RU2807802C1 - Composition for culturing regulatory t-cells and its application - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method for culturing regulatory T cells is proposed, which includes culturing isolated regulatory T cells in a medium containing a fusion protein dimer. Said fusion protein dimer contains IL-2 protein or a variant thereof and CD80 protein or a fragment thereof.

EFFECT: invention increases the efficiency of proliferation and viability of the resulting Foxp3+ regulatory T cells.

9 cl, 41 dwg, 14 tbl, 2 ex

Description

TECHNICAL FIELD

The present invention relates to a composition for culturing T cells and a method for culturing regulatory T cells using it.

BACKGROUND ART

T cells play a central role in cell-mediated immunity. T cells are distinguished from other lymphocytes, including B cells, by receptors on the surface of T cells, such as T cell receptors (TCRs). In addition, T cells include different types of T cells, such as helper T cells (TH cells), cytotoxic T cells (TC cells or CTL), memory T cells (TM cells), including central T cells -memory cells (SCM cells) and effector memory T cells (TEM cells), natural killer T cells (NKT cells), gamma delta T cells (γδ T cells) and regulatory T cells (Treg -cells).

Among them, regulatory T cells (Treg) are T cells that act to suppress the immune response of other cells. For example, Treg cells act to suppress T cell-mediated immunity during an immune response and to suppress autoreactive T cells that have escaped negative selection in the thymus. Treg cells can mainly be classified into natural regulatory T cells (nTreg) and induced regulatory T cells (iTreg). Natural regulatory T cells, known as CD4+CD25+FoxP3+ regulatory T cells, develop in the thymus. Induced regulatory T cells share a number of characteristics with naturally occurring Treg cells, but the characteristic of conversion from CD4+CD25-FoxP3- T cells to CD4+CD25+FoxP3+ regulatory T cells is known as a representative difference.

There has been active research into the importance of regulatory T cells per se in relation to disease caused by disturbances in various autoimmune systems. Since the introduction and first proposal by Gershon of the idea of suppressor T cells (R K Gershon and K Kondo, immunology, 1970, 18: 723-37) in the early 1970s. Research has been conducted in many areas of immunology to elucidate the biological properties and functions of regulatory T cells. In particular, since Sakaguchi proposed in 1995 that CD25 may function as an important phenotypic marker for naturally occurring CD4+ regulatory T cells (S Sakaguchi et, al., J Immunol, 1995, 155: 1151-1164) , conducted studies focusing on the role and importance of regulatory T cells in inducing peripheral tolerance to cautoantigens.

It is known that regulatory T cells are capable of secreting IL-10 (interleukin-10), TGF-β (transforming growth factor-beta), IL-35 or the like, known as immunosuppressive cytokines (Nishikawa et al., Int. J Cancer 2010, 127: 759-767). A study was conducted that confirms that regulatory T cells that secrete immunosuppressive cytokines can secrete IL-10 or the like to induce antigen-specific T cells that cause autoimmune disease in immunotolerant antigen-presenting cells, thereby inducing immunological tolerance.

The use of regulatory cells per se for the treatment of autoimmune disease is widely known, but specific methods that can amplify natural killer cells for their effective use are still lacking.

Detailed Description of the Invention

Technical problem

Accordingly, the inventors of the present invention investigated a method for effectively culturing regulatory T cells and, as a result, found that a novel fusion protein dimer containing IL-2 protein and CD80 protein in one molecule can effectively proliferate regulatory T cells, and carried out the present invention.

Solution

To achieve the above object, according to a typical embodiment, the composition or medium for culturing regulatory T cells contains, as an active ingredient, a dimer fusion protein comprising an IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof.

In another exemplary embodiment, a method is provided for culturing regulatory T cells using a fusion protein dimer comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof.

According to yet another typical embodiment, the pharmaceutical composition contains, as an active ingredient, regulatory T cells cultured in a medium containing a fusion protein dimer comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof.

Invention effect

The culture composition of the present invention can not only effectively proliferate T cells, but in particular effectively proliferate regulatory T cells. In particular, it was confirmed that the viability of regulatory T cells increased significantly compared to the traditionally used culture method with IL-2. It was also confirmed that the expression level of Foxp3+ increased in the obtained regulatory T cells. Therefore, this proliferation method can be used in the field of cell therapy using regulatory T cells.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Exemplary embodiments can be understood in greater detail from the following description, taken in conjunction with the accompanying drawings, in which:

FIG. 1A is a schematic diagram of a fusion protein dimer used in the present invention;

In FIG. 1B shows an SDS-PAGE gel image confirming the resulting fusion protein dimer (GI-101);

In FIG. 1C shows the dimer content of the fusion protein (GI-101) according to absorbance;

In FIG. 1D shows gel filtration analysis (SEC) of the resulting fusion protein dimer (GI-101);

In FIG. 2A shows an SDS-PAGE gel image confirming the resulting hCD80-Fc fusion protein dimer;

In FIG. 2B shows gel filtration analysis (SEC) of the resulting hCD80-Fc fusion protein dimer;

In FIG. 3A shows an SDS-PAGE gel image confirming the resulting Fc-IL2v2 fusion protein dimer;

In FIG. 3B shows gel filtration analysis (SEC) of the resulting Fc-IL2v2 fusion protein dimer;

In FIG. 3C shows an SDS-PAGE gel image confirming the resulting Fc-IL2wt fusion protein dimer;

In FIG. 3D shows gel filtration analysis (SEC) of the resulting Fc-IL2wt fusion protein dimer;

In FIG. 4A shows an SDS-PAGE gel image confirming the resulting hCD80-Fc-IL2wt fusion protein dimer;

In FIG. 4B shows gel filtration analysis (SEC) of the resulting hCD80-Fc-IL2wt fusion protein dimer;

In FIG. 5 is a schematic diagram of a method for culturing Treg cells using a fusion protein dimer;

In FIG. 6 shows the number of regulatory T cells cultured in a composition containing RPMI1640 medium;

In FIG. 7 shows the viability of regulatory T cells cultured in a composition containing RPMI1640 medium;

In FIG. 8 shows the number of regulatory T cells cultured in a composition containing TexMACS medium;

In FIG. 9 shows the viability of regulatory T cells cultured in a composition containing TexMACS medium;

In FIG. 10 shows the IL-10 secreting ability of regulatory T cells cultured using a composition containing RPMI1640 medium;

In FIG. 11 shows the IL-10 secreting ability of regulatory T cells cultured using a composition containing TexMACS medium;

In FIG. 12A and 12B show the number of proliferated regulatory T cells when cultured in a composition containing RPMI1640 medium in an optimized method;

In FIG. 13A and 13B show the viability of regulatory T cells when cultured in a composition containing RPMH640 medium in an optimized method;

In FIG. 14A and 14B show the number of regulatory T cells when cultured in a composition containing TexMACS medium in an optimized method;

In FIG. 15A and 15B show the viability of regulatory T cells when cultured in a composition containing TexMACS medium in an optimized method;

In FIG. 16A-16C show the results of FACS (fluorescent cell sorting) analysis of the properties of cells cultured in a composition containing RPMI1640 medium in an optimized method;

In FIG. 17A-17C show the number of regulatory T cells expressing Foxp3 when cultured in a composition containing RPMH640 medium in an optimized method;

In FIG. 18A-18C show the results of FACS analysis of the properties of cells cultured in a composition containing TexMACS medium in an optimized method;

In FIG. 19A-19C show the number of regulatory T cells expressing Foxp3 when cultured in a composition containing TexMACS medium in an optimized method;

In FIG. 20 shows the IL-10 secretion capacity of regulatory T cells when cultured in a composition containing RPMH640 medium in an optimized method; And

In FIG. 21 shows the ability of regulatory T cells to secrete IL-10 when cultured in a composition containing TexMACS medium.

Best Mode for Carrying Out the Invention

Composition and medium for proliferation of regulatory T cells

According to one aspect of the present invention, there is provided a composition for the proliferation of regulatory T cells, containing, as an active ingredient, a dimer of a fusion protein comprising an IL-2 protein or a variant thereof and a CD80 protein or a fragment thereof. In addition, a culture medium for regulatory T cells is provided. cells containing a fusion protein dimer as an active ingredient.

The regulatory T cell proliferation medium may be a medium in which a fusion protein dimer comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof is added to a T cell culture medium. In this case, the T cell culture medium may contain any component selected from the group consisting of amino acids, sugars, inorganic salts and vitamins. Preferably, the T cell culture medium may contain any amino acid, any sugar, any inorganic salt and any vitamin. In addition, the medium may additionally contain fetal bovine serum (FBS), hydroxyethylpiperazine ethanesulfonic acid (HEPES), proteins, carbohydrates, mercaptoethanol and growth factors. Also, the culture medium of regulatory T cells may additionally contain retinoic acid. In one embodiment, the regulatory T cell proliferation medium may contain the basic components described in the following Table 3 or Table 4.

