IFN- γ) compared to VB5052 and elicited a more antigen-specific tolerogenic response compared to VB 5051.

¹ Sugimoto MA,Sousa LP,Pinho V,Perretti M,Teixeira MM.Resolution of Inflammation:What Controls Its OnsetFront Immunol.2016Apr26；7:160.doi:10.3389/fimmu.2016.00160。

Example 3: assessment of tolerance-inducing ability of VB5042

The tolerance induction capacity of VB5042 (described in table 1) was determined and compared to the response induced by the VB5052 pro-inflammatory control vaccine (described in table 2) and the tolerance induction capacity of VB5051 (described in table 2), as described in example 2.

As can be seen from fig. 15A, IL-10 production was detected in unstimulated spleen cells harvested from mice vaccinated with all three constructs VB5042, VB5051 and VB5052, whereas only low background levels of IFN- γ were observed. As shown in FIG. 15B, after restimulation of splenocytes with MOG (35-55), elevated IFN- γ levels were detected after vaccination with VB5052, which were significantly higher than those elicited by VB5042 and VB 5051. The increased IL-10 levels observed in response to VB5052 can be explained by a potential feedback mechanism controlling the inflammatory response, as described in example 2. Spleen cells from mice vaccinated with one of VB5042 and VB5051 showed similar levels of IL-10 and IFN- γ, both with (fig. 15A) and without (fig. 15B) MOG (35-55) peptide restimulation. As shown in fig. 15C, VB5042 was detected to have a significantly higher IL-10/IFN- γ ratio compared to VB5052, indicating that VB5042 has a higher immunosuppressive potential compared to VB 5052.

As shown in FIG. 16, a higher percentage of MOG (38-49) -specific Foxp3+ cells was detected in response to VB5042 compared to VB 5051.

Thus, example 3 shows that vaccination with VB5042 (encoding a construct with scFv anti-DEC 205 and IL-10 as targeting units and MOG (27-63) as antigenic units) resulted in a higher ratio of non-inflammatory cytokines to inflammatory cytokines (IL-10/IFN-gamma) and a lack of inflammatory IFN-gamma compared to the pro-inflammatory vaccine version VB 5052. In addition, scFv anti-DEC 205 and IL-10 targeting proteins induced higher frequency MOG (38-49) -specific foxp3+ cells compared to VB 5051. Taken together, these results demonstrate that vaccination with scFv anti-DEC 205 and IL-10 bispecific constructs showed a lack of pro-inflammatory cytokine production (IFN- γ) compared to VB5052 and elicited a stronger antigen-specific tolerogenic response compared to VB 5051.

Example 4: evaluation of tolerance-inducing ability of VB5073

The tolerance induction capacity of VB5073 (described in table 1) was determined and compared to the response induced by the pro-inflammatory control vaccine VB5052 (described in table 2) and the tolerance induction capacity of VB5051 (described in table 2), as described in example 2.

As can be seen from fig. 17A, IL-10 production was detected in unstimulated spleen cells harvested from mice vaccinated with all three constructs VB5073, VB5051 and VB5052, whereas only low background levels of IFN- γ were observed. As shown in figure 17B, after MOG (35-55) re-stimulation of splenocytes, elevated IFN- γ levels were detected after inoculation with VB5052, which were significantly higher than those elicited by VB5073 and VB 5051. The observed increase in IL-10 levels in response to VB5052 can be explained by a potential feedback mechanism controlling the inflammatory response, as described in example 2. Spleen cells from mice vaccinated with either VB5073 or VB5051 showed similar levels of IL-10 and IFN-gamma, both with (FIG. 17B) or without (FIG. 17A) MOG (35-55) peptide restimulation. As shown in fig. 17C, VB5073 was detected to have a significantly higher IL-10/IFN- γ ratio compared to VB5052, indicating that VB5073 has a higher immunosuppressive potential compared to VB 5052.

As shown in FIG. 18, a higher percentage of MOG (38-49) -specific Foxp3+ cells was detected in response to VB5073 than VB 5051.

Thus, example 4 shows that vaccination with VB5073 (encoding a construct with SCGB3A2 and IL-10 as targeting units and MOG (38-49) as antigenic units) resulted in a higher ratio of non-inflammatory cytokines to inflammatory cytokines (IL-10/IFN-gamma) compared to the pro-inflammatory construct VB5052, indicating a lack of inflammatory IFN-gamma production and inducing a higher frequency of MOG (38-49) -specific Foxp3+ cells compared to VB 5051. Taken together, these results indicate that vaccination with SCGB3A2 and IL-10 bispecific constructs showed a lack of pro-inflammatory cytokine production (IFN- γ) compared to VB5052 and could elicit a stronger antigen-specific tolerogenic response compared to VB 5051.

Example 5: design, production and in vitro characterization of tolerance-inducible constructs of the invention-with use in therapy Shellfish allergy six T cell epitopes

Tropomyosin is the major allergen in shellfish. In the Balb/c mouse model of Met e 1 hypersensitivity, 6 major T cell epitopes of tropomyosin from the penaeus vannamei (metapenaeus ensis, met e 1) species were identified. Oral immunotherapy with peptides of these six T cell epitopes is effective in reducing the allergic response of shrimp tropomyosin (Wai, CYY et al 2015).

Design of DNA vector

DNA vectors VB5077 and VB5078 were designed and produced, comprising nucleic acid sequences encoding the elements/units listed in table 3 below. All the gene sequences described were ordered from GenScript (Genscript Biotech BV, netherlands) and cloned into the expression vector pALD-CV 77.

TABLE 3 Table 3

Vector DNA vectors VB5077 (SEQ ID NO: 37) and VB5078 (SEQ ID NO: 38) of the present disclosure encode constructs comprising targeting units, dimerization units, and antigenic units as set forth in the above tables.

Met e 1 (241-260), (210-230), (136-155), (76-95), (46-65), (16-35) antigenic units (SEQ ID NO: 22) contain GGGGSGGGGS (SEQ ID NO: 80) linkers between T cell epitopes.

In vitro characterization of protein expression and secretion of tolerance-inducing constructs containing Met e 1

The aim of this experiment was to characterize the expression and secretion of the proteins encoded by the Met e 1-containing DNA vectors VB5077 and VB5078 after transient transfection of mammalian cells.

The Expi293F cells were obtained from Thermo Fisher sci and transiently transfected with DNA vectors VB5077 and VB 5078. Briefly, expi293F cells (1.7x10 ⁶ Individual cells/ml, 1 ml) were inoculated into 96-well culture plates. Cells were transfected with 0.64 μg/ml plasmid DNA using the ExpiFectamine 293 reagent (Thermo Fisher Sci.) and the plates were humidified in CO ₂ Cell incubator (8% CO) ₂ Incubate on an orbital shaker (diameter 3mm,900 rpm) at 37 ℃. Plates were incubated for 72 hours and then supernatants were harvested.

Secreted proteins encoded by the Mete 1-containing vector were evaluated in the supernatant of transiently transfected cells by sandwich ELISA using anti-murine IL-10 antibodies (capture antibodies: mouse anti-murine IL-10 antibody, 2. Mu.g/ml, 100. Mu.l/well, MAB417, R & D Systems, detection antibodies: goat anti-murine IL-10 biotinylated antibodies, 0.8. Mu.g/ml, 100. Mu.l/well, BAF417, R & D Systems). The results are shown in figure 19, which shows that both Met e 1 containing constructs are expressed and secreted at high levels.

Characterization of intact proteins expressed by VB5077 and VB5078

Western blot analysis was performed on supernatant samples of transfected Expi293F cells to further characterize the proteins encoded by VB5077 and VB 5078.

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) (scale up of total sample volume according to the given ratio). The sample (reduced or non-reduced) was heated at 70℃for 10 minutes and then added (the added sample volume is shown in the drawing heading) to a 4% -20%Criterion TGX Stain-Free pre-gel (Bio-Rad). SDS-PAGE was performed in 1 XTris/glycine/SDS running buffer (Bio-Rad) using Precision Plus Protein All Blue pre-stained protein standards (Bio-Rad). Proteins were transferred from the gel onto EtOH-activated Low Fluorescence (LF) 0.45 μm PVDF membrane (Bio-Rad) using a Tran-Blot Turbo semi-dry transfer system (Bio-Rad). PVDF membrane was blocked in EveryBlot buffer (Bio-Rad) for 5 min and was used with rat anti-murine IL-10 (MAB 417, R) &D Systems) detection to detect IL-10. The membrane was incubated with the fluorescent dye conjugated species-specific secondary antibody for 1 hour at room temperature, then washed and dried. Using ChemiDoc ^TM The MP imaging system acquires an image.

The results are shown in FIG. 20.

Western blot analysis using anti-murine IL-10 antibodies showed that constructs containing six Met e 1T cell epitopes were secreted as full-length fusion proteins.

Sequence(s)

SEQ ID NO:1

Amino acid sequences of hinge exon h1 (amino acids 1-12) from IgG3 and hinge exon h4 (amino acids 13-27) from human IgG3

E ¹ LKTPLGDTTHT ¹² E ¹³ PKSCDTPPPCPRCP ²⁷

SEQ ID NO:2

Amino acid sequence of the hinge region of human IgG 1: upper hinge region (amino acids 1-4), middle hinge region (amino acids 5-15) and lower hinge region (amino acids 16-23)

E ¹ PKS ⁴ C ⁵ DKTHTCPPCP ¹⁵ A ¹⁶ PELLGGP ²³

SEQ ID NO:3

Amino acid sequence of CH3 domain of human IgG3

GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

SEQ ID NO:4

Amino acid sequence of CH3 Domain of human IgG1

GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO:5

Amino acid sequence of CREB bZIP motif

VKCLENRVAVLENQNKTLIEELKALKDLY

SEQ ID NO:6

Mouse immunoglobulin heavy chain signal sequence (Ig VH signal sequence)

MNFGLRLIFLVLTLKGVQC

SEQ ID NO:7

Single chain variable fragment (scFv) of mouse against DEC205

DIQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLELKGGGGSGGGGSGGGGSEVKLLESGGGLVQPGGSLRLSCAASGFTFNDFYMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISRDNTQNILYLQMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSS

SEQ ID NO:8

Hinge region from human IgG 1: upper hinge region hIgG1 (1-5), middle hinge region hIgG1 (6-20)

GLQGLEPKSCDKTHTCPPCP

SEQ ID NO:9

Murine IL-10

SRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS

SEQ ID NO:10

Mature murine TGF beta 1

ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASASPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS

SEQ ID NO:11

Murine CTLA-4 extracellular domain

EAIQVTQPSVVLASSHGVASFPCEYSPSHNTDEVRVTVLRQTNDQMTEVCATTFTEKNTVGFLDYPFCSGTFNESRVNLTIQGLRAVDTGLYLCKVELMYPPPYFVGMGNGTQIYVIDPEPCPDSD

SEQ ID NO:15

Murine MARCO ligand SCGB3A2 Signal sequence

MKLVSIFLLVTIGICGYSATA

SEQ ID NO:16

Murine MARCO ligand SCGB3A2

LLINRLPVVDKLPVPLDDIIPSFDPLKMLLKTLGISVEHLVTGLKKCVDELGPEASEAVKKLLEALSHLV

SEQ ID NO:17

Murine VISTA ligand VSIG-3 signal sequence

MTRRRSAPASWLLVSLLGVATS

SEQ ID NO:18

Murine VISTA ligand VSIG-3 extracellular domain

LEVSESPGSVQVARGQTAVLPCAFSTSAALLNLNVIWMVIPLSNANQPEQVILYQGGQMFDGALRFHGRVGFTGTMPATNVSIFINNTQLSDTGTYQCLVNNLPDRGGRNIGVTGLTVLVPPSAPQCQIQGSQDLGSDVILLCSSEEGIPRPTYLWEKLDNTLKLPPTATQDQVQGTVTIRNISALSSGLYQCVASNAIGTSTCLLDLQVISPQPRSV

SEQ ID NO:19

Hinge h1 hIgG3

ELKTPLGDTTHT

SEQ ID NO:20

Human CCL3L1 signal sequence

MQVSTAALAVLLCTMALCNQVLS

SEQ ID NO:21

Human CCL3L1

APLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSA

SEQ ID NO:23

The amino acid sequence of VB 5050. Mouse immunoglobulin heavy chain signal sequence "Ig VH signal sequence"

(1-19), the mouse single chain variable fragment "scFv" was anti-DEC 205 (20-265), hinge h1 hIgG3 (266-277), hinge h4 hIgG3 (278-292),joint(293-297), MOG amino acids 27-63 (298-334), upper hinge region hIgG1 (335-339), middle hinge region hIgG1 (340-354), murine IL-10 (355-514). MNFGLRLIFLVLTLKGVQCDIQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLELKGGGGSGGGGSGGGGSEVKLLESGGGLVQPGGSLRLSCAASGFTFNDFYMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISRDNTQNILYLQMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSSELKTPLGDTTHTEPKSCDTPPPCPRCPGLGGLSPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAPGLQGLEPKSCDKTHTCPPCPSRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS

SEQ ID NO:24

The amino acid sequence of VB 5038. The mouse immunoglobulin heavy chain signal sequence "Ig VH signal sequence" (1-19), the mouse single chain variable fragment "scFv" anti-DEC 205 (20-265), hinge h1 hIgG3 (266-277), hinge h4 hIgG3 (278-292), linker (293-297), MOG amino acids 27-63 (298-334), upper hinge region hIgG1 (335-339), middle hinge region hIgG1 (340-354), murine IL-10 (355-514). A partial MOG (27-63) sequence was obtained from Krienke et al 2021.MNFGLRLIFLVLTLKGVQCDIQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLELKGGGGSGGGGSGGGGSEVKLLESGGGLVQPGGSLRLSCAASGFTFNDFYMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISRDNTQNILYLQMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSSELKTPLGDTTHTEPKSCDTPPPCPRCP GLGGLSPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEAQPGLQGLEPKSCDKTHTCPPCPSRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS

SEQ ID NO:25

The amino acid sequence of VB 5042. The mouse immunoglobulin heavy chain signal sequence "Ig VH signal sequence" (1-19), the mouse single chain variable fragment "scFv" anti-DEC 205 (20-265), hinge h1hIgG3 (266-277), hinge h4 hIgG3 (278-292),joint(293-297), MOG amino acids 27-63 (298-334), upper hinge region hIgG1 (335-339), middle hinge region hIgG1 (340-354), hinge h1hIgG3 (355-366), murine IL-10 (367-526).