The term “cell culture medium” as used herein means a medium used for culturing cells, particularly regulatory T cells, and more specifically means a medium for culturing CD4+CD25+CD127- cells. It contains components required by cells for cell growth and survival in vitro, or contains components that assist cell growth and survival. In particular, these components may be vitamins, essential or non-essential amino acids and microelements. The medium may be a medium used for culturing cells, preferably eukaryotic cells, most preferably regulatory T cells, and much more preferably CD4+CD25+CD127- T cells or CD4+CD25+Foxp3+ T cells.

The cell culture medium of the present invention contains any amino acid component, any vitamin component, any inorganic salt component, any other component and purified water, where:

a) the amino acid component is at least one amino acid selected from the group consisting of glycine, L-alanine, L-valine, L-leucine, L-isoleucine, L-threonine, L-serine, L-cysteine, L- methionine, L-aspartic acid, L-glutamic acid, L-glutamine, L-lysine, L-arginine, L-histidine, L-phenylalanine, L-tyrosine, L-tryptophan, L-proline, β-alanine, γ- aminobutyric acid, ornithine, citrulline, homoserine, triiodothyronine, thyroxine and dioxyphenylalanine or a combination thereof, and preferably at least one amino acid selected from the group consisting of glycine, L-alanine, L-arginine, L-cysteine, L-glutamine, L-histidine, L-lysine, L-methionine, L-proline, L-serine, L-threonine and L-valine or combinations thereof;

b) the vitamin component is at least one vitamin selected from the group consisting of biotin, calcium D-pantothenate, folic acid, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, choline chloride, i-inositol and ascorbic acid or a combination thereof, and preferably at least one vitamin selected from the group consisting of i-inositol, thiamine hydrochloride, niacinamide and pyridoxine hydrochloride or a combination thereof;

c) the inorganic salt component is at least one inorganic salt selected from the group consisting of calcium chloride (CaCl ₂ ) (anhydrous), copper sulfate pentahydrate (CuSO ₄ -5H ₂ O), iron (II) sulfate heptahydrate (FeSO ₄ -7H ₂ O), magnesium chloride (anhydrous), magnesium sulfate (MgSO ₄ ) (anhydrous), potassium chloride (KCl), sodium chloride (NaCl), disodium hydrogen phosphate (Na ₂ HPO ₄ ), sodium dihydrogen phosphate monohydrate ( NaH ₂ PO ₄ -H ₂ O), zinc sulfate heptahydrate (ZnSO ₄ -7H ₂ O), iron (III) nitrate nonahydrate (Fe(NO ₃ ) ₃ ⋅9H ₂ O) and sodium bicarbonate (NaHCO ₃ ) or a combination thereof and preferably at least one inorganic salt selected from the group consisting of sodium chloride (NaCl), sodium hydrogen carbonate (NaHCO ₃ ), potassium chloride (KCl), calcium chloride (CaCl ₂ ) (anhydrous) and sodium dihydrogen phosphate monohydrate (NaH ₂ PO ₄ -H ₂ O) or combinations thereof;

d) the other component is at least one other component selected from the group consisting of D-glucose (dextrose), sodium pyruvate, Na hypoxanthine, thymidine, linoleic acid, lipoic acid, adenosine, cytidine, guanosine, uridine, 2' α-deoxyadenosine, 2'-deoxycytidine HCl and 2'-deoxyguanosine or combinations thereof, and it may preferably be sodium pyruvate; And

e) Purified water is used to dissolve amino acids, vitamins, inorganic salts and other components, and can be obtained through one or more than one distillation process, or purified through a filter.

In addition, the cell culture medium of the present invention may further contain growth factors or cytokines. This growth factor may be IGF (insulin-like growth factor), bFGF (basic fibroblast growth factor), TGF (transforming growth factor), HGF (hepatocyte growth factor), EGF (epidermal growth factor), VEGF (vascular endothelial growth factor), PDGF (platelet-derived growth factor) or the like, one or at least two of them, but not limited to them. The cytokine may be IL-1 (interleukin-1), IL-4, IL-6, IFN-γ (interferon-gamma), IL-10, IL-17 or the like, one or at least two of them, but not limited to them.

The term "T cell" as used herein refers to one of the lymphocytes responsible for antigen-specific adaptive immunity. T cells are classified into naïve T cells, which have not yet encountered antigen, mature T cells, and memory T cells, which have encountered antigen. In this case, mature effector T cells contain helper T cells, cytotoxic T cells and natural killer T cells.

The term “helper T cell or Th cell” as used herein refers to a cell that stimulates humoral immunity by regulating the differentiation and activation of other leukocytes. It is also called a CD4+ T cell because it has the CD4 protein on the cell surface. Helper T cells can be further classified into Th1, Th2, Th17, and Tregs according to their detailed functions. Th1 cells secrete interferon-gamma (IFN-γ) and tumor necrosis factor beta (TNF-β), thereby inducing the fusion of endosomes and lysosomes to form endolysosomes within macrophages. Meanwhile, Th2 cells secrete several types of interleukin (IL), allowing B cells to differentiate into plasma cells. Th17 cells secrete interleukin-17 (IL-17) to recruit neutrophils.

The term “regulatory T cells (Treg)” as used herein includes natural regulatory T cells (nTreg) or induced regulatory T cells (iTreg). Regulatory T cells as used herein include CD4+CD25+ T cells, CD4+CD25+CD127 low T cells, or CD4+CD25+Foxp3+ T cells. Regulatory T cells maintain immune homeostasis and block autoimmune responses and the like by inhibiting the immune response.

The term "cytotoxic T cell" as used herein refers to a cell that kills virus-infected cells or tumor cells or the like by secreting cytotoxic substances such as granzyme or perforin. It is also called a CD8 T cell because it has the CD8 protein on the surface of the cell. Unlike helper T cells, it eliminates the virus and cancer cells by mediating cellular immunity.

The term "natural killer T cell" as used herein refers to one of the effector T cells, which is distributed in a small proportion compared to helper T cells and cytotoxic T cells. Natural killer T cells have the same T cell receptors (TCRs) on the cell surface as T cells, but also have natural killer cell-specific molecules such as NK1.1. Natural killer T cells secrete interferon gamma, interleukin-4, or the like to regulate the immune response.

The term "memory T cell" as used herein refers to a T cell that has the potential ability to function as an effector T cell because it has recognized an antigen and has survived long-term differentiation processes and selection, and later, upon re-invasion of the antigen, is quickly activated. Naïve T cells differentiate into activated cells through antigen recognition, or effector T cells differentiate into long-lived memory T cells through the influence of interleukin-7 and interleukin-15.

In this case, a fusion protein dimer comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof may be contained in the culture medium in an amount of from 1 nM to 2000 nM. Moreover, the dimer may be present in an amount of 1 nM to 1000 nM or 1 nM to 500 nM. In addition, the dimer may be present in an amount of 2 nM to 300 nM, 5 nM to 100 nM, 10 nM to 80 nM, 20 nM to 70 nM, or 40 nM to 50 nM. In particular, the dimer fusion protein may be present in the medium in an amount of 1 nM, 3.2 nM, 10 nM or 50 nM.

A fusion protein dimer comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof

The term "IL-2" or "interleukin-2" as used herein, unless otherwise stated, refers to any wild-type IL-2 derived from any vertebrate source, including mammals, such as primates (such as humans) and rodents (such as mice and rats). IL-2 can be derived from animal cells and also includes IL-2 derived from recombinant cells capable of producing IL-2. In addition, IL-2 may be wild-type IL-2 or a variant thereof.

In the present specification, IL-2 or a variant thereof may be collectively expressed by the term "IL-2 protein" or "IL-2 polypeptide". IL-2, IL-2 protein, IL-2 polypeptide, and IL-2 variant bind specifically to, for example, the IL-2 receptor. This specific binding can be identified by methods known to those skilled in the art.

One embodiment of IL-2 may have the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36. Here, IL-2 may also be in a mature form. In particular, mature IL-2 may not contain a signal sequence and may have the amino acid sequence of SEQ ID NO: 10. Here, IL-2 may be used according to a configuration comprising a wild-type IL-2 fragment in which a portion of the N-terminus or C-terminus is truncated. end of wild-type IL-2.

In addition, a given IL-2 fragment may be in a form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 19, 20, 21, 22, 23, 24, or 25 contiguous amino acids are truncated from the N-terminus of the protein having the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36. In addition, the IL-2 fragment may be in the form in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 contiguous amino acids are truncated from the C-terminus of the protein having the amino acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36.

The term “IL-2 variant” as used herein refers to a form in which a portion of the amino acids in full-length IL-2 or an IL-2 fragment as described above is replaced. That is, the IL-2 variant may have a different amino acid sequence from wild-type IL-2 or a fragment thereof. However, the IL-2 variant may have equivalent or similar activity to wild-type IL-2. As used herein, “IL-2 activity” may, for example, refer to specific binding to an IL-2 receptor, which specific binding may be measured by methods known to those skilled in the art.

In particular, a variant of IL-2 can be obtained by replacing a portion of the amino acids in wild-type IL-2. One embodiment of the IL-2 amino acid substitution variant can be generated by substituting at least one of the 38th, 42nd, 45th, 61st and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10.

In particular, the IL-2 variant can be obtained by replacing at least one of the 38th, 42nd, 45th, 61st or 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 with another amino acid. In addition, when IL-2 is in a form in which part of the N-terminus of the amino acid sequence SEQ ID NO: 35 is truncated, an amino acid at a position complementary to the position in the amino acid sequence SEQ ID NO: 10 can be replaced by another amino acid. For example, when IL-2 has the amino acid sequence of SEQ ID NO: 35, an IL-2 variant thereof may be produced by replacing at least one of the 58th, 62nd, 65th, 81st, or 92nd amino acids in amino acid sequence SEQ ID NO: 35 with another amino acid. These amino acid residues correspond to amino acid residues 38, 42, 45, 61 and 72 in the amino acid sequence SEQ ID NO: 10, respectively. In one embodiment, one, two, three, four, five, six, seven, eight, nine or ten amino acids may be substituted, provided that the IL-2 variant retains IL-2 activity. According to another embodiment, from one to five amino acids can be replaced.

In one embodiment, the IL-2 variant may be in a form in which two amino acids are replaced. Specifically, the IL-2 variant may be produced by substituting the 38th and 42nd amino acids in the amino acid sequence of SEQ ID NO: 10. Additionally, in one embodiment, the IL-2 variant may be prepared by substituting the 38th and 45th amino acids in the amino acid sequence of SEQ ID NO: 10. Additionally, in one embodiment, an IL-2 variant may be produced by substituting the 38th and 61st amino acids in the amino acid sequence of SEQ ID NO: 10. Additionally, in one embodiment, an IL-2 variant may be prepared by substituting the 38th and 72nd amino acids in the amino acid sequence SEQ ID NO: 10. Additionally, in one embodiment, the IL-2 variant may be prepared by substituting the 42nd and 45th amino acids in the amino acid sequence SEQ ID NO: 10. Additionally, in one embodiment, an IL-2 variant may be produced by substituting the 42nd and 61st amino acids in the amino acid sequence of SEQ ID NO: 10. Additionally, in one embodiment, an IL-2 variant may be prepared by substituting the 42nd and the 72nd amino acid in the amino acid sequence of SEQ ID NO: 10. Additionally, in one embodiment, an IL-2 variant may be generated by replacing the 45th and 61st amino acids in the amino acid sequence of SEQ ID NO: 10. Additionally, in one embodiment, In one embodiment, the IL-2 variant can be made by replacing the 45th and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10. Additionally, in one embodiment, the IL-2 variant can be made by replacing the 61st and 72nd amino acids in the amino acid sequence sequence SEQ ID NO: 10.

In addition, the IL-2 variant may be in a form in which three amino acids are changed. Specifically, the IL-2 variant may be produced by substituting amino acids 38, 42, and 45 in the amino acid sequence of SEQ ID NO: 10. Additionally, in one embodiment, the IL-2 variant may be prepared by substituting the 38, The 42nd and 61st amino acids in the amino acid sequence SEQ ID NO: 10. Additionally, in one embodiment, an IL-2 variant can be prepared by replacing the 38th, 42nd and 72nd amino acids in the amino acid sequence SEQ ID NO: 10. Additionally, in one embodiment, an IL-2 variant may be produced by substituting amino acids 38, 45, and 61 in the amino acid sequence of SEQ ID NO: 10. Additionally, in one embodiment, an IL-2 variant may be produced by substituting the 38th, 45th and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10. Additionally, in one embodiment, an IL-2 variant may be prepared by substituting the 38th, 61st and 72nd amino acids in the amino acid sequence sequence of SEQ ID NO: 10. Additionally, in one embodiment, an IL-2 variant may be generated by substituting the 42nd, 45th, and 61st amino acids in the amino acid sequence of SEQ ID NO: 10. Additionally, in one embodiment, an IL-2 variant -2 can be made by substituting amino acids 42, 45, and 72 in the amino acid sequence SEQ ID NO: 10. Additionally, in one embodiment, an IL-2 variant can be made by substituting amino acids 45, 61, and 72 th amino acid in the amino acid sequence SEQ ID NO: 10.

In addition, the IL-2 variant may be in a form in which four amino acids are changed. Specifically, the IL-2 variant may be produced by substituting amino acids 38, 42, 45 and 61 in the amino acid sequence of SEQ ID NO: 10. Additionally, in one embodiment, the IL-2 variant may be prepared by substituting The 38th, 42nd, 45th and 72nd amino acids in the amino acid sequence SEQ ID NO: 10. Additionally, in one embodiment, an IL-2 variant can be generated by replacing the 38th, 45th, 61st and the 72nd amino acid in the amino acid sequence SEQ ID NO: 10. Additionally, in one embodiment, an IL-2 variant can be generated by replacing the 38th, 42nd, 61st and 72nd amino acids in the amino acid sequence SEQ ID NO: : 10. Additionally, in one embodiment, an IL-2 variant can be generated by replacing the 42nd, 45th, 61st and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10.

In addition, the IL-2 variant may be in a form in which five amino acids are changed. In particular, an IL-2 variant may be prepared by replacing each of the 38th, 42nd, 45th, 61st and 72nd amino acids in the amino acid sequence of SEQ ID NO: 10 with a different amino acid.

Here, the “other amino acid” introduced by the substitution may be any amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline , serine, threonine, tryptophan, tyrosine and valine. However, regarding the amino acid substitution for the IL-2 variant in the amino acid sequence SEQ ID NO: 10, the 38th amino acid cannot be replaced with arginine, the 42nd amino acid cannot be replaced with phenylalanine, the 45th amino acid cannot be replaced with tyrosine, 61- The 1st amino acid cannot be replaced by glutamic acid, and the 72nd amino acid cannot be replaced by leucine.

Regarding the amino acid substitution for the IL-2 variant in the amino acid sequence of SEQ ID NO: 10, the 38th amino acid, arginine, may be replaced with an amino acid other than arginine. Preferably, with respect to the amino acid substitution for the IL-2 variant in the amino acid sequence of SEQ ID NO: 10, the 38th amino acid, arginine, may be replaced with alanine (R38A).

Regarding the amino acid substitution for the IL-2 variant in the amino acid sequence of SEQ ID NO: 10, the 42nd amino acid phenylalanine may be replaced with an amino acid other than phenylalanine. Preferably, with respect to the amino acid substitution for the IL-2 variant in the amino acid sequence of SEQ ID NO: 10, the 42nd amino acid, phenylalanine, may be replaced with alanine (F42A).

Regarding the amino acid substitution for the IL-2 variant in the amino acid sequence of SEQ ID NO: 10, the 45th amino acid tyrosine may be replaced with an amino acid other than tyrosine. Preferably, with respect to the amino acid substitution for the IL-2 variant in the amino acid sequence of SEQ ID NO: 10, the 45th amino acid, tyrosine, may be replaced with alanine (Y45A).

Regarding the amino acid substitution for the IL-2 variant in the amino acid sequence SEQ ID NO: 10, the 61st amino acid, glutamic acid, may be replaced with an amino acid other than glutamic acid. Preferably, with respect to the amino acid substitution for the IL-2 variant in the amino acid sequence of SEQ ID NO: 10, the 61st amino acid - glutamic acid - can be replaced with arginine (E61R).

Regarding the amino acid substitution for the IL-2 variant in the amino acid sequence of SEQ ID NO: 10, the 72nd amino acid leucine may be replaced with an amino acid other than leucine. Preferably, with respect to the amino acid substitution for the IL-2 variant in the amino acid sequence of SEQ ID NO: 10, the 72nd amino acid leucine may be replaced with glycine (L72G).

In particular, the IL-2 variant can be obtained by at least one substitution selected from the group consisting of R38A, F42A, Y45A, E61R and L72G in the amino acid sequence of SEQ ID NO: 10.

In particular, the IL-2 variant can be obtained by amino acid substitutions at two, three, four or five positions among the positions selected from the group consisting of R38A, F42A, Y45A, E61R and L72G.

In addition, the IL-2 variant may be in a form in which two amino acids are replaced. In particular, the IL-2 variant can be obtained by substitutions R38A and F42A. Additionally, in one embodiment, the IL-2 variant can be generated by R38A and Y45A substitutions. Additionally, in one embodiment, the IL-2 variant can be generated by substitutions R38A and E61R. Additionally, in one embodiment, the IL-2 variant can be generated by substitutions R38A and L72G. Additionally, in one embodiment, the IL-2 variant can be generated by substitutions F42A and Y45A. Additionally, in one embodiment, the IL-2 variant can be generated by substitutions F42A and E61R. Additionally, in one embodiment, the IL-2 variant can be generated by substitutions F42A and L72G. Additionally, in one embodiment, the IL-2 variant can be generated by E61R and L72G substitutions.