MNFGLRLIFLVLTLKGVQCDIQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLELKGGGGSGGGGSGGGGSEVKLLESGGGLVQPGGSLRLSCAASGFTFNDFYMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISRDNTQNILYLQMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSSELKTPLGDTTHTEPKSCDTPPPCPRCPGLGGLSPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAPGLQGLEPKSCDKTHTCPPCPELKTPLGDTTHTSRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS

SEQ ID NO:26

The amino acid sequence of VB 5066. The mouse immunoglobulin heavy chain signal sequence "Ig VH signal sequence" (1-19), the mouse single chain variable fragment "scFv" anti-DEC 205 (20-265), hinge h1hIgG3 (266-277), hinge h4 hIgG3 (278-292),joint(293-297), MOG amino acids 27-63 (298-334), upper hinge region hIgG1 (335-339), middle hinge region hIgG1 (340-354), murine TGF beta 1 mature sequence (355-466).

MNFGLRLIFLVLTLKGVQCDIQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLELKGGGGSGGGGSGGGGSEVKLLESGGGLVQPGGSLRLSCAASGFTFNDFYMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISRDNTQNILYLQMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSSELKTPLGDTTHTEPKSCDTPPPCPRCPGLGGLSPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAPGLQGLEPKSCDKTHTCPPCPALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASASPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS

SEQ ID NO:27

The amino acid sequence of VB 5043. The mouse immunoglobulin heavy chain signal sequence "Ig VH signal sequence" (1-19), the mouse single chain variable fragment "scFv" anti-DEC 205 (20-265), hinge h1hIgG3 (266-277), hinge h4 hIgG3 (278-292),joint(293-297), MOG amino acids 27-63 (298-334), upper hinge region hIgG1 (335-339), middle hinge region hIgG1 (340-354), hinge h1hIgG3 (355-366), murine TGF beta 1 mature sequence (355-478).

MNFGLRLIFLVLTLKGVQCDIQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLELKGGGGSGGGGSGGGGSEVKLLESGGGLVQPGGSLRLSCAASGFTFNDFYMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISRDNTQNILYLQMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSSELKTPLGDTTHTEPKSCDTPPPCPRCPGLGGLSPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAPGLQGLEPKSCDKTHTCPPCPELKTPLGDTTHTALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALYNQHNPGASASPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS

SEQ ID NO:28

The amino acid sequence of VB 5067. The mouse immunoglobulin heavy chain signal sequence "Ig VH signal sequence" (1-19), the mouse single chain variable fragment "scFv" anti-DEC 205 (20-265), hinge h1 hIgG3 (266-277), hinge h4 hIgG3 (278-292),joint(293-297), MOG amino acids 27-63 (298-334), upper hinge region hIgG1 (335-339), middle hinge region hIgG1 (340-354), murine CTLA-4 (355-480).

MNFGLRLIFLVLTLKGVQCDIQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLELKGGGGSGGGGSGGGGSEVKLLESGGGLVQPGGSLRLSCAASGFTFNDFYMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISRDNTQNILYLQMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSSELKTPLGDTTHTEPKSCDTPPPCPRCPGLGGLSPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAPGLQGLEPKSCDKTHTCPPCPEAIQVTQPSVVLASSHGVASFPCEYSPSHNTDEVRVTVLRQTNDQMTEVCATTFTEKNTVGFLDYPFCSGTFNESRVNLTIQGLRAVDTGLYLCKVELMYPPPYFVGMGNGTQIYVIDPEPCPDSD

SEQ ID NO:29

The amino acid sequence of VB 5072. A signal sequence of a murine MARCO ligand SCGB3A2 (1-21), a murine MARCO ligand SCGB3A2 (22-91), a hinge h1 hIgG3 (92-103), a hinge h4 hIgG3 (104-118),joint(119-123), MOG amino acids 27-63 (124-160), upper hinge region hIgG1 (161-165), middle hinge region hIgG1 166-180), murine IL-10 (181-340)

MKLVSIFLLVTIGICGYSATALLINRLPVVDKLPVPLDDIIPSFDPLKMLLKTLGISVEHLVTGLKKCVDELGPEASEAVKKLLEALSHLVELKTPLGDTTHTEPKSCDTPPPCPRCPGLGGLSPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAPGLQGLEPKSCDKTHTCPPCPSRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS

SEQ ID NO:30

The amino acid sequence of VB 5073. A signal sequence of a murine MARCO ligand SCGB3A2 (1-21), a murine MARCO ligand SCGB3A2 (22-91), a hinge h1 hIgG3 (92-103), a hinge h4 hIgG3 (104-118),joint(119-123), MOG amino acids 27-63 (124-160), upper hinge region hIgG1 (161-165), middle hinge region hIgG1 166-180), hinge h1 hIgG3 (181-192), murine IL-10 (193-352)

MKLVSIFLLVTIGICGYSATALLINRLPVVDKLPVPLDDIIPSFDPLKMLLKTLGISVEHLVTGLKKCVDELGPEASEAVKKLLEALSHLVELKTPLGDTTHTEPKSCDTPPPCPRCPGLGGLSPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAPGLQGLEPKSCDKTHTCPPCPELKTPLGDTTHTSRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS

The amino acid sequence of SEQ ID NO. 31dB 5074. Murine VISTA ligand VSIG-3 signal sequence (1-22), murine VISTA ligand VSIG-3 extracellular domain (23-240), hinge h1 hIgG3 (241-252), hinge h4 hIgG3 (253-267), linker (268-272), MOG amino acids 27-63 (273-309), upper hinge region hIgG1 (310-314), middle hinge region hIgG1 (315-329), murine IL-10 (330-489).

MTRRRSAPASWLLVSLLGVATSLEVSESPGSVQVARGQTAVLPCAFSTSAALLNLNVIWMVIPLSNANQPEQVILYQGGQMFDGALRFHGRVGFTGTMPATNVSIFINNTQLSDTGTYQCLVNNLPDRGGRNIGVTGLTVLVPPSAPQCQIQGSQDLGSDVILLCSSEEGIPRPTYLWEKLDNTLKLPPTATQDQVQGTVTIRNISALSSGLYQCVASNAIGTSTCLLDLQVISPQPRSVELKTPLGDTTHTEPKSCDTPPPCPRCPGLGGLSPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAPGLQGLEPKSCDKTHTCPPCPSRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS

The amino acid sequence of SEQ ID NO. 32VB 5075. Murine VISTA ligand VSIG-3 signal sequence (1-22), murine VISTA ligand VSIG-3 extracellular domain (23-240), hinge h1 hIgG3 (241-252), hinge h4 hIgG3 (253-267), linker (268-272), MOG amino acids 27-63 (273-309), upper hinge region hIgG1 (310-314), middle hinge region hIgG1 (315-329), hinge h1 hIgG3 (330-341), murine IL-10 (342-501).

MTRRRSAPASWLLVSLLGVATSLEVSESPGSVQVARGQTAVLPCAFSTSAALLNLNVIWMVIPLSNANQPEQVILYQGGQMFDGALRFHGRVGFTGTMPATNVSIFINNTQLSDTGTYQCLVNNLPDRGGRNIGVTGLTVLVPPSAPQCQIQGSQDLGSDVILLCSSEEGIPRPTYLWEKLDNTLKLPPTATQDQVQGTVTIRNISALSSGLYQCVASNAIGTSTCLLDLQVISPQPRSVELKTPLGDTTHTEPKSCDTPPPCPRCPGLGGLSPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAPGLQGLEPKSCDKTHTCPPCPELKTPLGDTTHTSRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS

SEQ ID NO:33

The amino acid sequence of VB 5052. Human CCL3L1 signal sequence "Mip1a" (1-23), human CCL3L1"hMip1a" (24-93), hinge h1 hIgG3 (94-105), hinge h4 hIgG3 (106-120),joint(121-130)，hCH3 IgG3(131-237)，Joint(238-242), MOG amino acids 27-63 (243-279). ###GGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLSPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAP

SEQ ID NO:34

The amino acid sequence of VB5002 b. Human CCL3L1 signal sequence "Mip1a" (1-23), human CCL3L1"hMip1a" (24-93),hinge h1 hIgG3 (94-105), hinge h4 hIgG3 (106-120),joint(121-130)，hCH3 IgG3(131-237)，Joint(238-242), MOG amino acids 27-63 (243-279). A partial MOG (27-63) sequence was obtained from Krienke et al 2021.

MQVSTAALAVLLCTMALCNQVLSAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEEWVQKYVSDLELSAELKTPLGDTTHTEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGKGLGGLSPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAP

SEQ ID NO:35

The amino acid sequence of VB 5051. Mouse immunoglobulin heavy chain signal sequence "Ig VH signal sequence" (1-19), MOG amino acids 27-63 (20-56).

MNFGLRLIFLVLTLKGVQCSPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEQAP

SEQ ID NO:36

The amino acid sequence of VB5001 b. Mouse immunoglobulin heavy chain signal sequence "Ig VH signal sequence" (1-19), MOG amino acids 27-63 (20-56). A partial MOG (27-63) sequence was obtained from Krienke et al 2021.

MNFGLRLIFLVLTLKGVQCSPGKNATGMEVGWYRSPFSRVVHLYRNGKDQDAEAQP

SEQ ID NO:37

The amino acid sequence of VB 5077. The mouse immunoglobulin heavy chain signal sequence "Ig VH signal sequence" (1-19), the mouse single chain variable fragment "scFv" anti-DEC 205 (20-265), hinge h1 hIgG3 (266-277), hinge h4 hIgG3 (278-292),joint(293-297), met e 1 "241-260", "210-230", "136-155", "76-95", "46-65", "16-35" (293-468), upper hinge region hIgG1 (469-473), middle hinge region hIgG1 (474-488), murine IL-10 (489-648).

MNFGLRLIFLVLTLKGVQCDIQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLELKGGGGSGGGGSGGGGSEVKLLESGGGLVQPGGSLRLSCAASGFTFNDFYMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISRDNTQNILYLQMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSSELKTPLGDTTHTEPKSCDTPPPCPRCPGLGGLKEVDRLEDELVNEKEKYKSIGGGGSGGGGSAYKEQIKTLTNKLKAAEARAEGGGGSGGGGSNQLKEARFLAEEADRKYDEVGGGGSGGGGSAALNRRIQLLEEDLERSEERGGGGSGGGGSDLDQVQESLLKANNQLVEKDGGGGSGGGGSEQQNKEANNRAEKSEEEVHNGLQGLEPKSCDKTHTCPPCPSRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS

SEQ ID NO:38

The amino acid sequence of VB 5078. The mouse immunoglobulin heavy chain signal sequence "Ig VH signal sequence" (1-19), the mouse single chain variable fragment "scFv" anti-DEC 205 (20-265), hinge h1 hIgG3 (266-277), hinge h4 hIgG3 (278-292),joint(293-297), met e 1 "241-260", "210-230", "136-155", "76-95", "46-65", "16-35" (293-468), upper hinge region hIgG1 (469-473), middle hinge region hIgG1 (474-488), hinge h1 hIgG3 (489-500), murine IL-10 (501-660).

MNFGLRLIFLVLTLKGVQCDIQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLELKGGGGSGGGGSGGGGSEVKLLESGGGLVQPGGSLRLSCAASGFTFNDFYMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISRDNTQNILYLQMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSSELKTPLGDTTHTEPKSCDTPPPCPRCPGLGGLKEVDRLEDELVNEKEKYKSIGGGGSGGGGSAYKEQIKTLTNKLKAAEARAEGGGGSGGGGSNQLKEARFLAEEADRKYDEVGGGGSGGGGSAALNRRIQLLEEDLERSEERGGGGSGGGGSDLDQVQESLLKANNQLVEKDGGGGSGGGGSEQQNKEANNRAEKSEEEVHNGLQGLEPKSCDKTHTCPPCPELKTPLGDTTHTSRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS

SEQ ID NO:41

The amino acid sequence of VB 5041. The mouse immunoglobulin heavy chain signal sequence "Ig VH signal sequence" (1-19), the mouse single chain variable fragment "scFv" anti-DEC 205 (20-265), hinge h1hIgG3 (266-277), hinge h4 hIgG3 (278-292),joint(293-297), MOG amino acids 35-55 (298-318), upper hinge region hIgG1 (319-323), middle hinge region hIgG1 (323-338), hinge h1hIgG3 (339-350), murine IL-10 (315-510).

MNFGLRLIFLVLTLKGVQCDIQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLELKGGGGSGGGGSGGGGSEVKLLESGGGLVQPGGSLRLSCAASGFTFNDFYMNWIRQPPGQAPEWLGVIRNKGNGYTTEVNTSVKGRFTISRDNTQNILYLQMNSLRAEDTAIYYCARGGPYYYSGDDAPYWGQGVMVTVSSELKTPLGDTTHTEPKSCDTPPPCPRCPGLGGLMEVGWYRSPFSRVVHLYRNGKGLQGLEPKSCDKTHTCPPCPELKTPLGDTTHTSRGQYSREDNNCTHFPVGQSHMLLELRTAFSQVKTFFQTKDQLDNILLTDSLMQDFKGYLGCQALSEMIQFYLVEVMPQAEKHGPEIKEHLNSLGEKLKTLRMRLRRCHRFLPCENKSKAVEQVKSDFNKLQDQGVYKAMNEFDIFINCIEAYMMIKMKS

Description of the embodiments

1. A tolerance-inducing construct comprising:

a. a first targeting unit, a first junction region (first joint region);

b. an antigenic unit (antigenic unit);

c. a second joining zone (second joint region); a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

2. A tolerance-inducing construct comprising:

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

3. A tolerance-inducing construct comprising:

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

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

4. The tolerance-inducing construct according to any of the preceding embodiments, wherein the multimeric protein, such as a dimeric protein, consists of a plurality of polypeptides, such as two polypeptides, which are linked to each other via their junction regions, preferably via their respective first junction regions and via their respective second junction regions.

5. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the multimeric protein consists of a plurality of polypeptides linked to each other via their junction regions, preferably via their respective first junction regions and via their respective second junction regions.

6. The tolerance-inducing construct according to any one of the preceding embodiments, wherein the dimeric protein consists of two polypeptides linked to each other via their junction regions, preferably via their respective first junction regions and via their respective second junction regions.

7. The tolerance-inducing construct of any one of the preceding embodiments, wherein the first and second junction regions comprise a flexible unit and a binding unit.

8. The tolerance-inducing construct of any one of the preceding embodiments, wherein the first and/or second junction region comprises a binding unit that is a non-covalent binding unit.

9. The tolerance inducing construct of any one of the preceding embodiments, wherein the non-covalent binding unit is a trimerization unit.

10. The tolerance inducing construct of any one of embodiments 1-9, wherein the trimerization unit is a collagen-derived trimerization unit.

11. The tolerance-inducing construct of embodiment 10, wherein the collagen-derived trimerization unit is a human collagen-derived XVIII trimerization domain.

12. The tolerance-inducing construct of embodiment 10, wherein the collagen-derived trimerization unit is a human collagen XV trimerization domain.

13. The tolerance inducing construct of any one of embodiments 1-8, wherein the non-covalent binding unit is a tetramerization unit.

14. The tolerance-inducing construct of embodiment 13, wherein the tetramerization domain is a domain derived from p 53.

15. The tolerance inducing construct of any one of embodiments 1-8, wherein the non-covalent binding unit is a dimerization unit.

16. The tolerance-inducing construct of embodiment 15, wherein the dimerization unit comprises a hinge region and an immunoglobulin domain.

17. The tolerance-inducing construct of embodiment 16, wherein the dimerization unit is an immunoglobulin constant domain.

18. The tolerance-inducing construct of embodiment 15, wherein the dimerization unit comprises a dHLX protein.

19. The tolerance-inducing construct of any one of the preceding embodiments, wherein the first and/or second junction region comprises a binding unit that is a covalent binding unit.

20. The tolerance-inducing construct of any one of the preceding embodiments, wherein the first junction region and/or the second junction region comprises or consists of a naturally occurring sequence.

21. The tolerance-inducing construct of any one of embodiments 1-19, wherein the first junction region and/or the second junction region comprises or consists of an artificial sequence.

22. The tolerance inducing construct of any one of embodiments 19-21, wherein the first and second junction regions comprise a covalent binding unit comprising one or more cysteine residues.

23. The tolerance-inducing construct of embodiment 22, wherein the covalent binding unit comprises at least 2 cysteine residues.

24. The tolerance-inducing construct of embodiments 22-23, wherein the covalent binding unit comprises at least 2 cysteine residues, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 cysteine residues.

25. The tolerance-inducing construct of embodiments 19 through 22, wherein the covalent binding unit comprises a cysteine-rich sequence.

26. The tolerance-inducing construct of any one of embodiments 19-25, wherein the covalent binding unit of the first junction region comprises a different number of cysteine residues than the covalent binding unit of the second junction region.

27. The tolerance-inducing construct of any one of embodiments 22-26, wherein the position of the cysteine residue comprised in the covalent binding unit of the first junction region is different from the position of the cysteine residue comprised in the covalent binding unit of the second junction region.

28. The tolerance-inducing construct of embodiments 22-27, wherein the number of amino acid residues between cysteine residues of the covalent binding unit of the first junction region is different from the number of amino acid residues between cysteine residues of the covalent binding unit of the second junction region.

29. The tolerance inducing construct of any one of embodiments 22-28, wherein the number of cysteine residues is based on the length of the antigenic unit.

30. The tolerance inducing construct of any one of embodiments 19-29, wherein at least one covalent binding unit is derived from an immunoglobulin.

31. The tolerance-inducing construct of embodiment 30, wherein the covalent binding unit is a hinge region derived from an immunoglobulin, e.g., exon h4 of IgG3 or the middle hinge of IgG 1.

32. The tolerance-inducing construct of embodiment 30, wherein the hinge region is derived from Ig, e.g., derived from IgG1, igG2 or IgG3.

33. The tolerance-inducing construct of embodiment 30, wherein the hinge region is derived from IgM.

34. The tolerance-inducing construct of embodiment 30, wherein the hinge region comprises SEQ ID NO:157 or an amino acid sequence encoded by said nucleotide sequence, or a combination thereof.

35. The tolerance-inducing construct of embodiment 30, wherein the covalent binding unit comprises or consists of an amino acid sequence having at least 40% sequence identity, e.g. at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity, to the amino acid sequence of 13-27 of SEQ ID No. 1.

36. The tolerance-inducing construct of embodiment 30, wherein the covalent binding unit comprises or consists of the amino acid sequence of 13-27 of SEQ ID No. 1, wherein any one amino acid of the flexible unit has been substituted, deleted or inserted with another amino acid, provided that NO more than 6 amino acids, such as NO more than 5 amino acids, such as NO more than 4 amino acids, such as NO more than 3 amino acids, such as NO more than 2 amino acids or NO more than 1 amino acid have been so substituted, deleted or inserted.

37. The tolerance-inducing construct of embodiment 30, wherein the covalent binding unit consists of the amino acid sequence of 13-23 of SEQ ID NO. 1.

38. The tolerance inducing construct of embodiment 31, wherein the covalent binding unit is hinge exon h4 of IgG 3.

39. The tolerance-inducing construct of embodiment 30, wherein the covalent binding unit comprises the sequence EPKSCDTPPPCPRCP (SEQ ID NO:156; amino acids 13-27 corresponding to SEQ ID NO: 1).

40. The tolerance-inducing construct according to any of embodiments 1-30, wherein the covalent binding unit comprises or consists of an amino acid sequence having at least 40% sequence identity, e.g. at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity, with the amino acid sequence of 5-15 of SEQ ID No. 2, provided that the number and position of cysteine residues are preserved.

41. The tolerance-inducing construct of embodiments 1-30, wherein the covalent binding unit comprises or consists of the amino acid sequence of 5-15 of SEQ ID No. 2, wherein any one amino acid of the flexible unit has been substituted, deleted or inserted with another amino acid, provided that NO more than 5 amino acids, such as NO more than 4 amino acids, such as NO more than 3 amino acids, such as NO more than 2 amino acids or NO more than 1 amino acid have been so substituted, deleted or inserted.

42. The tolerance-inducing construct of embodiment 41, wherein the covalent binding unit consists of the amino acid sequence of 5-15 of SEQ ID NO. 2 or comprises the amino acid sequence of 5-15 of SEQ ID NO. 2.

43. The tolerance-inducing construct of embodiment 30, wherein the covalent binding unit is the mid-hinge region of IgG 1.

44. The tolerance inducing construct of any one of embodiments 19-43, wherein the covalent binding unit is a non-immunogenic sequence.

45. The tolerance inducing construct of any one of embodiments 19-44, wherein the covalent binding unit is a naturally occurring peptide sequence.

46. The tolerance-inducing construct of any one of embodiments 19-45, wherein the covalent binding unit consists of 2 to 100 amino acids, e.g. 3 to 70 amino acids, e.g. 4 to 50 amino acids or 5 to 30 amino acids.

47. The tolerance inducing construct of any one of embodiments 19-46, wherein the covalent binding unit consists of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

48. The tolerance inducing construct of any one of embodiments 19-47, wherein at least one covalent binding unit is an artificial sequence.

49. The tolerance inducing construct of any one of embodiments 8-48, wherein the first and second junction regions comprise binding units that are non-covalent binding units.

50. The tolerance inducing construct of any one of embodiments 8-49, wherein the non-covalent binding units promote multimerization by non-covalent interactions, e.g., hydrophobic interactions.

51. The tolerance inducing construct of any one of embodiments 8-50, wherein the non-covalent binding unit promotes dimerization via a non-covalent interaction, e.g., a hydrophobic interaction.

52. The tolerance inducing construct of any one of embodiments 8-51, wherein the non-covalent binding unit has the ability to form a multimeric protein by non-covalent interactions.

53. The tolerance inducing construct of any one of embodiments 8-52, wherein the non-covalent binding unit has the ability to form a dimer by non-covalent interactions.

54. The tolerance inducing construct of any one of embodiments 8-53, wherein at least one non-covalent binding unit is a naturally occurring sequence.

55. The tolerance inducing construct of any one of embodiments 8-54, wherein at least one non-covalent binding unit is an artificial sequence.

56. The tolerance inducing construct of any one of embodiments 8-55, wherein the non-covalent binding unit is or comprises an immunoglobulin.

57. The tolerance inducing construct of any one of embodiments 8-56, wherein the non-covalent binding unit consists of or comprises: immunoglobulin domains, such as immunoglobulin constant domains (C domains), e.g., carboxy-terminal constant domains (i.e., CH3 domains), CH1 domains, or CH2 domains, or sequences substantially identical to the C domains, or variants thereof.

58. The tolerance-inducing construct of any one of embodiments 8-57, wherein the non-covalent binding unit comprises or consists of a CH3 domain derived from IgG, e.g. derived from IgG3 or IgG1, preferably derived from IgG 1.

59. The tolerance inducing construct of any one of embodiments 8-58, wherein the non-covalent binding unit comprises or consists of a CH3 domain derived from IgG 3.

60. The tolerance inducing construct of any one of embodiments 8-59, wherein the non-covalent binding unit comprises or consists of a carboxy-terminal C domain derived from IgG3 having a sequence identical to SEQ ID NO:3, having at least 80% sequence identity to the amino acid sequence of seq id no.

61. The tolerance-inducing construct of any one of embodiments 8-60, wherein the non-covalent binding unit comprises or consists of a carboxy-terminal C domain derived from IgG3 having 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. 3.

62. The tolerance-inducing construct of any one of embodiments 8-61, wherein the non-covalent binding unit comprises or consists of a carboxy-terminal C domain derived from IgG3 having the amino acid sequence of SEQ ID NO:3, wherein any one amino acid of the flexible unit has been substituted, deleted or inserted with another amino acid, provided that NO more than 21 amino acids have been so substituted, deleted or inserted, e.g. NO more than 20 amino acids, e.g. NO more than 19 amino acids, e.g. NO more than 18 amino acids, e.g. NO more than 17 amino acids, e.g. NO more than 16 amino acids, e.g. NO more than 15 amino acids, e.g. NO more than 14 amino acids, e.g. NO more than 13 amino acids, e.g. NO more than 12 amino acids, e.g. NO more than 11 amino acids, e.g. NO more than 10 amino acids, e.g. NO more than 9 amino acids, e.g. NO more than 8 amino acids, e.g. NO more than 7 amino acids, e.g. NO more than 6 amino acids, e.g. NO more than 5 amino acids, e.g. NO more than 4 amino acids, e.g. NO more than 3 amino acids, e.g. NO more than 1, such as deleted or NO more than 1 amino acid.

63. The tolerance inducing construct of any one of embodiments 8-62, wherein the non-covalent binding unit comprises a CH3 domain from IgG1 or consists of a CH3 domain from IgG 1.

64. The tolerance inducing construct of any one of embodiments 8-63, wherein the non-covalent binding unit comprises or consists of a CH3 domain derived from IgGl having a sequence identical to SEQ ID NO:4 has an amino acid sequence having at least 80% sequence identity.