In addition, the IL-2 variant may be in a form in which three amino acids are changed. In particular, the IL-2 variant can be obtained by substitutions R38A, F42A and Y45A. Additionally, in one embodiment, the IL-2 variant can be generated by substitutions R38A, F42A and E61R. Additionally, in one embodiment, the IL-2 variant can be generated by substitutions R38A, F42A and L72G. Additionally, in one embodiment, the IL-2 variant can be made with R38A, Y45A, and E61R substitutions. Additionally, in one embodiment, the IL-2 variant can be made with R38A, Y45A, and E61R substitutions. Additionally, in one embodiment, the IL-2 variant can be made with R38A, Y45A, and L72G substitutions. Additionally, in one embodiment, the IL-2 variant can be generated by substitutions F42A, Y45A and E61R. Additionally, in one embodiment, the IL-2 variant can be generated by substitutions F42A, Y45A and L72G. Additionally, in one embodiment, the IL-2 variant can be generated by substitutions F42A, E61R and L72G. Additionally, in one embodiment, the IL-2 variant can be made with Y45A, E61R, and L72G substitutions.

In addition, the IL-2 variant may be in a form in which four amino acids are changed. In particular, the IL-2 variant can be obtained by substitutions R38A, F42A, Y45A and E61R. Additionally, in one embodiment, the IL-2 variant can be produced by substitutions R38A, F42A, Y45A and L72G. Additionally, in one embodiment, the IL-2 variant can be made with R38A, F42A, E61R, and L72G substitutions. Additionally, in one embodiment, the IL-2 variant can be made with R38A, Y45A, E61R, and L72G substitutions. Additionally, in one embodiment, the IL-2 variant can be generated by substitutions F42A, Y45A, E61R, and L72G.

In addition, the IL-2 variant can be obtained by substitutions R38A, F42A, Y45A, E61R and L72G.

Preferably, one embodiment of the IL-2 variant may contain a substitution that is any substitution selected from the following substitution combinations (a)-(d) in the amino acid sequence of SEQ ID NO: 10:

(a) R38A/F42A

(b) R38A/F42A/Y45A

(c) R38A/F42A/E61R

(d) R38A/F42A/L72G

Here, when IL-2 has an amino acid substitution of SEQ ID NO: 35, the amino acid substitution may be present at a position complementary to the position in the amino acid sequence of SEQ ID NO: 10. Moreover, even when IL-2 is a fragment of the amino acid sequence of SEQ ID NO: 10 : 35, the amino acid substitution may be present at a position complementary to the position in the amino acid sequence SEQ ID NO: 10.

In particular, the IL-2 variant may have the amino acid sequence of SEQ ID NO: 6, 22, 23 or 24.

In addition, the IL-2 variant may have low toxicity in vivo. Here, the low toxicity in vivo may represent a side effect caused by the binding of IL-2 to the alpha chain of the IL-2 receptor (IL-2Rα). Various variants of IL-2 have been developed to reduce the intensity of the side effect caused by the binding of IL-2 to IL-2Rα, and such variants of IL-2 may be those disclosed in US Patent No. 5,229,109 and Korean Patent No. 1,667,096. In particular, the IL-2 variants described herein have low binding affinity for the IL-2 receptor alpha chain (IL-2Rα) and thus have lower in vivo toxicity than wild-type IL-2.

The term "CD80" as used herein, also referred to as "B7-1", refers to a membrane protein present in dendritic cells, activated B cells and monocytes. CD80 provides costimulatory signals important for T cell activation and survival. CD80 is known as a ligand for two different proteins, CD28 and CTLA-4, present on the surface of T cells. CD80 consists of 288 amino acids and may specifically have the amino acid sequence SEQ ID NO: 11. Moreover, the term “CD80 protein” as used herein refers to full-length CD80 or a fragment of CD80.

The term “CD80 fragment” as used herein refers to a truncated form of CD80. In addition, the CD80 fragment may be an extracellular domain of CD80. One embodiment of the CD80 fragment can be obtained by eliminating the 1st to 34th amino acids from the N-terminus, which constitute the CD80 signal sequence. In particular, one embodiment of the CD80 fragment may be a protein consisting of amino acids 35 to 288 in SEQ ID NO: 11. Additionally, one embodiment of the CD80 fragment may be a protein consisting of amino acids 35 to 242 amino acids in SEQ ID NO: 11. Additionally, one embodiment of the CD80 fragment may be a protein consisting of amino acids 35 to 232 of SEQ ID NO: 11. Additionally, one embodiment of the CD80 fragment may be a protein consisting of of amino acids 35 to 139 of SEQ ID NO: 11. Additionally, one embodiment of the CD80 fragment may be a protein consisting of amino acids 142 to 242 of SEQ ID NO: 11. In one embodiment, the CD80 fragment may have the amino acid sequence SEQ ID NO: 2.

In addition, the IL-2 protein and the CD80 protein can be attached to each other through a linker or carrier. In particular, IL-2 or a variant thereof and CD80 (B7-1) or a fragment thereof can be attached to each other via a linker or carrier. In the present description, the terms “linker” and “carrier” may be used interchangeably.

A linker connects two proteins. One embodiment of the linker may contain from 1 to 50 amino acids, albumin or a fragment thereof, an immunoglobulin Fc domain, or the like. Here, an immunoglobulin Fc domain refers to a protein that contains the heavy chain constant region 2 (CH2) and the heavy chain constant region 3 (CH3) of an immunoglobulin, and does not contain the heavy and light chain variable regions, and the light chain constant region 1 (CL1) of an immunoglobulin. The immunoglobulin may be IgG, IgA, IgE, IgD or IgM and preferably may be IgG4. Here, the wild-type immunoglobulin G4 Fc domain may have the amino acid sequence of SEQ ID NO: 4.

In addition, the Fc domain of an immunoglobulin may be a variant Fc domain as well as a wild-type Fc domain. In addition, the term “Fc domain variant” as used herein can refer to a form that differs from the wild-type Fc domain in terms of glycosylation pattern, is highly glycosylated compared to the wild-type Fc domain, or has low glycosylation compared to the wild type Fc domain, or is a deglycosylated form. In addition, a non-glycosylated Fc domain is included herein. The Fc domain or variant thereof can be adapted to have an adjusted number of sialic acids, fucosylations or glycosylations through cultural conditions or genetic manipulation of the host.

In addition, the glycosylation of the Fc domain of an immunoglobulin can be modified by conventional methods such as chemical methods, enzymatic methods and genetic engineering methods using microorganisms. In addition, the Fc domain variant may be in a mixed form of the corresponding Fc regions of immunoglobulins - IgG, IgA, IgE, IgD and IgM. In addition, the Fc domain variant may be in a form in which some amino acids of the Fc domain are replaced with other amino acids. One embodiment of the Fc domain variant may have the amino acid sequence of SEQ ID NO: 12.

The fusion protein may have a structure in which, using the Fc domain as a linker (or carrier), the CD80 protein and the IL-2 protein, or the IL-2 protein and the CD80 protein, are linked to the N-terminus and C-terminus of the linker or carrier, respectively (Fig. .1A). Linkage between the N-terminus or C-terminus of the Fc domain and CD-80 or IL-2 can possibly be achieved through a linker peptide.

In particular, the fusion protein may consist of the following structural formula (I) or (II):

Here in structural formulas (I) and (II)

N' represents the N-terminus of the fusion protein,

C represents the C-terminus of the fusion protein,

X represents CD80 protein,

Y is IL-2 protein,

linkers (1) and (2) are peptide linkers and

n and m are each independently equal to 0 or 1.

Preferably, the fusion protein may consist of structural formula (I). The IL-2 protein is as described above. In addition, the CD80 protein is as described above. In one embodiment, the IL-2 protein may be a variant of IL-2 with one to five amino acid substitutions compared to wild-type IL-2. The CD80 protein may be a fragment obtained by truncation of up to about 34 contiguous amino acid residues from the N-terminus or C-terminus of wild-type CD80. Alternatively, the CD protein may be an extracellular immunoglobulin-like domain having binding activity to the T cell surface receptors CTLA-4 and CD28.

Specifically, the fusion protein may have an amino acid sequence of SEQ ID NO: 9, 26, 28, or 30. In another embodiment, the fusion protein comprises a polypeptide having a sequence identity of 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with amino acid sequence SEQ ID NO: 9, 26, 28 or 30. Here, identity is , for example, percent homology and can be determined by a homology comparison program such as the National Center for Biotechnology Information (NCBI) BlastN program.

The peptide linker (1) may be contained between the CD80 protein and the Fc domain. The peptide linker (1) may consist of 5-80 contiguous amino acids, 20-60 contiguous amino acids, 25-50 contiguous amino acids, or 30-40 contiguous amino acids. In one embodiment, the peptide linker (1) may consist of 30 amino acids. In addition, the peptide linker (1) may contain at least one cysteine. In particular, the peptide linker (1) may contain one, two or three cysteines. In addition, the peptide linker (1) may be derived from an immunoglobulin hinge. In one embodiment, the peptide linker (1) may be a peptide linker consisting of the amino acid sequence SEQ ID NO: 3.

The peptide linker (2) may consist of 1-50 contiguous amino acids, 3-30 contiguous amino acids, or 5-15 contiguous amino acids. In one embodiment, the peptide linker (2) may be (G4S) _n (where n is an integer from 1 to 10). Here in (G4S), _n n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, peptide linker (2) may be a peptide linker consisting of the amino acid sequence SEQ ID NO: 5.