65. The tolerance-inducing construct of any one of embodiments 8-64, wherein the non-covalent binding unit comprises or consists of a CH3 domain from IgG1 having an amino acid sequence with 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. 4.

66. The tolerance-inducing construct according to any of embodiments 8 to 65, wherein the non-covalent binding unit comprises or consists of a CH3 domain derived from IgGl having the amino acid sequence of SEQ ID No. 3, wherein any one amino acid of the flexible unit has been substituted, deleted or inserted by another amino acid, provided that NO more than 21 amino acids have been so substituted, deleted or inserted, such as NO more than 20 amino acids, such as NO more than 19 amino acids, such as NO more than 18 amino acids, such as NO more than 17 amino acids, such as NO more than 16 amino acids, such as NO more than 15 amino acids, such as NO more than 14 amino acids, such as NO more than 13 amino acids, such as NO more than 12 amino acids, such as NO more than 11 amino acids, such as NO more than 10 amino acids, such as NO more than 9 amino acids, such as NO more than 8 amino acids, such as NO more than 7 amino acids, such as NO more than 6 amino acids, such as NO more than 5 amino acids, such as NO more than 4 amino acids, such as NO more than 3, such as NO more than 2 amino acids, such as NO more than 1 amino acid has been substituted.

67. The tolerance inducing construct of any one of embodiments 8-66, wherein the non-covalent binding unit comprises or consists of a leucine zipper motif.

68. The tolerance-inducing construct of embodiment 67, wherein the leucine zipper motif is derived from a bZIP-like eukaryotic transcription factor.

69. The tolerance inducing construct of any one of embodiments 8-67, wherein the non-covalent binding unit comprises or consists of a Jun/Fos-based leucine zipper.

70. The tolerance inducing construct of any one of embodiments 8-67, wherein the leucine zipper motif comprises or consists of the amino acid sequence of SEQ ID No. 5.

71. The tolerance-inducing construct of any one of embodiments 8-67, wherein the non-covalent binding unit comprises or consists of an amino acid sequence having at least 80%, e.g. at least 81% or at least 81%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID No. 5.

72. The tolerance-inducing construct according to any one of embodiments 8-67, wherein the non-covalent binding unit comprises or consists of the amino acid sequence of SEQ ID NO 5, wherein any amino acid of the flexible unit has been substituted, deleted or inserted with another amino acid, provided that NO more than 12, such as NO more than 11, such as NO more than 10, such as NO more than 9, such as NO more than 8, such as NO more than 7, such as NO more than 6, such as NO more than 5, such as NO more than 4 amino acids, such as NO more than 3 amino acids, such as NO more than 2 amino acids, such as NO more than 1 amino acid.

73. The tolerance-inducing construct of any one of embodiments 8-72, wherein the non-covalent binding unit binds a plurality of polypeptides, such as two, three, four or more polypeptides, to a multimeric protein, such as a dimeric protein, a trimeric protein or a tetrameric protein.

74. The tolerance-inducing construct of any one of embodiments 8-73, wherein the non-covalent binding unit is or comprises 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.

75. The tolerance-inducing construct according to any one of embodiments 8-74, wherein the non-covalent binding unit is a trimerization unit comprising or consisting of the nucleotide sequence of SEQ ID No. 158 or an amino acid sequence encoded by said nucleotide sequence.

76. The tolerance inducing construct according to any one of embodiments 8-75, wherein the trimerization unit comprises or consists of the C-terminal domain of T4 fibritin.

77. The tolerance-inducing construct according to any one of embodiments 8-76, wherein the non-covalent binding unit is a trimerisation unit comprising or consisting of the amino acid sequence of SEQ ID No. 159 or a nucleotide sequence encoding said amino acid sequence.

78. The tolerance inducing construct of any one of embodiments 8-77, wherein the non-covalent binding unit comprises or consists of a tetramerisation unit, such as a domain derived from p 53.

79. The tolerance-inducing construct according to any one of embodiments 8-78, wherein the non-covalent binding unit is a tetramerisation unit comprising or consisting of the nucleotide sequence of SEQ ID No. 160 or an amino acid sequence encoded by said nucleotide sequence.

80. The tolerance-inducing construct of any one of the preceding embodiments, wherein the first junction region and/or the second junction region comprises or consists of a naturally occurring sequence.

81. The tolerance-inducing construct of any one of the preceding embodiments, wherein the first junction region and/or the second junction region comprises or consists of an artificial sequence.

82. The tolerance-inducing construct of any one of the preceding embodiments, wherein the number of cysteine residues in the first and/or second junction regions is based on the length of the antigenic unit.

83. The tolerance-inducing construct of any one of the preceding embodiments, wherein the junction region comprises a binding unit comprising a covalent binding unit and a non-covalent binding unit.

84. The tolerance-inducing construct of any one of the preceding embodiments, wherein the junction region is non-immunogenic.

85. The tolerance inducing construct of any one of embodiments 7 to 84, wherein the flexible unit is located between the targeting unit and the binding unit.

86. The tolerance inducing construct of any one of embodiments 7 to 85, wherein the flexible unit is a non-immunogenic sequence.

87. The tolerance inducing construct of any one of embodiments 7 to 86, wherein at least one flexible unit is a naturally occurring peptide sequence.

88. The tolerance inducing construct of any one of embodiments 7 to 87, wherein the flexible unit is derived from an immunoglobulin.

89. The tolerance inducing construct of any one of embodiments 7 to 88, wherein the flexible unit is a hinge region derived from an immunoglobulin, e.g., exon h1 of IgG3 or the lower hinge of IgG 1.

90. The tolerance inducing construct of any one of embodiments 7 to 89, wherein the flexible unit comprises or consists of hinge exon h1 of IgG 3.

91. The tolerance inducing construct of any one of embodiments 7 to 90, wherein the flexible unit comprises or consists of the lower hinge region of IgG 1.

92. The tolerance inducing construct of any one of embodiments 7 to 91, wherein the flexible unit comprises or consists of the amino acid sequence of 1-12 of SEQ ID No. 1.

93. The tolerance-inducing construct according to any one of embodiments 7 to 92, wherein the flexible unit comprises or consists of an amino acid sequence having at least 50% sequence identity, e.g. 60% or e.g. 70% or e.g. 80% or e.g. 90% sequence identity, with the amino acid sequence of 16-23 of SEQ ID No. 2.

94. The tolerance-inducing construct of any one of embodiments 7 to 93, wherein any one amino acid of the flexible unit has been substituted, deleted or inserted with another amino acid, provided that no more than 5 amino acids, such as no more than 4 amino acids, such as no more than 3 amino acids, such as no more than 2 amino acids, or no more than 1 amino acid have been so substituted, deleted or inserted.

95. The tolerance-inducing construct of any one of embodiments 7 to 94, wherein the flexible unit is derived from an immunoglobulin, e.g., a hinge region of an immunoglobulin that does not comprise a cysteine residue.

96. The tolerance inducing construct of any one of embodiments 7 to 95, wherein at least one flexible unit is an artificial sequence.

97. The tolerance inducing construct of any one of embodiments 7-83 and 96, wherein the flexible unit is a serine and/or glycine rich linker.

98. The tolerance inducing construct of any one of embodiments 7-84 and 97-98, wherein the flexible unit is a glycine-serine linker, e.g., GGGGSGGGGS (SEQ ID NO: 80).

99. The tolerance inducing construct of any one of embodiments 7 to 99, wherein the flexible unit is not a target of a protease.

100. The tolerance-inducing construct of any one of embodiments 7 to 99, wherein the flexible unit consists of up to 20 amino acids, e.g. up to 15 amino acids, e.g. 12 amino acids or 10 amino acids.

101. The tolerance-inducing construct according to any one of embodiments 7 to 100, wherein the flexible unit comprised in the second junction region consists of 5 to 60 amino acids, e.g. 7 to 55 amino acids or 8 to 50 amino acids or 9 to 45 amino acids or 10 to 40 amino acids or 11 to 35 amino acids or 12 to 30 amino acids or 13 to 20 amino acids.

102. The tolerance-inducing construct of any one of embodiments 7 to 101, wherein the flexible unit comprises a small non-polar amino acid, such as glycine, alanine, or leucine, or a polar amino acid, such as serine or threonine.

103. The tolerance-inducing construct of any one of the preceding embodiments, wherein the junction region is non-immunogenic.

104. The tolerance-inducing construct of any one of the preceding embodiments, wherein at least one of the first or second targeting units comprises a moiety that interacts with a surface molecule on an antigen presenting cell, preferably wherein both the first and second targeting units comprise a moiety that interacts with a surface molecule on an antigen presenting cell.

105. The tolerance-inducing construct of embodiment 104, wherein the surface molecule is selected from the group consisting of tgfβ receptor (tgfβr1, tgfβr2, or tgfβr3), IL10R (e.g., IL-10RA and IL 10-RB), IL2R, IL4R, IL6R, IL11R and IL13R, IL27R, IL35R, IL R, 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, HVEM, aryl hydrocarbon receptor, and vitamin D receptor.

106. The tolerance-inducing construct of embodiment 105, wherein the targeting unit comprises a moiety that is a natural ligand, an antibody or a portion thereof (e.g., scFv), or a synthetic ligand.

107. The tolerance-inducing construct of embodiment 106, wherein the natural ligand is selected from the group consisting of TGF-beta, IL-10, IL1RA, 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, 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).

108. The tolerance-inducing construct of any one of the preceding embodiments, wherein the targeting unit comprises or consists of IL-10 or tgfβ, preferably human IL-10 or human tgfβ.

109. The tolerance-inducing construct of 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 tgfβ.

110. The tolerance-inducing construct of any one of the preceding embodiments, wherein the targeting unit comprises or consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence of human tgfβ, 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%, e.g. at least 99% or e.g. 100%.

111. The tolerance-inducing construct of any one of the preceding embodiments, wherein the targeting unit comprises or consists of the amino acid sequence of human tgfβ, except that up to 22 amino acids, e.g. up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid has been substituted, deleted or inserted.

112. The tolerance-inducing construct of 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.

113. The tolerance-inducing construct of any one of the preceding embodiments, wherein the targeting unit comprises or consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence of human IL-10, 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%, e.g. at least 99% or e.g. 100%.

114. The tolerance-inducing construct of any one of the preceding embodiments, wherein the targeting unit comprises or consists of the amino acid sequence of human IL-10, except that up to 22 amino acids, e.g. up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid has been substituted, deleted or inserted.

115. The tolerance-inducing construct of any one of the preceding embodiments, wherein the targeting unit comprises or consists of an amino acid sequence of human IL-10 or a nucleotide sequence encoding human IL-10.

116. The tolerance-inducing construct of any one of the preceding embodiments, wherein the targeting unit is or comprises SCGB3A2 or VSIG-3, preferably human VSIG-3 or human SCGB3A2.

117. The tolerance-inducing construct of 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.

118. The tolerance-inducing construct of any one of the preceding embodiments, wherein the targeting unit comprises or consists of an amino acid sequence having at least 85% sequence identity with the amino acid sequence of human SCGB3A2, 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%.

119. The tolerance-inducing construct of any one of the preceding embodiments, wherein the targeting unit comprises or consists of the amino acid sequence of human SCGB3A2, except that up to 22 amino acids, e.g. up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid has been substituted, deleted or inserted.

120. The tolerance-inducing construct of any one of the preceding embodiments, wherein the targeting unit comprises or consists of the amino acid sequence of human SCGB3A2 or the nucleotide sequence encoding human SCGB3 A2.

121. The tolerance-inducing construct of 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.

122. The tolerance-inducing construct of any one of the preceding embodiments, wherein the targeting unit comprises or consists of an amino acid sequence having at least 85% sequence identity to the amino acid sequence of human VSIG-3, 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%, e.g., at least 99%, or e.g., 100%.

123. The tolerance-inducing construct of any one of the preceding embodiments, wherein the targeting unit comprises or consists of the amino acid sequence of human VSIG-3, except that up to 22 amino acids, e.g., up to 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid have been substituted, deleted or inserted.

124. The tolerance-inducing construct of any one of the preceding embodiments, wherein the targeting unit comprises or consists of the amino acid sequence of human VSIG-3 or the nucleotide sequence encoding human VSIG-3.

125. The tolerance-inducing construct of any one of the preceding embodiments, wherein the targeting unit comprises or consists of an antibody or a portion thereof, e.g. an scFv, specific for CD 205.

126. The tolerance-inducing construct of any one of the preceding embodiments, wherein the first targeting unit and the second targeting unit are the same.

127. The tolerance inducing construct of any one of embodiments 1 to 125, wherein the first targeting unit and the second targeting unit are different.

128. The tolerance inducing construct of any one of embodiments 104 to 127, wherein the surface molecules are present on the same cell.

129. The tolerance inducing construct of any one of embodiments 104 to 128, wherein binding of the first targeting unit or the second targeting unit results in internalization of the construct.

130. The tolerance-inducing construct of any one of the preceding embodiments, wherein the antigenic unit is located between the first and second junction regions.

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

132. The tolerance-inducing construct of embodiment 131, wherein the plurality of T cell epitopes are T cell epitopes of the same autoantigen, e.g., comprised in the same autoantigen.

133. The tolerance inducing construct according to any one of embodiments 131-132, wherein the plurality of T cell epitopes are T cell epitopes of different autoantigens, e.g. comprised in different autoantigens.

134. The tolerance-inducing construct of any one of the preceding embodiments, wherein the antigenic unit comprises more than one T cell epitope and the antigenic unit comprises one or more linkers separating the T cell epitopes.

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

136. The tolerance-inducing construct of any one of the preceding embodiments, wherein 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.

137. The tolerance-inducing construct of any one of the preceding embodiments, wherein the antigenic unit comprising n antigens comprises n-1 subunits (wherein each subunit comprises a T cell epitope and a linker of a self antigen, allergen, alloantigen or xenogeneic antigen), and further comprises a terminal T cell epitope.

138. The tolerance-inducing construct of embodiment 137, wherein n is an integer from 1 to 50, e.g., 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.

139. The tolerance-inducing construct of any one of the preceding embodiments, wherein the antigenic unit comprises a linker designed to be non-immunogenic.

140. The tolerance-inducing construct of any one of embodiments 131-139, wherein the antigenic unit comprises one or more T cell epitopes of an allergen, e.g., one T cell epitope of an allergen or more than one T cell epitope of an allergen, e.g., a plurality of T cell epitopes of an allergen.

141. The tolerance inducing construct of any one of embodiments 131-140, wherein the plurality of T cell epitopes are T cell epitopes of the same allergen, e.g., are comprised in the same allergen.

142. The tolerance inducing construct according to any one of embodiments 131-141, wherein the plurality of T cell epitopes are T cell epitopes of different allergens, i.e. comprised in different allergens.

143. The tolerance-inducing construct of 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 more than one T cell epitope of an alloantigen/xenogeneic antigen, e.g. a plurality of T cell epitopes of an alloantigen/xenogeneic antigen.

144. The tolerance inducing construct according to any one of embodiments 131-143, wherein the plurality of T cell epitopes are T cell epitopes of the same alloantigen/xenogeneic antigen, i.e. comprised in the same alloantigen/xenogeneic antigen.

145. The tolerance inducing construct according to any one of embodiments 131-144, wherein the plurality of T cell epitopes are T cell epitopes of different alloantigens/xenogeneic antigens, e.g. comprised in different alloantigens/xenogeneic antigens.

146. The tolerance-inducing construct of any one of the preceding embodiments, wherein the antigenic unit comprises a T cell epitope.

147. The tolerance-inducing construct of any one of the preceding embodiments, wherein the antigenic unit comprises more than one T cell epitope, e.g., a plurality of T cell epitopes.

148. The tolerance-inducing construct of any one of the preceding embodiments, wherein the antigenic unit comprises a length of 7 to 150 amino acids, preferably 7 to 100 amino acids, e.g. about 10 to about 100 amino acids or about 15 to about 100 amino acids or about 20 to about 75 amino acids or about 25 to about 50 amino acids, such as a T cell epitope of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids.

149. The tolerance inducing construct of any one of embodiments 131-148, wherein one T cell epitope is of a length such that the protein is incorrectly folded.

150. The tolerance inducing construct of any one of embodiments 131-149, wherein the T cell epitope has a length suitable for presentation by MHC (major histocompatibility complex).

151. The tolerance-inducing construct of any one of the preceding embodiments, wherein the antigenic unit comprises a T cell epitope having a length suitable for specific presentation on MHC class I or MHC class II.

152. The tolerance inducing construct of any one of embodiments 131-151, wherein the T cell epitope has a length of 7 to 11 amino acids for MHC class I presentation. In another embodiment, the T cell epitope sequence has a length 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 for MHC class II presentation.

153. The tolerance inducing construct of any one of embodiments 131-152, wherein the T cell epitope has a length of 15 amino acids for MHC class II presentation.

154. The tolerance-inducing construct of 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.

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

156. The tolerance-inducing construct of any one of embodiments 137-155, wherein the subunit antigenic unit comprises 1 to 3T cell epitopes, e.g., 1, 2, 3T cell epitopes, or 1 to 5T cell epitopes, e.g., 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.

157. The tolerance inducing construct of any one of embodiments 131-156, wherein the T cell epitope is randomly arranged in an antigenic unit.

158. The tolerance inducing construct according to any one of embodiments 131-157, wherein the T cell epitopes are arranged in order of higher antigenicity to lower antigenicity along the direction from the multimerization/dimerization unit to the terminus of the antigenic unit.

159. The tolerance inducing construct according to any one of embodiments 131-158, wherein the T cell epitopes are arranged in order of higher antigenicity to lower antigenicity along the direction from the first junction region to the second junction region.

160. The tolerance inducing construct according to any one of embodiments 131-158, wherein the most hydrophobic T cell epitope is located substantially in the middle of the first antigenic unit and the most hydrophilic T cell epitope is located towards the junction region.

161. The tolerance inducing construct of any one of embodiments 131-158, wherein the T cell epitope alternates between a hydrophilic T cell epitope and a hydrophobic T cell epitope.

162. The tolerance inducing construct according to any one of embodiments 131-158, wherein the GC-rich sequence encoding the T cell epitope is arranged in a manner that avoids GC clusters.

163. The tolerance-inducing construct of embodiment 162, wherein the GC-rich T cell sequences are arranged in a manner such that there is at least one non-GC-rich T cell sequence therebetween.

164. The tolerance-inducing construct of any one of the preceding embodiments, wherein the construct is a polynucleotide further comprising a nucleotide sequence encoding a signal peptide.

165. The tolerance-inducing construct of 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.

166. The tolerance-inducing construct of 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.

167. The tolerance-inducing construct of 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.

168. The tolerance-inducing construct of 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.

169. The tolerance-inducing construct according to any of the preceding embodiments, wherein the signal peptide comprises or consists of the amino acid sequence of SEQ ID No. 6, wherein any one amino acid of the signal peptide has been substituted, deleted or inserted with another amino acid, provided that NO more than 5 amino acids, e.g. NO more than 4 amino acids, e.g. NO more than 3 amino acids, e.g. NO more than 2 amino acids or NO more than 1 amino acid have been so substituted, deleted or inserted.

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

171. A vector comprising the polynucleotide of embodiment 170.

172. A host cell comprising the polynucleotide of any one of embodiments 169-170 and/or the vector of any one of embodiments 169-170.

173. A polypeptide encoded by a nucleotide sequence nucleic acid as defined in any one of embodiments 1 to 169.

174. Multimeric proteins, such as dimeric proteins, are defined in any one of embodiments 1 to 169, wherein a plurality of polypeptides, such as two polypeptides, are linked to each other via their respective first junction regions and via their respective second junction regions.

175. The multimeric protein of any one of embodiments 1-169, wherein said plurality of polypeptides are linked to each other via their respective first junction regions and via their respective second junction regions.

176. The dimeric protein as defined in any one of the preceding embodiments, wherein the two polypeptides are linked to each other via their respective first junction regions and via their respective second junction regions.

177. A method of preparing a pharmaceutical composition, the method comprising:

a) Providing a polynucleotide, polypeptide, or multimeric protein of any one of embodiments 1 to 176, e.g., a dimeric protein; and

178. A method of preparing a pharmaceutical composition, the method comprising:

179. A method of preparing a pharmaceutical composition, the method comprising:

a) Providing the polynucleotide, polypeptide, or dimeric protein of any one of embodiments 1-176; and

180. A pharmaceutical composition comprising a polynucleotide, polypeptide or multimeric protein, such as a dimeric protein, as defined in any one of embodiments 1 to 176 and a pharmaceutically acceptable carrier.

181. A pharmaceutical composition comprising a polynucleotide, polypeptide or multimeric protein as defined in any one of embodiments 1 to 175 and a pharmaceutically acceptable carrier.

182. A pharmaceutical composition comprising a polynucleotide, polypeptide or dimeric protein as defined in any one of embodiments 1 to 176 and a pharmaceutically acceptable carrier.

183. A pharmaceutical composition comprising the polynucleotide of any one of embodiments 1 to 176, further comprising one or more pharmaceutically acceptable excipients and/or diluents.

184. A pharmaceutical composition comprising the polynucleotide of any one of embodiments 1 to 176, 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.

185. The pharmaceutical compositions of embodiments 180 to 185 are for use as a medicament.

186. The pharmaceutical compositions of embodiments 180-185 are useful for treating conditions involving undesired immune responses, such as for prophylactic or therapeutic treatment of autoimmune diseases, allergic diseases, and graft rejection.

187. The pharmaceutical compositions of embodiments 180-186 are useful for treating autoimmune diseases.

188. The pharmaceutical compositions of embodiments 180 to 187 are for use in treating allergy.

189. The pharmaceutical compositions of embodiments 180-188 are useful for treating graft rejection.

190. A method for treating a subject having or suspected of having an immune disorder selected from the group consisting of autoimmune disorders, allergic disorders, and graft rejection, or in need of prevention of such immune disorder, the method comprising administering to the subject the pharmaceutical composition of any one of embodiments 180 to 189, the pharmaceutical composition comprising a pharmaceutically acceptable carrier.

191. A pharmaceutical composition for use in the prophylactic or therapeutic treatment of an immune disorder selected from the group consisting of an autoimmune disorder, an allergic disorder, and graft rejection, wherein the pharmaceutical composition is the pharmaceutical composition of any one of embodiments 180 to 189.

192. A pharmaceutical composition for use in the prophylactic or therapeutic treatment of a subject having or suspected of having an immune disease selected from the group consisting of an autoimmune disease, an allergic disease, and graft rejection, wherein the pharmaceutical composition is a pharmaceutical composition of any one of embodiments 180 to 189.

193. Use of 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, wherein the pharmaceutical composition is the pharmaceutical composition of any one of embodiments 180 to 189.

194. Use of a pharmaceutical composition in the manufacture of a medicament for the 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 transplant rejection, wherein the pharmaceutical composition is the pharmaceutical composition of any one of embodiments 180 to 189.

195. Use of a pharmaceutical composition for the prophylactic or therapeutic treatment of a subject suffering from an immune disorder selected from the group consisting of autoimmune disorders, allergic disorders, and graft rejection, wherein the pharmaceutical composition is the pharmaceutical composition of any one of embodiments 180 to 189.

196. Use of the pharmaceutical composition of any one of embodiments 180 to 189 in the manufacture of a medicament for the prophylactic or therapeutic treatment of a subject having or suspected of having an immune disorder selected from the group consisting of autoimmune disorders, allergic disorders, and transplant rejection, wherein the medicament is administered to the subject.

197. Use of the pharmaceutical composition of any of embodiments 180-189 for the prophylactic or therapeutic treatment of a subject having or suspected of having an immune disorder selected from the group consisting of autoimmune disorders, allergic disorders, and transplant rejection, wherein a medicament is administered to the subject.

198. The pharmaceutical composition of any of embodiments 180 to 189 when used for the prophylactic or therapeutic treatment of an immune disorder selected from the group consisting of autoimmune disorders, allergic disorders, and transplant rejection.

199. A medicament for prophylactic or therapeutic treatment of a subject suffering from or suspected of suffering from an immune disorder selected from the group consisting of autoimmune disorders, allergic disorders and graft rejection by administering to said subject a pharmaceutical composition according to any one of embodiments 180 to 189.

200. A method of making a polypeptide or multimeric protein, such as a dimeric protein, the method comprising:

a) Transfecting a cell with the vector defined in embodiment 171 or the polynucleotide of any one of embodiments 1 to 170;

b) Culturing the cell such that the cell expresses the polypeptide encoded by the polynucleotide; and

c) Multimeric proteins, such as dimeric proteins, and/or polypeptides expressed by the cells are obtained and purified.

201. A method of producing a polypeptide or multimeric protein, the method comprising:

a) Transfecting a cell with the vector defined in embodiment 171 or the polynucleotide of any one of embodiments 1 to 170 and 173;

c) Multimeric proteins and/or polypeptides expressed by the cells are obtained and purified.

202. A method of producing a polypeptide or a dimeric protein, the method comprising:

c) The dimeric proteins and/or polypeptides expressed by the cells are obtained and purified.

203. The method of any one of embodiments 200 to 202, wherein step c) comprises a step of purifying a fraction comprising said plurality of proteins, e.g. dimeric proteins, wherein said plurality of polypeptides, e.g. two polypeptides, are linked to each other via their respective first junction region and via their respective second junction region.

204. A method of treating a disorder involving an undesired immune response, for example in the prophylactic or therapeutic treatment of autoimmune diseases, allergic diseases and graft rejection, the method comprising administering to a subject in need thereof the polynucleotide, polypeptide, multimeric protein or dimeric protein of any one of embodiments 1 to 170 and 173, the vector of embodiment 171 or the pharmaceutical composition of any one of embodiments 180 to 189.