In another aspect of the present invention, a dimer is provided by linking two fusion proteins, each of which contains an IL-2 protein and a CD80 protein. The fusion protein comprising IL-2 or a variant thereof and CD80 or a fragment thereof is as described above.

Here, binding between the fusion proteins constituting a given dimer may be achieved through, but not limited to, a disulfide bond formed by cysteines present in the linker. The fusion proteins making up the dimer may be the same or different fusion proteins. Preferably, the dimer may be a homodimer. One embodiment of the fusion protein constituting the dimer may be a protein having the amino acid sequence of SEQ ID NO: 9.

Method for culturing regulatory T cells

According to another aspect of the present invention, there is provided a method for culturing regulatory T cells, comprising culturing CD4+CD25+CD127- T cells in a medium containing a fusion protein dimer comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof.

Currently, CD4+CD25+CD127- T cells can be obtained from blood cells. Currently, CD4+CD25+CD127- T cells can be isolated from peripheral blood mononuclear cells (PBMCs). Alternatively, CD4+CD25+CD127- T cells can be obtained by specifically proliferating CD4+CD25+CD127- T cells from blood cells. Alternatively, CD4+CD25+CD127- T cells can be derived from CD4+ cells after removal of PBMCs from CD4- T cells. Additionally, CD4+ T cells can be isolated using anti-CD4 antibodies, and in one embodiment they have been isolated using beads to which anti-CD4 antibodies bind. Additionally, CD25+ T cells can be isolated using anti-CD25+ antibodies. In particular, regulatory T cells can be isolated by isolating CD4+, CD25+ and CD127- T cells.

The medium may be a traditionally used medium. Preferably, it may be a medium optimized for CD4+CD25+CD127- T cells. In a specific embodiment, it may be a medium in which FBS, HEPES, L-glutamine and 2-mercaptoethanol are added to RPMI1640 medium or TexMACS medium, as disclosed in Tables 3 and 4. In addition, this medium may further contain retinoic acid. In addition, this medium may additionally contain penicillin and/or streptomycin.

In this case, CD4+ regulatory T cells can be cultured in the medium for 1-30 days or 2-20 days. In addition, they can be cultivated for 3-10 days or 4-6 days.

Meanwhile, CD4+CD25+CD127- T cells can be obtained through a CD4+ T cell culture step or a CD25+ T cell culture step.

Currently, CD4+ T cells and CD25+ T cells can be derived from blood cells, respectively. Currently, CD4+ T cells and CD25+ T cells can be isolated from peripheral blood mononuclear cells (PBMCs) or can be obtained from blood cells through specific proliferation of CD4+ T cells or CD25+ T cells, respectively. Alternatively, CD4+ T cells or CD25+ T cells can be obtained after removing CD4- T cells or CD25- T cells from PBMCs. In addition, CD4+ T cells can be isolated using anti-CD4 antibodies, and in one embodiment, they can be isolated using beads to which anti-CD4 antibodies bind. Additionally, CD25+ T cells can be isolated using anti-CD25+ antibodies. In particular, regulatory T cells can be isolated by isolating CD4+, CD25+ and CD127- T cells from CD4+ T cells or CD25+ T cells.

Derived regulatory T cells and their applications

According to another aspect of the present invention, regulatory T cells obtained by this culture method are provided.

According to yet another aspect of the present invention, there is provided a composition for the treatment of a disease mediated by regulatory T cells, containing regulatory T cells obtained by the above method as an active ingredient.

Currently, regulatory T cells produced by this culture method may have increased amounts of Foxp3+ expression. The term "Foxp3" as used herein refers to a protein also called scurfin. This protein is a protein involved in the regulatory pathway of regulatory T cells and is known as a marker of regulatory T cells. Preferably, the regulatory T cells obtained above may be CD4+CD25+CD127-Foxp3+ T cells.

The term “regulatory T cell-mediated disease” as used herein refers to a disease induced by abnormality or deficiency of regulatory T cells and may be specifically characterized as an inflammatory disease or an autoimmune disease.

A specific embodiment of the present invention may be characterized in that the inflammatory disease is at least one selected from the group consisting of lupus, Sjögren's syndrome, rheumatoid arthritis, fibromyositis, scleroderma, ankylosing spondylitis, Behçet's disease, autosous stomatitis, Guillain-Barre syndrome , alopecia areata, polymyositis, Crohn's disease, colitis, polyarteritis nodosa, relapsing polychondritis and autoimmune thrombocytopenia. In addition, a specific embodiment of the present invention may be characterized in that the autoimmune disease is at least one selected from the group consisting of rheumatoid arthritis, systemic sclerosis, insulin-dependent juvenile diabetes caused by antibodies against pancreatic cells, alopecia areata, psoriasis, pemphigus , asthma, oral thrush, chronic thyroiditis, some acquired aplastic anemia, primary cirrhosis, ulcerative colitis, Behçet's disease, Crohn's disease, silicosis, asbestosis, IgA nephropathy, post-streptococcal glomerulonephritis, Sjögren's syndrome, Guillain-Barre syndrome, polymyositis, multiple myositis, multiple sclerosis, autoimmune hemolytic anemia, autoimmune encephalomyelitis, myasthenia gravis, Graves' hyperthyroidism, polyarteritis nodosa, ankylosing spondylitis, fibromyalgia, temporal arteritis, Wilson's disease, Fanconi syndrome, multiple myeloma and systemic lupus erythematosus.

The composition of the present invention may contain a pharmaceutically acceptable carrier and/or additive or the like. For example, it may contain sterile water, normal saline, a conventional buffer (eg, phosphoric acid, citric acid, or other organic acid), stabilizer, salt, antioxidant, surfactant, suspending agent, isotonic agent, or preservative. In addition, it may contain an organic substance such as a biopolymer and an inorganic substance such as hydroxyapatite, in particular, a collagen matrix, a polylactic acid polymer or a copolymer thereof, a polyethylene glycol polymer or a copolymer thereof, a chemical derivative thereof, and a mixture thereof, but not limited to them. Examples of the stabilizer may include dextran 40, methyl cellulose, gelatin, sodium sulfite, sodium metasulfate or the like. Examples of the antioxidant may include a chelator such as erythorbic acid, dibutylated hydroxytoluene, butylated hydroxyanisole, α-tocopherol, tocopheryl acetate, L-ascorbic acid palmitate, L-ascorbic acid stearate, sodium hydrosulfite, sodium sulfite, triamyl gallic acid, propyl gallic acid, or sodium ethylenediaminetetraacetic acid (EDTA), sodium pyrophosphate, sodium metaphosphate and the like. Examples of the suspending agent may include methylcellulose, polysorbate 80, hydroxyethylcellulose, gum acacia, gum tragacanth, sodium carboxymethylcellulose, polyoxyethylene sorbitan monolaurate and the like. Examples of the isotonic agent may include D-mannitol and sorbitol. Examples of the preservative may include methyl paraoxybenzoate, ethyl paraoxybenzoate, sorbic acid, phenol, cresol, chlorocresol or the like.

Method of treatment using obtained regulatory T cells

According to another aspect of the present invention, there is provided a method of treating a disease mediated by regulatory T cells, comprising administering regulatory T cells to an individual having a disease mediated by regulatory T cells. Currently, regulatory T cells and regulatory T cell-mediated disease are as described above.

According to yet another aspect of the present invention, the use of regulatory T cells for the treatment of a disease mediated by regulatory T cells is provided.

Method for carrying out this invention

Below, the present invention will be described in more detail by means of the following examples. However, the following examples are intended only to illustrate the present invention, and the scope of the present invention is not limited to them.

Preparation example 1. Preparation of hCD80-Fc-IL-2 (2M) variant: GI-101

In order to obtain a fusion protein containing a fragment of human CD80, an Fc domain and an IL-2 variant, a polynucleotide containing a nucleotide sequence (SEQ ID NO: 8) encoding a fusion protein containing a signal peptide (SEQ ID NO: 1) was synthesized. CD80 fragment (SEQ ID NO: 2), Ig hinge linked to linker (SEQ ID NO: 3), Fc domain (SEQ ID NO: 4), linker (SEQ ID NO: 5) and IL-2 variant (2M) , in which two amino acids (R38A, F42A) are replaced (SEQ ID NO: 6), in this order from the N-terminus through the GeneArt Gene Synthesis Service of Invitrogen Thermo Fisher Scientific Inc. and cloned into the pcDNA3_4 vector. In addition, this vector was introduced into CHO cells (Chinese hamster eggs) (EXPI-CHO™) to express the fusion protein SEQ ID NO: 9. After the introduction of the vector, the culture solution was cultured in an environment of 37°C, 125 rpm and 8% CO ₂ for 7 days and then selected for purification of the fusion protein. The purified fusion protein dimer was named "GI-101)).