205. The method of embodiment 204, wherein the subject is a mammal.

206. The method of embodiment 205, wherein the mammal is a human.

Sequence listing

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<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(29)

<223> amino acid sequence of CREB bZIP motif

<400> 5

Val Lys Cys Leu Glu Asn Arg Val Ala Val Leu Glu Asn Gln Asn Lys

1 5 10 15

Thr Leu Ile Glu Glu Leu Lys Ala Leu Lys Asp Leu Tyr

20 25

<210> 6

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(19)

<223> mouse immunoglobulin heavy chain signal sequence (Ig VH signal sequence)

<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> 246

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(246)

<223> mouse Single chain variable fragment (scFv) anti-DEC205

<400> 7

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> 8

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(20)

<223> hinge region of human IgG1

<400> 8

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

1 5 10 15

Pro Pro Cys Pro

20

<210> 9

<211> 160

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(160)

<223> murine IL-10

<400> 9

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> 10

<211> 112

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(112)

<223> mature murine TGFbeta1

<400> 10

Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser Thr Glu Lys Asn Cys Cys

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 11

<211> 126

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(126)

<223> mature murine CTLA-4

<400> 11

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> 12

<211> 37

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(37)

<223> MOG(27-63)

<400> 12

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> 13

<211> 37

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(37)

<223> MOG (27-63) sequences obtained from Krienke et al 2021 Patent

US2020061166A1

<400> 13

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> 14

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(21)

<223> MOG(35-55)

<400> 14

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> 15

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(21)

<223> murine MARCO ligand SCGB3A2 Signal sequence

<400> 15

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> 16

<211> 70

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(70)

<223> murine MARCO ligand SCGB3A2

<400> 16

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> 17

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(22)

<223> murine VISTA ligand VSIG-3 Signal sequence

<400> 17

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> 18

<211> 218

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(218)

<223> murine VISTA ligand VSIG-3 extracellular domain

<400> 18

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> 19

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(12)

<223> hinge h1 hIgG3

<400> 19

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

1 5 10

<210> 20

<211> 23

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(23)

<223> human CCL3L1 Signal sequence

<400> 20

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> 21

<211> 70

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(70)

<223> human CCL3L1

<400> 21

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> 22

<211> 171

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(171)

<223> Met e 1 (241-260), (210-230), (136-155), (76-95), (46-65),

(16-35)

<400> 22

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> 23

<211> 514

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(514)

<223> amino acid sequence of VB5050

<400> 23

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Gly Glu Lys Leu Lys Thr Leu Arg Met Arg Leu Arg Arg Cys His Arg

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

Lys Ser

<210> 24

<211> 514

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(514)

<223> amino acid sequence of VB5038

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Gly Glu Lys Leu Lys Thr Leu Arg Met Arg Leu Arg Arg Cys His Arg

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

Lys Ser

<210> 25

<211> 526

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(526)

<223> amino acid sequence of VB5042

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

<210> 26

<211> 466

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(466)

<223> amino acid sequence of VB5066

<400> 26

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 Leu Gly Gly Leu Ser Pro Gly Lys Asn Ala Thr

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Cys Pro Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser Thr Glu Lys Asn

355 360 365

Cys Cys Val Arg Gln Leu Tyr Ile Asp Phe Arg Lys Asp Leu Gly Trp

370 375 380

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

385 390 395 400

Pro Cys Pro Tyr Ile Trp Ser Leu Asp Thr Gln Tyr Ser Lys Val Leu

405 410 415

Ala Leu Tyr Asn Gln His Asn Pro Gly Ala Ser Ala Ser Pro Cys Cys

420 425 430

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

435 440 445

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

450 455 460

Cys Ser

465

<210> 27

<211> 478

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(478)

<223> amino acid sequence of VB5043

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475

<210> 28

<211> 480

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(480)

<223> amino acid sequence of VB5067

<400> 28

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 Leu Gly Gly Leu Ser Pro Gly Lys Asn Ala Thr

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Ser His Gly Val Ala Ser Phe Pro Cys Glu Tyr Ser Pro Ser His Asn

370 375 380

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

385 390 395 400

Thr Glu Val Cys Ala Thr Thr Phe Thr Glu Lys Asn Thr Val Gly Phe

405 410 415

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

420 425 430

Leu Thr Ile Gln Gly Leu Arg Ala Val Asp Thr Gly Leu Tyr Leu Cys

435 440 445

Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Phe Val Gly Met Gly Asn

450 455 460

Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp

465 470 475 480

<210> 29

<211> 340

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(340)

<223> amino acid sequence of VB5072

<400> 29

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 Leu Gly Gly Leu Ser Pro Gly Lys Asn

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Pro Pro Cys Pro Ser Arg Gly Gln Tyr Ser Arg Glu Asp Asn Asn Cys

180 185 190

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

195 200 205

Ala Phe Ser Gln Val Lys Thr Phe Phe Gln Thr Lys Asp Gln Leu Asp

210 215 220

Asn Ile Leu Leu Thr Asp Ser Leu Met Gln Asp Phe Lys Gly Tyr Leu

225 230 235 240

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Val Glu Val

245 250 255

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

260 265 270

Ser Leu Gly Glu Lys Leu Lys Thr Leu Arg Met Arg Leu Arg Arg Cys

275 280 285

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val

290 295 300

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

305 310 315 320

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

325 330 335

Lys Met Lys Ser

340

<210> 30

<211> 352

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(352)

<223> amino acid sequence of VB5073

<400> 30

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 Leu Gly Gly Leu Ser Pro Gly Lys Asn

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

<210> 31

<211> 489

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(489)

<223> amino acid sequence of VB5074

<400> 31

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 Leu Gly Gly Leu

260 265 270

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

275 280 285

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

290 295 300

Ala Glu Gln Ala Pro Gly Leu Gln Gly Leu Glu Pro Lys Ser Cys Asp

305 310 315 320

Lys Thr His Thr Cys Pro Pro Cys Pro Ser Arg Gly Gln Tyr Ser Arg

325 330 335

Glu Asp Asn Asn Cys Thr His Phe Pro Val Gly Gln Ser His Met Leu

340 345 350

Leu Glu Leu Arg Thr Ala Phe Ser Gln Val Lys Thr Phe Phe Gln Thr

355 360 365

Lys Asp Gln Leu Asp Asn Ile Leu Leu Thr Asp Ser Leu Met Gln Asp

370 375 380

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

385 390 395 400

Tyr Leu Val Glu Val Met Pro Gln Ala Glu Lys His Gly Pro Glu Ile

405 410 415

Lys Glu His Leu Asn Ser Leu Gly Glu Lys Leu Lys Thr Leu Arg Met

420 425 430

Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys

435 440 445

Ala Val Glu Gln Val Lys Ser Asp Phe Asn Lys Leu Gln Asp Gln Gly

450 455 460

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

465 470 475 480

Ala Tyr Met Met Ile Lys Met Lys Ser

485

<210> 32

<211> 501

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(501)

<223> amino acid sequence of VB5075

<400> 32

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 Leu Gly Gly Leu

260 265 270

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

275 280 285

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

290 295 300

Ala Glu Gln Ala Pro Gly Leu Gln Gly Leu Glu Pro Lys Ser Cys Asp

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Thr Ala Phe Ser Gln Val Lys Thr Phe Phe Gln Thr Lys Asp Gln Leu

370 375 380

Asp Asn Ile Leu Leu Thr Asp Ser Leu Met Gln Asp Phe Lys Gly Tyr

385 390 395 400

Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Val Glu

405 410 415

Val Met Pro Gln Ala Glu Lys His Gly Pro Glu Ile Lys Glu His Leu

420 425 430

Asn Ser Leu Gly Glu Lys Leu Lys Thr Leu Arg Met Arg Leu Arg Arg

435 440 445

Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln

450 455 460

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

465 470 475 480

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

485 490 495

Ile Lys Met Lys Ser

500

<210> 33

<211> 279

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(279)

<223> amino acid sequence of VB5052

<400> 33

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

1 5 10 15

Leu Cys Asn Gln Val Leu Ser Ala Pro Leu Ala Ala Asp Thr Pro Thr

20 25 30

Ala Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile

35 40 45

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

50 55 60

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

65 70 75 80

Trp 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> 34

<211> 279

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(279)

<223> VB5002b amino acid sequence

<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 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> 35

<211> 56

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(56)

<223> amino acid sequence of VB5051

<400> 35

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

1 5 10 15

Val Gln Cys 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> 36

<211> 56

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(56)

<223> VB5001b amino acid sequence

<400> 36

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

1 5 10 15

Val Gln Cys 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> 37

<211> 648

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(648)

<223> amino acid sequence of VB5077

<400> 37

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

Pro Arg Cys Pro Gly Leu Gly Gly Leu Lys Glu Val Asp Arg Leu Glu

290 295 300

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

305 310 315 320

Gly Ser Gly Gly Gly Gly Ser Ala Tyr Lys Glu Gln Ile Lys Thr Leu

325 330 335

Thr Asn Lys Leu Lys Ala Ala Glu Ala Arg Ala Glu Gly Gly Gly Gly

340 345 350

Ser Gly Gly Gly Gly Ser Asn Gln Leu Lys Glu Ala Arg Phe Leu Ala

355 360 365

Glu Glu Ala Asp Arg Lys Tyr Asp Glu Val Gly Gly Gly Gly Ser Gly

370 375 380

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

385 390 395 400

Asp Leu Glu Arg Ser Glu Glu Arg Gly Gly Gly Gly Ser Gly Gly Gly

405 410 415

Gly Ser Asp Leu Asp Gln Val Gln Glu Ser Leu Leu Lys Ala Asn Asn

420 425 430

Gln Leu Val Glu Lys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

435 440 445

Glu Gln Gln Asn Lys Glu Ala Asn Asn Arg Ala Glu Lys Ser Glu Glu

450 455 460

Glu Val His Asn Gly Leu Gln Gly Leu Glu Pro Lys Ser Cys Asp Lys

465 470 475 480

Thr His Thr Cys Pro Pro Cys Pro Ser Arg Gly Gln Tyr Ser Arg Glu

485 490 495

Asp Asn Asn Cys Thr His Phe Pro Val Gly Gln Ser His Met Leu Leu

500 505 510

Glu Leu Arg Thr Ala Phe Ser Gln Val Lys Thr Phe Phe Gln Thr Lys

515 520 525

Asp Gln Leu Asp Asn Ile Leu Leu Thr Asp Ser Leu Met Gln Asp Phe

530 535 540

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

545 550 555 560

Leu Val Glu Val Met Pro Gln Ala Glu Lys His Gly Pro Glu Ile Lys

565 570 575

Glu His Leu Asn Ser Leu Gly Glu Lys Leu Lys Thr Leu Arg Met Arg

580 585 590

Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala

595 600 605

Val Glu Gln Val Lys Ser Asp Phe Asn Lys Leu Gln Asp Gln Gly Val

610 615 620

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

625 630 635 640

Tyr Met Met Ile Lys Met Lys Ser

645

<210> 38

<211> 660

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(660)

<223> amino acid sequence of VB5078

<400> 38

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 Leu Gly Gly Leu Lys Glu Val Asp Arg Leu Glu

290 295 300

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

305 310 315 320

Gly Ser Gly Gly Gly Gly Ser Ala Tyr Lys Glu Gln Ile Lys Thr Leu

325 330 335

Thr Asn Lys Leu Lys Ala Ala Glu Ala Arg Ala Glu Gly Gly Gly Gly

340 345 350

Ser Gly Gly Gly Gly Ser Asn Gln Leu Lys Glu Ala Arg Phe Leu Ala

355 360 365

Glu Glu Ala Asp Arg Lys Tyr Asp Glu Val Gly Gly Gly Gly Ser Gly

370 375 380

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

385 390 395 400

Asp Leu Glu Arg Ser Glu Glu Arg Gly Gly Gly Gly Ser Gly Gly Gly

405 410 415

Gly Ser Asp Leu Asp Gln Val Gln Glu Ser Leu Leu Lys Ala Asn Asn

420 425 430

Gln Leu Val Glu Lys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

435 440 445

Glu Gln Gln Asn Lys Glu Ala Asn Asn Arg Ala Glu Lys Ser Glu Glu

450 455 460

Glu Val His Asn Gly Leu Gln Gly Leu Glu Pro Lys Ser Cys Asp Lys

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

Ala Phe Ser Gln Val Lys Thr Phe Phe Gln Thr Lys Asp Gln Leu Asp

530 535 540

Asn Ile Leu Leu Thr Asp Ser Leu Met Gln Asp Phe Lys Gly Tyr Leu

545 550 555 560

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Val Glu Val

565 570 575

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

580 585 590

Ser Leu Gly Glu Lys Leu Lys Thr Leu Arg Met Arg Leu Arg Arg Cys

595 600 605

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val

610 615 620

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

625 630 635 640

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

645 650 655

Lys Met Lys Ser

660

<210> 39

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(6)

<223> joint

<220>

<221> MISC_FEATURE

<222> (1)..(4)

The <223> sequence may be repeated m times, m being an integer from 1 to 5, e.g. 1, 2, 3, 4 or 5

<400> 39

Glu Ala Ala Lys Gly Ser

1 5

<210> 40

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 40

Gly Leu Ser Gly Leu

1 5

<210> 41

<211> 510

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(510)

<223> amino acid sequence of VB5041

<400> 41

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 Leu Gly Gly Leu Met Glu Val Gly Trp Tyr Arg

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

<210> 42

<211> 36

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(36)

<223> joint

<400> 42

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> 43

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(18)

<223> Natural leader sequence, murine IL-10

<400> 43

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

1 5 10 15

Arg Ile

<210> 44

<211> 35

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(35)

<223> Natural leader sequence, murine CTLA4

<400> 44

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> 45

<211> 29

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(29)

<223> Natural leader sequence, murine transforming growth factor beta-1

<400> 45

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> 46

<211> 361

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(361)

<223> murine transforming growth factor beta-1 proprotein

<400> 46

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> 47

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(24)

<223> Natural leader sequence, murine PD-1

<400> 47

Met Trp Val Arg Gln Val Pro Trp Ser Phe Thr Trp Ala Val Leu Gln

1 5 10 15

Leu Ser Trp Gln Ser Gly Trp Leu

20

<210> 48

<211> 145

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(145)

<223> murine PD-1 extracellular Domain

<400> 48

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

1 5 10 15

Trp Leu Thr Val Ser Glu Gly Ala Asn Ala Thr Phe Thr Cys Ser Leu

20 25 30

Ser Asn Trp Ser Glu Asp Leu Met Leu Asn Trp Asn Arg Leu Ser Pro

35 40 45

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

50 55 60

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

65 70 75 80

Asp Phe His Met Asn Ile Leu Asp Thr Arg Arg Asn Asp Ser Gly Ile

85 90 95

Tyr Leu Cys Gly Ala Ile Ser Leu His Pro Lys Ala Lys Ile Glu Glu

100 105 110

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

115 120 125

Thr Arg Tyr Pro Ser Pro Ser Pro Lys Pro Glu Gly Arg Phe Gln Gly

130 135 140

Met

145

<210> 49

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(20)

<223> GAD65 (202-221)

<400> 49

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> 50

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(20)

<223> Mete1 (16-35)

<400> 50

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> 51

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(20)

<223> Mete1(46-65)

<400> 51

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> 52

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(20)

<223> Met e 1(76-95)

<400> 52

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> 53

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(20)

<223> Met e 1(136-155)

<400> 53

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> 54

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(21)

<223> Met e 1(210-230)

<400> 54

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> 55

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(20)

<223> Met e 1(241-260)

<400> 55

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> 56

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(21)

<223> joint

<400> 56

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> 57

<211> 30

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(30)

<223> joint

<400> 57

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> 58

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(14)

<223> joint

<400> 58

Asn Thr Trp Thr Thr Cys Gln Ser Ile Ala Phe Pro Ser Lys

1 5 10

<210> 59

<211> 1173

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<222> (1)..(1173)

<223> human transforming growth factor beta-1

<400> 59

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> 60

<211> 390

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(390)

<223> human transforming growth factor beta-1

<400> 60

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> 61

<211> 1329

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<222> (1)..(1329)

<223> human transforming growth factor beta 2 (TGFB 2)

<400> 61

atgcactact gtgtgctgag cgcttttctg atcctgcatc tggtcacggt cgcgctcagc 60

ctgtctacct gcagcacact cgatatggac cagttcatgc gcaagaggat cgaggcgatc 120

cgcgggcaga tcctgagcaa gctgaagctc accagtcccc cagaagacta tcctgagccc 180

gaggaagtcc ccccggaggt gatttccatc tacaacagca ccagggactt gctccaggag 240

aaggcgagcc ggagggcggc cgcctgcgag cgcgagagga gcgacgaaga gtactacgcc 300

aaggaggttt acaaaataga catgccgccc ttcttcccct ccgaaactgt ctgcccagtt 360

gttacaacac cctctggctc agtgggcagc ttgtgctcca gacagtccca ggtgctctgt 420

gggtaccttg atgccatccc gcccactttc tacagaccct acttcagaat tgttcgattt 480

gacgtctcag caatggagaa gaatgcttcc aatttggtga aagcagagtt cagagtcttt 540

cgtttgcaga acccaaaagc cagagtgcct gaacaacgga ttgagctata tcagattctc 600

aagtccaaag atttaacatc tccaacccag cgctacatcg acagcaaagt tgtgaaaaca 660

agagcagaag gcgaatggct ctccttcgat gtaactgatg ctgttcatga atggcttcac 720

cataaagaca ggaacctggg atttaaaata agcttacact gtccctgctg cacttttgta 780

ccatctaata attacatcat cccaaataaa agtgaagaac tagaagcaag atttgcaggt 840

attgatggca cctccacata taccagtggt gatcagaaaa ctataaagtc cactaggaaa 900

aaaaacagtg ggaagacccc acatctcctg ctaatgttat tgccctccta cagacttgag 960

tcacaacaga ccaaccggcg gaagaagcgt gctttggatg cggcctattg ctttagaaat 1020

gtgcaggata attgctgcct acgtccactt tacattgatt tcaagaggga tctagggtgg 1080

aaatggatac acgaacccaa agggtacaat gccaacttct gtgctggagc atgcccgtat 1140

ttatggagtt cagacactca gcacagcagg gtcctgagct tatataatac cataaatcca 1200

gaagcatctg cttctccttg ctgcgtgtcc caagatttag aacctctaac cattctctac 1260

tacattggca aaacacccaa gattgaacag ctttctaata tgattgtaaa gtcttgcaaa 1320

tgcagctaa 1329

<210> 62

<211> 442

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(442)

<223> human transforming growth factor beta-2

<400> 62

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> 63

<211> 930

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<222> (1)..(930)

<223> human transforming growth factor beta 3 (TGFB 3)

<400> 63

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> 64

<211> 309

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(309)

<223> human transforming growth factor beta-3

<400> 64

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> 65

<211> 537

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<222> (1)..(537)

<223> human interleukin 10 (IL 10)

<400> 65

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> 66

<211> 178

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(178)

<223> human interleukin-10 isoform 1 precursor

<400> 66

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> 67

<211> 282

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<222> (1)..(282)

<223> human secretoglobin 3A Member 2 (SCGB 3A 2)

<400> 67

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> 68

<211> 93

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(93)

<223> human secretoglobin 3A member 2 precursor

<400> 68

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> 69

<211> 723

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<222> (1)..(723)

<223> human immunoglobulin superfamily member 11 (IGSF 11) extracellular domain

<400> 69

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> 70

<211> 241

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(431)

<223> human immunoglobulin superfamily member 11 i (IGSF 11) extracellular domain

<400> 70

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> 71

<211> 483

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<222> (1)..(483)

<223> human cytotoxic T-lymphocyte-associated protein 4 (CTLA 4) extracellular domain

<400> 71

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> 72

<211> 161

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(161)

<223> CTLA4 human cytotoxic T-lymphocyte protein 4 extracellular domain

<400> 72

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> 73

<211> 510

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<222> (1)..(510)

<223> human programmed cell death 1 (PDCD 1) extracellular Domain

<400> 73

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

<210> 74

<211> 170

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(170)

<223> human apoptosis protein 1

<400> 74

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> 75

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 75

Gly Gly Gly Gly Ser Gly Gly Gly Ser Ser

1 5 10

<210> 76

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 76

Gly Gly Gly Ser Gly

1 5

<210> 77

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 77

Gly Gly Ser Gly Gly

1 5

<210> 78

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(6)

<223> joint

<400> 78

Ser Gly Ser Ser Gly Ser

1 5

<210> 79

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 79

Gly Gly Gly Gly Ser

1 5

<210> 80

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 80

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 81

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 81

Gly Gly Gly Gly Ser

1 5

<210> 82

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<220>

<221> MISC_FEATURE

<222> (1)..(5)

The <223> sequence may be repeated m times, where m is an integer from 1 to 5, such as 1, 2, 3, 4 or 5

<400> 82

Gly Gly Gly Ser Ser

1 5

<210> 83

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<400> 83

Gly Gly Gly Ser Gly

1 5

<210> 84

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(6)

<223> joint

<220>

<221> MISC_FEATURE

<222> (1)..(6)

<400> 84

Ser Gly Ser Ser Gly Ser

1 5

<210> 85

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 85

Leu Gly Gly Gly Ser

1 5

<210> 86

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 86

Gly Leu Gly Gly Ser

1 5

<210> 87

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 87

Gly Gly Leu Gly Ser

1 5

<210> 88

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 88

Gly Gly Gly Leu Ser

1 5

<210> 89

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 89

Gly Gly Gly Gly Leu

1 5

<210> 90

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 90

Leu Gly Gly Ser Gly

1 5

<210> 91

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 91

Gly Leu Gly Ser Gly

1 5

<210> 92

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 92

Gly Gly Leu Ser Gly

1 5

<210> 93

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 93

Gly Gly Gly Leu Gly

1 5

<210> 94

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 94

Gly Gly Gly Ser Leu

1 5

<210> 95

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 95

Leu Gly Gly Ser Ser

1 5

<210> 96

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 96

Gly Leu Gly Ser Ser

1 5

<210> 97

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 97

Gly Gly Leu Ser Ser

1 5

<210> 98

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 98

Leu Gly Leu Gly Ser

1 5

<210> 99

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 99

Gly Leu Gly Leu Ser

1 5

<210> 100

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 100

Gly Leu Leu Gly Ser

1 5

<210> 101

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 101

Leu Gly Gly Leu Ser

1 5

<210> 102

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 102

Gly Leu Gly Gly Leu

1 5

<210> 103

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 103

Leu Gly Leu Ser Gly

1 5

<210> 104

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 104

Gly Leu Leu Ser Gly

1 5

<210> 105

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 105

Gly Gly Leu Ser Leu

1 5

<210> 106

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 106

Gly Gly Leu Leu Gly

1 5

<210> 107

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 107

Gly Leu Gly Ser Leu

1 5

<210> 108

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 108

Leu Gly Leu Ser Ser

1 5

<210> 109

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<400> 109

Gly Gly Leu Leu Ser

1 5

<210> 110

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 110

Leu Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 111

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 111

Gly Leu Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 112

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 112

Gly Gly Leu Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 113

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 113

Gly Gly Gly Leu Ser Gly Gly Gly Gly Ser

1 5 10

<210> 114

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 114

Gly Gly Gly Gly Leu Gly Gly Gly Gly Ser

1 5 10

<210> 115

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 115

Leu Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10

<210> 116

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 116

Gly Leu Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10

<210> 117

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 117

Gly Gly Leu Ser Gly Gly Gly Gly Ser Gly

1 5 10

<210> 118

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 118

Gly Gly Gly Leu Gly Gly Gly Gly Ser Gly

1 5 10

<210> 119

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 119

Gly Gly Gly Ser Leu Gly Gly Gly Ser Gly

1 5 10

<210> 120

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 120

Leu Gly Gly Ser Ser Gly Gly Gly Ser Ser

1 5 10

<210> 121

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 121

Gly Leu Gly Ser Ser Gly Gly Gly Ser Ser

1 5 10

<210> 122

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 122

Gly Gly Leu Ser Ser Gly Gly Gly Ser Ser

1 5 10

<210> 123

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 123

Gly Gly Gly Leu Ser Gly Gly Gly Ser Ser

1 5 10

<210> 124

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 124

Gly Gly Gly Ser Leu Gly Gly Gly Ser Ser

1 5 10

<210> 125

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 125

Leu Gly Gly Gly Ser Leu Gly Gly Gly Ser

1 5 10

<210> 126

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 126

Gly Leu Gly Gly Ser Gly Leu Gly Gly Ser

1 5 10

<210> 127

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 127

Gly Gly Leu Gly Ser Gly Gly Leu Gly Ser

1 5 10

<210> 128

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 128

Gly Gly Gly Leu Ser Gly Gly Gly Leu Ser

1 5 10

<210> 129

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 129

Gly Gly Gly Gly Leu Gly Gly Gly Gly Leu

1 5 10

<210> 130

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 130

Leu Gly Gly Ser Gly Leu Gly Gly Ser Gly

1 5 10

<210> 131

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 131

Gly Leu Gly Ser Gly Gly Leu Gly Ser Gly

1 5 10

<210> 132

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 132

Gly Gly Leu Ser Gly Gly Gly Leu Ser Gly

1 5 10

<210> 133

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 133

Gly Gly Gly Leu Gly Gly Gly Gly Leu Gly

1 5 10

<210> 134

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 134

Gly Gly Gly Ser Leu Gly Gly Gly Ser Leu

1 5 10

<210> 135

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 135

Leu Gly Gly Ser Ser Leu Gly Gly Ser Ser

1 5 10

<210> 136

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 136

Gly Leu Gly Ser Ser Gly Leu Gly Ser Ser

1 5 10

<210> 137

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 137

Gly Gly Leu Ser Ser Gly Gly Leu Ser Ser

1 5 10

<210> 138

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(6)

<223> joint

<400> 138

Gly Ser Gly Gly Gly Ala

1 5

<210> 139

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(12)

<223> joint

<400> 139

Gly Ser Gly Gly Gly Ala Gly Ser Gly Gly Gly Ala

1 5 10

<210> 140

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(18)

<223> joint

<400> 140

Gly Ser Gly Gly Gly Ala Gly Ser Gly Gly Gly Ala Gly Ser Gly Gly

1 5 10 15

Gly Ala

<210> 141

<211> 24

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(24)

<223> joint

<400> 141

Gly Ser Gly Gly Gly Ala Gly Ser Gly Gly Gly Ala Gly Ser Gly Gly

1 5 10 15

Gly Ala Gly Ser Gly Gly Gly Ala

20

<210> 142

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 142

Gly Glu Asn Leu Tyr Phe Gln Ser Gly Gly

1 5 10

<210> 143

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 143

Ser Gly Gly Gly Ser Ser Gly Gly Gly Ser

1 5 10

<210> 144

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 144

Ser Ser Gly Gly Gly Ser Ser Gly Gly Gly

1 5 10

<210> 145

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 145

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

1 5 10

<210> 146

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 146

Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser

1 5 10

<210> 147

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> joint

<400> 147

Gly Gly Gly Ser Ser Gly Gly Gly Ser Gly

1 5 10

<210> 148

<211> 6

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(6)