Purification was performed using chromatography containing MabSelect SuRe protein A resin. The fusion protein was bound under conditions of 25 mM Tris, 25 mM NaCl and pH 7.4. It was then eluted with 100 mM NaCl and 100 mM acetic acid at pH 3. After placing 20% 1 M Tris-HCl pH 9 in a collection tube, the fusion protein was collected. The selected fusion protein was dialyzed into PBS buffer for 16 hours to exchange the buffer.

Absorbance was then measured at 280 nm over time using gel filtration with a TSKgel G3000SWXL column (TOSOH Bioscience) to obtain a high concentration of the fusion protein. The isolated and purified fusion protein was subjected to SDS-PAGE under reducing (R) or non-reducing (NR) conditions and stained with Coomassie blue to confirm its purity (FIG. 1B). The fusion protein was confirmed to be present at a concentration of 2.78 mg/ml when detected using NanoDrop (FIG. 1C). Also, the result analyzed using gel filtration is as shown in FIG. 1D.

Preparation Example 2: Preparation of Fc-IL-2 Variant Dimer (2M): Fc-IL-2v2

In order to obtain a fusion protein containing an Fc domain and an IL-2 variant, a polynucleotide was synthesized containing a nucleotide sequence (SEQ ID NO: 45) encoding a fusion protein containing a signal peptide (SEQ ID NO: 1), Ig hinge (SEQ ID NO: 38), Fc domain (SEQ ID NO: 4), linker (SEQ ID NO: 5) and IL-2 variant (2M) in which two amino acids are replaced (R38A, F42A) (SEQ ID NO: 6) , in this order from the N-terminus via the GeneArt Invitrogen ThermoFisher Scientific Inc. gene synthesis service. and cloned into the pcDNA3_4 vector. In addition, this vector was introduced into CHO cells (EXPI-CHO™) to express the fusion protein SEQ ID NO: 44. After introduction of this vector, the culture solution was cultured in an environment of 37°C, 125 rpm and 8% CO ₂ for 7 days and then selected for purification of the fusion protein dimer. The purified fusion protein dimer was named "Fc-IL2v2".

Purification and selection of this fusion protein was carried out in the same manner as in Preparation Example 1. The isolated and purified fusion protein was subjected to SDS-PAGE under reducing (R) or non-reducing (NR) conditions and stained with Coomassie blue to confirm its purity (FIG. 3A ). As a result, it was confirmed that the fusion protein forms a dimer. Also, the result analyzed using gel filtration is as shown in FIG. 3B.

Preparation Example 3: Preparation of Fc-IL-2 dimer: Fc-IL-2wt

In order to obtain a fusion protein containing the Fc domain and wild-type IL-2, a polynucleotide was synthesized containing the nucleotide sequence (SEQ ID NO: 43) encoding a fusion protein containing the signal peptide (SEQ ID NO: 1), Ig hinge ( SEQ ID NO: 38), Fc domain (SEQ ID NO: 4), linker (SEQ ID NO: 5) and wild type IL-2 (SEQ ID NO: 10), in this order from N-terminus via gene synthesis service GeneArt Invitrogen Thermo Fisher Scientific inc. and cloned into the pcDNA3_4 vector. In addition, this vector was introduced into CHO cells (EXPI-CHO™) to express the fusion protein SEQ ID NO: 42. After introduction of the vector, the culture solution was cultured in an environment of 37°C, 125 rpm and 8% CO ₂ in for 7 days and then selected for purification of the fusion protein dimer. The purified fusion protein dimer was named "Fc-IL2wt".

Purification and selection of this fusion protein was carried out in the same manner as in Preparation Example 1. The isolated and purified fusion protein was subjected to SDS-PAGE under reducing (R) or non-reducing (NR) conditions and stained with Coomassie blue to confirm its purity (FIG. 3C ). As a result, it was confirmed that the fusion protein forms a dimer. Also, the result analyzed using gel filtration is as shown in FIG. 3D.

Preparation Example 4: Preparation of wild type hCD80-Fc-IL-2 dimer: hCD80-Fc-IL-2wt

In order to obtain a fusion protein containing a fragment of human CD80, an Fc domain and a wild-type IL-2 protein, a polynucleotide containing a nucleotide sequence (SEQ ID NO: 41) encoding a fusion protein containing a signal peptide (SEQ ID NO: 1) was synthesized ), CD80 fragment (SEQ ID NO: 2), Ig hinge linked to linker (SEQ ID NO: 3), Fc domain (SEQ ID NO: 4), linker (SEQ ID NO: 5) and wild-type IL-2 (SEQ ID NO: 10) in this order from the N-terminus, through the GeneArt Gene Synthesis Service of Invitrogen ThermoFisher Scientific Inc. and cloned into the pcDNA3_4 vector. In addition, this vector was introduced into CHO cells (EXPI-CHO™) to express the fusion protein SEQ ID NO: 46. After introduction of the vector, the culture solution was cultured in an environment of 37°C, 125 rpm and 8% CO ₂ in for 7 days and then selected for purification of the fusion protein dimer. The purified fusion protein dimer was named "hCD80-Fc-IL2wt".

Purification was performed using chromatography containing MabSelect SuRe protein A resin. The fusion protein was bound under conditions of 25 mM Tris, 25 mM NaCl and pH 7.4. It was then eluted with 100 mM NaCl and 100 mM acetic acid at pH 3. After placing 20% 1 M Tris-HCl pH 9 in a collection tube, the fusion protein was collected. The selected fusion protein was dialyzed into PBS buffer for 16 hours to exchange the buffer.

Absorbance was then measured at 280 nm over time using gel filtration with a TSKgel G3000SWXL column (TOSOH Bioscience) to obtain a high concentration of the fusion protein. Here, the isolated and purified fusion protein was subjected to SDS-PAGE under reducing (R) or non-reducing (NR) conditions and stained with Coomassie blue to confirm its purity (FIG. 4A). As a result, it was confirmed that this fusion protein forms a dimer. Also, the result analyzed using gel filtration is as shown in FIG. 4B.

Preparation Example 5: Preparation of hCD80-Fc dimer: hCD80-Fc

In order to obtain a fusion protein containing a human CD80 fragment and an Fc domain, a polynucleotide (SEQ ID NO: 39) was synthesized containing a nucleotide sequence encoding a fusion protein containing a signal peptide (SEQ ID NO: 1), a CD80 fragment (SEQ ID NO: 2), Ig hinge linked to linker (SEQ ID NO: 3), and Fc domain (SEQ ID NO: 4) in this order from the N-terminus, through GeneArt invitrogen ThermoFisher Scientific inc. and cloned into the pcDNA3_4 vector. In addition, this vector was introduced into CHO cells (EXPi-CHO™) to express the fusion protein SEQ ID NO: 40. After introduction of the vector, the culture solution was cultured in an environment of 37°C, 125 rpm and 8% CO ₂ in for 7 days and then selected for purification of the fusion protein dimer. The purified fusion protein dimer was named "hCD80-Fc".

Purification was performed using chromatography containing MabSelect SuRe protein A resin. The fusion protein was bound under conditions of 25 mM Tris, 25 mM NaCl and pH 7.4. It was then eluted with 100 mM NaCl and 100 mM acetic acid at pH 3. After placing 20% 1 M Tris-HCl pH 9 in a collection tube, the fusion protein was collected. The selected fusion protein was dialyzed into PBS buffer for 16 hours to exchange the buffer.

Absorbance was then measured at 280 nm over time using gel filtration with a TSKgel G3000SWXL column (TOSOH Bioscience) to obtain a high concentration of the fusion protein. Here, the isolated and purified fusion protein was subjected to SDS-PAGE under reducing (R) or non-reducing (NR) conditions and stained with Coomassie blue to confirm its purity (FIG. 2A). As a result, it was confirmed that this fusion protein forms a dimer. Also, the result analyzed using gel filtration is as shown in FIG. 2B.

Preparation example 1. Culture composition for cultivating regulatory T cells

Receipt example 1.1. Culture composition of CD4+ cells

CD4+ cell culture medium was prepared as the following composition. In this case, a medium was prepared from the main components, and then additional components Gl-101, Gl-101WT, hCD80-Fc, Fc-IL-2v2 or Fc-IL-2wt were added before use.

Receipt example 1.2. Cultural composition of CD4+CD25+CD127- cells

The cultural composition of CD4+CD25+CD127- cells was prepared as follows.

Example 1 Determination of the level of proliferation of regulatory T cells using a fusion protein dimer containing IL-2 protein and CD80 protein

Example 1.1. Preparation of beads to stimulate proliferation of regulatory T cells

In order to stimulate regulatory T cell proliferation in isolated CD4+ cells, regulatory T cell proliferation stimulation beads were prepared using the MACS GMP ExpAct Treg kit (Cat. No: 170-076-119) (Miltenyi Biotec, Bergisch Giadbach, Germany ). Specifically, the reagent in the MACS GMP ExpAct Treg kit, which contains beads to stimulate proliferation of regulatory T cells, was transferred to a new tube, the tube was allowed to sit on a magnet for 1 minute, and then the supernatant was removed to separate the beads. In this case, 1 μl of the reagent in the MACS GMP ExpAct Treg kit was used for 2×10 ⁵ cells. After isolation of the beads, 0.5 ml to 1 ml of the CD4+ cell culture composition in Table 1 or Table 2, which does not contain any additional component (containing only the main components), was added to resuspend the beads.

Example 1.2. Isolation of CD4+ T cells

The number of acquired PBMCs was measured (Cat. No.: SER-PBMC-200-F) (Zen-Bio. Inc, NC 27709, USA). They were then centrifuged at 300 g for 10 minutes. Then, after removing the supernatant buffer, 80 μl of MAC buffer per 1 x 10 ⁷ cells was added to resuspend the cell pellets. Then 20 μl of CD4 microbeads (cat. no. 130-045-101) (Miltenyi Biotec, Bergisch Gladbach, Germany) was dispensed onto 1×10 ⁷ cells, tapped and mixed sufficiently. Then the interaction was carried out at 4°C-8°C for 15 minutes.

For washing, 10 ml of MAC buffer was added and then centrifuged at 300 g for 10 minutes. After removing the supernatant, 500 μl of MAC buffer was then added to 1 x 10 ⁸ cells to resuspend the cell pellets. An LS column was then prepared, and then 3 mL of MAC buffer was passed through. The cell suspension obtained above was passed through an LS column (cat. no. 130-042-401) (Miltenyi Biotec, Bergisch Gladbach, Germany). 3 ml of MAC buffer was passed through 3 times so that the cells attached to the LS column could be sufficiently washed. Then, after removing the LS column from the magnetic stage, 3 ml of MACS buffer was added and piston pressure was applied to isolate CD4+ cells. Then centrifugation was carried out at 300 g for 5 minutes. The supernatant was then removed and the cell number was then measured.

In addition, the regulatory T cell culture solution containing the beads obtained in Example 1.1 was inoculated onto the CD4+ T cells isolated above.

Example 1.3. CD4+ T cell culture

In a 6-well plate, the CD4+ cells obtained in Example 1.2 were seeded at a concentration of 1×10 ⁷ cells/ml, and the CD4+ cells were cultured under the conditions of the cell culture composition in Table 1 or Table 2, respectively, containing GI-101 (50 nM) , GI-101_WT (50 nM), CD80-Fc dimer (50 nM) + Fc-IL-2-2v2 dimer (50 nM) or CD80-Fc dimer (50 nM) + Fc-IL-2wt dimer (50 nM) as additives. When cells showed greater than 80% confluency in a 6-well plate, they were subcultured in a 25T flask. When cells in a 25T flask demonstrated greater than 80% confluency, they were subcultured in a 75T flask. Finally, when cells in the 75T flask demonstrated more than 80% confluency, they were selected.

Example 1.4. Isolation of CD4+CD25+CD127- cells

CD4+ cells isolated in Example 1.3 were centrifuged at 1300 rpm and 4°C for 5 minutes. In addition, the supernatant was removed. Add 1 ml of Fc blocking reagent (biolegend, cat. no. 422302), diluted in FACS buffer to 1:200, and leave on ice for 10 minutes, and then add 50 μl of CD4-Pacific Blue (BioLegend, cat. no. 317429), 50 µl CD25-PE/Cy7 (BioLegend, cat. no. 356108) and 50 µl CD127-PE (BD, cat. no. 557938) and left on ice for 20 minutes. Then an additional 4 ml of FACS buffer was added and centrifuged at 1300 rpm and 4°C for 5 minutes. Then, after removing the supernatant, 3 ml of FACS buffer was added and centrifuged at 1300 rpm and 4°C for 5 minutes. The supernatant was then removed and 3 mL of FACS buffer was added to resuspend the cells. Finally, CD4+CD25+CD127− cells were isolated using BD FACS Aria.

Example 1.5. Cultivation of CD4+CD25+CD127- cells

The CD4+CD25+CD127- cells isolated in Example 1.4 were seeded at a concentration of 3x10 ⁵ cells/ml in a 48-well plate, and at the same time beads were isolated from 1 μl of the MACS GMP ExpAct Treg kit (cat. no.: 170-076-119) (Miltenyi Biotec, Bergisch Gladbach, Germany) at 2×10 ⁶ cells to stimulate the proliferation of regulatory T cells, as in Example 1.1. The isolated beads were resuspended in the cell culture composition (0.5 ml to 1 ml) in Table 3 or Table 4 (containing only the base components), which does not contain any additional component, and then added to the well plate into which the cells were seeded CD4+CD25+CD127- obtained in Example 1.4. Then CD4+CD25+CD127- cells were cultured under the conditions of the cell culture composition in Table 3 or Table 4, respectively, containing GI-101 (50 nM), GI-101_WT (50 nM), CD80-Fc (50 nM) + Fc- IL-2-2v2 (50 nM) or CD80-Fc (50 nM) + Fc-IL-2wt (50 nM) as a supplement.

In culture, when cells showed more than 80% confluency in a 48-well plate, they were subcultured in a 24-well plate. When cells showed more than 80% confluency in a 24-well plate, they were subcultured in a 12-well plate. When cells showed more than 80% confluency in a 12-well plate, they were subcultured in a 6-well plate. When cells demonstrated greater than 80% confluency in a 6-well plate, they were subcultured in a 25T flask. When cells demonstrated greater than 80% confluency in a 25T flask, they were subcultured in a 75T flask. Finally, cells were selected when they showed greater than 80% confluency after subculture in a 75T flask.

As a result, the proliferation results of regulatory T cells in the regulatory T cell culture medium composition containing RPMI1640 medium are as shown in Table 5 and FIG. 6, and cell viability are as shown in Table 6 and FIG. 7. In addition, the results of proliferation of regulatory T cells in the culture composition containing TexMACS medium are shown in Table 7 and FIG. 8 and cell viability are shown in Table 8 and FIG. 9.

Example 1.6. Determination of the secretory abilities of immunosuppressive cytokines: interleukin-10

In order to evaluate the secretory abilities of IL-10 regulatory T cells obtained in Example 1.5, these cells were cultured such that the cell number was adjusted to 1×10 ⁶ cells/ml, and then the culture supernatant was obtained for analysis by ELISA (enzyme-linked immunosorbent assay).

First, 100 μl of human IL-10 ELISA kit incubation buffer (Invitrogen, cat. no. KAC1321) was dispensed into each microplate, and then 100 μl of calibration, control, or regulatory T cell culture supernatant sample was added to dispense, respectively. These microplates were then coated with an adhesive film and reacted with shaking at 700 rpm at room temperature (18°C to 25°C) for 2 hours on a shaker. Rows of microwells were then washed 3 times with approximately 150 μL of wash buffer per well.

After washing, 100 μL of sample diluent was added. Next, 50 μl of anti-IL-10 antibody conjugated to HRP (horseradish peroxidase) was added, followed by shaking at 700 rpm at room temperature for 2 hours, followed by washing 3 times with approximately 150 μl of wash buffer. to the hole. After washing, 100 μl of TMB (tetramethylbenzidine) was added and then reacted at room temperature for 15 minutes, followed by termination of the reaction by adding 100 μl of stopping solution. The fluorescence values of each microwell were then measured at 450 nm.

As a result, the secretory abilities of interleukin-10 cells cultured in the composition containing RPMH640 medium are as shown in FIG. 10, and the interleukin-10 secretory abilities of cells cultured in the composition containing TexMACS medium are as shown in FIG. 11. In FIG. 10 and 11 confirmed that interleukin-10 was highly expressed in regulatory T cells cultured in a culture composition containing GI-101.

Example 2 Determination of Regulatory T Cell Proliferation by GH01 in an Optimized Culture Method

Example 2.1. Isolation of CD4+ T cells

The number of acquired PBMCs was measured (Cat. No: SER-PBMC-200-F) (Zen-Bio Inc, NC 27709, USA). They were then centrifuged at 300 g for 10 minutes. Then, after removing the supernatant buffer, 80 μl of MAC buffer per 1 x 10 ⁷ cells was added to resuspend the cell pellets. Then 20 μl of CD4 microbeads (cat. no. 130-045-101) (Miltenyi Biotec, Bergisch Gladbach, Germany) was dispensed onto 1×10 ⁷ cells, tapped and mixed sufficiently. Then the interaction was carried out at 4°C-8°C for 15 minutes.

For washing, 10 ml of MAC buffer was added and then centrifuged at 300 g for 10 minutes. After removing the supernatant, 500 μl of MAC buffer was then added to 1 x 10 ⁸ cells to resuspend the cell pellets. An LS column was then prepared, and then 3 mL of MAC buffer was passed through. The cell suspension obtained above was passed through an LS column (cat. no. 130-042-401) (Miltenyi Biotec, Bergisch Gladbach, Germany). 3 ml of MAC buffer was passed through 3 times so that the cells attached to the LS column could be sufficiently washed. Then, after removing the LS column from the magnetic stage, 3 ml of MAC buffer was added and piston pressure was applied to isolate CD4+ cells. Then centrifugation was carried out at 300 g for 5 minutes. After centrifugation, the supernatant was removed and cell numbers were measured.