<223> joint

<400> 148

Gly Gly Gly Ser Ser Ser

1 5

<210> 149

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(15)

<223> joint

<400> 149

Gly Gly Gly Ser Ser Gly Gly Gly Ser Ser Gly Gly Gly Ser Ser

1 5 10 15

<210> 150

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(8)

<223> joint

<400> 150

Gly Leu Gly Gly Leu Ala Ala Ala

1 5

<210> 151

<211> 16

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(16)

<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

<222> (1)..(13)

<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

<222> (1)..(5)

<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

<222> (1)..(36)

<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

<222> (1)..(36)

<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> 15

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(15)

<223> joint

<400> 156

Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

1 5 10 15

<210> 157

<211> 333

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<222> (1)..(333)

<223> IgM-derived hinge region

<400> 157

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> 158

<211> 180

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<222> (1)..(180)

<223> trimerization unit

<400> 158

gctgggcagg tgaggatctg ggccacatac cagaccatgc tggacaagat ccgggaggtg 60

ccggagggct ggctcatctt tgtggccgag agggaagagc tctatgtacg cgttagaaat 120

ggcttccgga aggtgctgct ggaggcccgg acagccctcc cgagaggcac gggcaatgag 180

<210> 159

<211> 27

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(27)

<223> trimerization unit

<400> 159

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> 160

<211> 117

<212> DNA

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> misc_feature

<222> (1)..(117)

<223> tetramerization Unit

<400> 160

aagcctctgg acggagagta tttcactctc cagatccggg gccccgaaag gttcgaaatg 60

ttccgggagc ttaacgaggc cttggagctg aaagacgcac aggccggaaa ggaaccg 117

<210> 161

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<400> 161

Gly Gly Ser Gly Gly

1 5

<210> 162

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> joint

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<400> 162

Gly Leu Gly Gly Leu

1 5

<210> 163

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(12)

<223> joint

<400> 163

Lys Pro Glu Pro Lys Pro Ala Pro Ala Pro Lys Pro

1 5 10

<210> 164

<211> 12

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(12)

<223> joint

<400> 164

Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala

1 5 10

<210> 165

<211> 7

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(7)

<223> joint

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<400> 165

Glu Ala Ala Ala Lys Gly Ser

1 5

<210> 166

<211> 15

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(15)

<223> joint

<400> 166

Pro Ser Arg Leu Glu Glu Glu Leu Arg Arg Arg Leu Thr Glu Pro

1 5 10 15

<210> 167

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(8)

<220>

<221> MISC_FEATURE

<222> (1)..(8)

<223> joint

<400> 167

Ser Ala Cys Tyr Cys Glu Leu Ser

1 5

<210> 168

<211> 8

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(8)

<223> joint

<400> 168

Ser Ile Ile Asn Phe Glu Lys Leu

1 5

<210> 169

<211> 13

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(13)

<223> joint

<400> 169

His Cys Leu Gly Lys Trp Leu Gly His Pro Asp Lys Phe

1 5 10

<210> 170

<211> 29

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(29)

<223> joint

<400> 170

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> 171

<211> 14

<212> PRT

<213> artificial sequence

<220>

<223> Artificial sequence

<220>

<221> MISC_FEATURE

<222> (1)..(14)

<223> joint

<400> 171

Asn Thr Trp Thr Thr Cys Gln Ser Ile Ala Phe Pro Ser Lys

1 5 10

Claims

1. A tolerance-inducing construct comprising:

a. a first targeting unit, a first junction region;

b. an antigenic unit;

c. a second land; and

d. a second targeting unit;

ii) a polypeptide encoded by the nucleotide sequence defined in i); or (b)

iii) A multimeric protein consisting of a plurality of polypeptides as defined in ii), for example a dimeric protein consisting of two polypeptides.

2. Tolerance-inducing construct according to claim 1, wherein the multimeric protein, e.g. dimeric protein, consists of a plurality of polypeptides, e.g. two polypeptides, linked to each other via their junction regions, preferably via their respective first junction regions and via their respective second junction regions.

3. The tolerance-inducible construct according to any one of the preceding claims, wherein the first and second junction regions comprise a flexible unit and a binding unit.

4. The tolerance-inducing construct according to any one of the preceding claims, wherein the first and/or second junction region comprises a binding unit, which is a non-covalent binding unit.

5. The tolerance-inducing construct according to any one of the preceding claims, wherein the non-covalent binding unit comprises or consists of: a trimerisation unit, such as the C-terminal domain of T4 fibritin, or e.g. a collagen-derived trimerisation unit, such as the human collagen-derived XVIII trimerisation domain or the human collagen XV trimerisation domain, or a tetramerisation unit, such as a domain derived from p 53.

6. The tolerance-inducing construct according to any one of the preceding claims, wherein the non-covalent binding unit comprises or consists of: dimerization units, for example dimerization units comprising a hinge region and an immunoglobulin domain, such as an immunoglobulin constant domain, or dimerization units comprising a dHLX protein.

7. The tolerance-inducing construct according to any one of the preceding claims, wherein the first and/or second junction region comprises a binding unit, which is a covalent binding unit.

8. The tolerance-inducing construct according to any one of the preceding claims, wherein the first and/or second junction region comprises or consists of a naturally occurring sequence.

9. The tolerance-inducing construct according to any one of claims 1-7, wherein the first and/or second junction region comprises or consists of an artificial sequence.

10. The tolerance-inducing construct according to any one of claims 7-9, wherein the first and second junction regions comprise a covalent binding unit comprising cysteine residues, such as at least 2 cysteine residues.

11. The tolerance-inducing construct of claim 10, wherein the covalent binding unit comprises a cysteine-rich sequence.

12. The tolerance-inducing construct according to any one of claims 10-11, wherein the covalent binding unit of the first junction region comprises a different number of cysteine residues than the covalent binding unit of the second junction region.

13. The tolerance-inducing construct according to any one of claims 10-12, wherein the position of the cysteine residue comprised in the covalent binding unit of the first junction region is different from the position of the cysteine residue comprised in the covalent binding unit of the second junction region, e.g. wherein the number of amino acid residues between the cysteine residues of the covalent binding unit of the first junction region is different from the number of amino acid residues between the cysteine residues of the covalent binding unit of the second junction region.

14. The tolerance-inducing construct according to any one of claims 10-13, wherein the number of cysteine residues is based on the length of the antigenic unit.

15. Tolerance-inducing construct according to any one of claims 7-14, wherein at least one of the covalent binding units is derived from an immunoglobulin, such as from the hinge region of an immunoglobulin, such as the middle hinge of exon h4 or IgGl of IgG 3.

16. The tolerance-inducing construct according to any one of claims 7-15, wherein at least one of the covalent binding units is an artificial sequence.

17. The tolerance-inducing construct according to any one of claims 1 to 4, wherein the first and second junction regions comprise non-covalent binding units.

18. The tolerance-inducing construct of claim 17, wherein at least one of the non-covalent binding units is a naturally occurring sequence.

19. The tolerance-inducible construct of claim 17, wherein at least one of the non-covalent binding units is an artificial sequence.

20. The tolerance-inducing construct according to any one of claims 17-19, wherein the non-covalent binding unit is or comprises an immunoglobulin or is derived from an immunoglobulin, such as a CH3 domain derived from IgG3 or IgG 1.

21. The tolerance-inducible construct according to any one of claims 17-19, wherein the non-covalent binding unit is or comprises a leucine zipper motif, a Jun/Fos-based leucine zipper, or is derived from a bZIP-like eukaryotic transcription factor, such as SEQ ID NO: 5.

22. The tolerance-inducing construct according to any one of claims 1-21, wherein the junction region comprises a binding unit comprising a covalent binding unit and a non-covalent binding unit.

23. The tolerance-inducing construct according to any one of claims 3 to 22, wherein at least one of the flexible units is a naturally occurring peptide sequence.

24. Tolerance-inducing construct according to any one of claims 3 to 23, wherein the flexible unit is derived from an immunoglobulin, such as from the hinge region of an immunoglobulin, such as exon h1 of IgG3 or the lower hinge of IgG 1.

25. The tolerance-inducing construct according to any one of claims 3 to 24, wherein at least one of the flexible units is an artificial sequence.

26. Tolerance-inducing construct according to any one of claims 3-22 and 25, wherein the flexible unit is a glycine-serine linker, such as GGGGSGGGGS (SEQ ID NO: 80).

27. The tolerance-inducing construct according to any one of claims 3 to 26, wherein the flexible unit is not a target of a protease.

28. The tolerance-inducing construct according to any one of claims 3 to 27, wherein the flexible unit consists of up to 20 amino acids, such as up to 15 amino acids, such as 12 amino acids or 10 amino acids.

29. The tolerance-inducing construct according to any one of claims 1 to 28, wherein the junction region is non-immunogenic.

30. The tolerance-inducible construct according to any one of the preceding claims, wherein at least one of the first targeting unit or the second targeting unit comprises a moiety that interacts with a surface molecule on an antigen presenting cell, preferably wherein both the first targeting unit and the second targeting unit comprise a moiety that interacts with a surface molecule on an antigen presenting cell.

31. The tolerance-inducing construct of claim 30, wherein the surface molecule is selected from the group consisting of tgfβ receptors (tgfβr1, tgfβr2 or tgfβr3), 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, CD83, SIGLEC, MGL, CD80, CD86, clec9A, clec12A, clec12B, DCIR2, langerin, MR, DC-Sign, treml4, dectin-1, PDL2, HVEM, aryl hydrocarbon receptors and vitamin D receptors.

32. The tolerance-inducing construct of claim 31, wherein the targeting unit comprises a moiety that is a natural ligand, an antibody or a portion thereof, such as an scFv, or a synthetic ligand.

33. The tolerance-inducing construct of claim 33, wherein the natural ligand is selected from the group consisting of tgfβ, IL-10, IL1RA, 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, CTLA-4, preferably extracellular domain of CTLA-4, PD-1, preferably extracellular domain of PD-1, and BTLA, preferably extracellular domain of BTLA.

34. The tolerance-inducible construct according to any one of the preceding claims, wherein the first targeting unit and the second targeting unit are identical.

35. The tolerance-inducible construct of any one of claims 1 to 33, wherein the first targeting unit and the second targeting unit are different.

36. The tolerance-inducing construct according to any one of claims 30 to 35, wherein the surface molecules are present on the same cell.

37. The tolerance-inducible construct of any one of claims 30 to 36, wherein binding of the first targeting unit or the second targeting unit results in internalization of the construct.

38. The tolerance-inducing construct according to any one of the preceding claims, wherein the first targeting unit comprises or consists of: IL-10 or TGF beta, preferably human IL-10 or TGF beta, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of human IL-10 or TGF beta.

39. The tolerance-inducing construct according to any one of the preceding claims, wherein the construct is the polynucleotide further comprising a nucleotide sequence encoding a signal peptide.

40. A multimeric protein, e.g. a dimeric protein, as defined in any one of the preceding claims, wherein said plurality of polypeptides, e.g. two polypeptides, are linked to each other via their respective first junction region and via their respective second junction region.

41. A method of preparing a pharmaceutical composition, the method comprising:

c) Providing a polynucleotide, polypeptide or multimeric protein, such as a dimeric protein, of any one of claims 1 to 40; and

d) The polynucleotide, polypeptide, or multimeric protein, such as a dimeric protein, is combined with a pharmaceutically acceptable carrier.

42. A pharmaceutical composition comprising a polynucleotide, polypeptide or multimeric protein, such as a dimeric protein, as defined in any one of claims 1 to 40, and a pharmaceutically acceptable carrier.

43. The pharmaceutical composition of claim 42 for use as a medicament.

44. The pharmaceutical composition of claim 42 for use in the prophylactic or therapeutic treatment of a condition involving an undesired immune response, such as autoimmune disease, allergic disease and graft rejection.

45. A vector comprising the polynucleotide of any one of claims 1-39.

46. A host cell comprising the vector of claim 45.

47. A method of producing a polypeptide or multimeric protein, such as a dimeric protein, the method comprising:

a) Transfecting a cell with a vector as defined in claim 45 or a polynucleotide as defined in any one of claims 1 to 39;

b) Culturing a cell such that the cell expresses a polypeptide encoded by the polynucleotide; and

c) Obtaining and purifying the multimeric protein, such as a dimeric protein, and/or the polypeptide expressed by the cell.

48. The method according to claim 47, wherein step c) comprises a step of purifying a fraction comprising said multimeric protein, such as a dimeric protein, wherein a plurality of polypeptides, e.g. two polypeptides, are linked to each other via their respective first junction region and via their respective second junction region.

49. A method for treating a condition involving an undesired immune response, for example in the prophylactic or therapeutic treatment of autoimmune diseases, allergic diseases and graft rejection, the method comprising administering to a subject in need thereof a polynucleotide, polypeptide or multimeric protein according to any one of claims 1 to 40, such as a dimeric protein, a vector according to claim 45 or a pharmaceutical composition according to any one of claims 42 to 44.

50. The method of claim 49, wherein the subject is a mammal, such as a human.