In order to stimulate the proliferation of regulatory T cells in the isolated CD4+ cells, 0.5 ml to 1 ml of the CD4+ cell culture compositions in Table 1 or Table 2 (containing only the basic components), which do not contain any additional component, were added to resuspending beads that were isolated and prepared as in Example 1.1, and this was inoculated into CD4+ cells isolated from PBMC.

Example 2.2. Cultivation of CD4+ T cells

In a 6-well plate, CD4+ cells obtained in Example 2.1 were seeded at a concentration of 1×10 ⁷ cells/ml and cultured under the conditions of the cell culture composition in Table 1 or Table 2, respectively, containing GI-101 (3.2 nM/50 nM), GI-101_WT (3.2 nM/50 nM), CD80-Fc dimer (3.2 nM/50 nM) + Fc-IL-2v2 dimer (3.2 nM/50 nM) or CD80-Fc dimer (3.2 nM/50 nM) + Fc-IL-2wt dimer (3.2 nM/50 nM) as a supplement. When cells demonstrated greater than 80% confluency in a 6-well plate, they were subcultured in a 25T flask. When cells in a 25T flask demonstrated greater than 80% confluency, they were subcultured in a 75T flask. Finally, when cells in the 75T flask demonstrated more than 80% confluency, they were selected.

Example 2.3. Isolation of T cells CD4+CD25+CD127-

CD4+ cells isolated in Example 2.2 were centrifuged at 1300 rpm and 4°C for 5 minutes. In addition, the supernatant was removed and 1 ml of Fc blocking reagent (biolegend, cat. no. 422302), diluted in FACS buffer to 1:200, was added, followed by leaving on ice for 10 minutes. Add 50 µl CD4-Pacific Blue (BioLegend, cat. no. 317429), 50 µl CD25-PE/Cy7 (BioLegend, cat. no. 356108) and 50 µl CD127-PE (BD, cat. no. 557938) to sample 2 and leave on ice for 20 minutes.

Then an additional 4 ml of FACS buffer was added and centrifuged at 1300 rpm and 4°C for 5 minutes. The supernatant was then removed and 3 ml of FACS buffer was added, followed by centrifugation at 1300 rpm and 4°C for 5 minutes. The supernatant was then removed and 3 mL of FACS buffer was added to resuspend the cells. Finally, CD4+CD25+CD127− cells were isolated using BD FACS Aria.

Example 2.4. Cultivation of T cells CD4+CD25+CD127-

All CD4+CD25+CD127- cells isolated in Example 2.3 were seeded in a 24-well plate and, at the same time, beads were isolated from 1 μl MACS GMP ExpAct Treg kit (cat. no.) to stimulate proliferation of regulatory T cells. :170-076-119) (Miltenyi Biotec, Bergisch Gladbach, Germany) into 2×10 ⁵ cells as in Example 2.1, and these beads were resuspended in the cell culture composition (0.5 ml to 1 ml) in Table 3 or Table 4 (containing only the basic components), which does not contain any additional component. They were then added to a well plate in which CD4+CD25+CD127- cells were seeded.

CD4+CD25+CD127- cells were then cultured under the conditions of the cell culture composition in Table 3 or Table 4, respectively, containing GI-101 (3.2 nM/50 nM), GI-101_WT (3.2 nM/50 nM), CD80-Fc dimer (3.2 nM/50 nM) + Fc-IL-2v2 dimer (3.2 nM/50 nM) or CD80-Fc dimer (3.2 nM/50 nM) + Fc-IL-2wt ( 3.2 nM/50 nM) as an additive. When cells showed more than 80% confluency in a 24-well plate, they were subcultured in a 12-well plate. When cells showed more than 80% confluency in a 12-well plate, they were subcultured in a 6-well plate. When cells demonstrated greater than 80% confluency in a 6-well plate, they were subcultured in a 25T flask. When cells in a 25T flask demonstrated greater than 80% confluency, they were subcultured in a 75T flask.

For the group treated with 3.2 nM of the supplements in Table 3 or Table 4, cells were finally selected when they demonstrated greater than 80% confluence in the 75T flask. For the group treated with 50 nM of the supplements in Table 3 or Table 4, cells were finally selected when they demonstrated greater than 80% confluency after subculture in a 175T flask.

As a result, in the culture composition containing RPMH640 medium, the cell proliferation results are as shown in Table 9, FIG. 12A and FIG. 12B, and cell viability are as shown in Table 10, FIG. 13A and FIG. 13V. In addition, in the culture composition containing TexMACS medium, the cell proliferation results are shown in Table 11, FIG. 14A and FIG. 14B, and cell viability are shown in Table 12, FIG. 15A and FIG. 15V.

Example 2.5. Cell characteristics

CD4+CD25+CD127- cells isolated in Example 2.4 were centrifuged at 1300 rpm and 4°C for 5 minutes. In addition, the supernatant was removed. Then, 100 μl of Fc blocking reagent (biolegend, cat. no. 422302), diluted in FACS buffer to 1:200, was added, left on ice for 10 minutes, and an additional 2.5 μl of CD4-PerCP-Cy5.5 (eBioscience, cat. no.) was added. 45-0048-42), 2.5 µl CD25-APC/Cy7 (BD, cat. no. 557753) and 2.5 µl CD127-Brilliant Violet 785 (Biolegend, cat. no. 351330) and left on ice for 20 minutes .

Then an additional 1 ml of FACS buffer was added and centrifuged at 1300 rpm and 4°C for 5 minutes. A 1:3 mixture solution of fixative/permeabilizer concentrate and fixative/permeabilizer diluent was prepared, and 100 μl was added after removing the supernatant from the centrifuged cells and left on ice for 30 minutes. Then, an additional 1 ml of FACS buffer was added, and centrifugation was repeated twice at 1300 rpm and 4°C for 5 minutes. The supernatant was then removed, 100 μl of FACS buffer and 2.5 μl of Foxp3-Pacific Blue (Biolegend, cat. no. 320116) were added and left on ice for 20 minutes. Then an additional 1 ml of FACS buffer was added and centrifugation was carried out at 1300 rpm and 4°C for 5 minutes. The supernatant was then removed and 200 μl of FACS buffer was added to resuspend the cells. Finally, cells were characterized using Cytek Aurora equipment.

As a result, the results of FACS analysis of the properties of cells cultured in the composition containing RPIVH1640 medium are as shown in FIG. 16A-16C, and the number of T cells expressing CD4+CD25+Foxp3+ confirmed under the above culture conditions is as shown in Table 13 and FIG. 17A-17C. In addition, the results of FACS analysis of the properties of cells cultured in the composition containing TexMACS medium are as shown in FIG. 18A-18C, and the number of CD4+CD25+Foxp3+ expressing T cells confirmed under the above culture conditions is as shown in Table 14 and FIG. 19A-19C.

Example 2.6. Determination of the secretory abilities of immunosuppressive cytokines: interleukin-10

In order to evaluate the secretory abilities of IL-10 regulatory T cells obtained in Example 2.4, these cells were cultured such that the cell number was adjusted to 1×10 ⁶ cells/ml, and then the culture supernatant was obtained for analysis by ELISA (solid phase linked immunosorbent assay).

First, 100 μl of human IL-10 ELiSA kit incubation buffer (Invitrogen, cat. no. KAC1321) was dispensed into each microplate, and then 100 μl of calibration, control, or regulatory T cell culture supernatant sample was added to dispense, respectively. These microplates were then coated with an adhesive film and reacted with shaking at 700 rpm at room temperature (18°C to 25°C) for 2 hours on a shaker. Rows of microwells were then washed 3 times with approximately 150 μL of wash buffer per well.

After washing, 100 μL of sample diluent was added. Next, 50 μl of HRP-conjugated anti-IL-10 antibody was added, followed by shaking at 700 rpm at room temperature for 2 hours, followed by washing 3 times with approximately 150 μl of wash buffer per well. After washing, 100 μl of TMB was added and then reacted at room temperature for 15 minutes, followed by termination of the reaction by adding 100 μl of stopping solution. The fluorescence values of each microwell were then measured at 450 nm.

As a result, the secretory abilities of interleukin-10 cells cultured in the composition containing RPMI1640 medium are as shown in FIG. 20, and the secretory abilities of interleukin-10 cells cultured in the composition containing TexMACS medium are as shown in FIG. 21. In FIG. 20 and 21 confirmed that interleukin-10 was highly expressed in regulatory T cells cultured in a culture composition containing GI-101.

--->

 LIST OF SEQUENCES

<110>GI Cell, Inc.

<120> COMPOSITION FOR CULTURING REGULATORY T-CELLS AND ITS APPLICATION

<130> SPO20-038-PCT-GIC

<150> KR 10-2019-0149779

<151> 2019-11-20

<150> KR 10-2020-0033229

<151> 2020-03-18

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Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

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Ala Val Phe Val Ser Pro Ser His Ala

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<211> 208

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<213> Artificial sequence

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<223> hB7-1:35-242

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Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys

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Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp

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

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Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn

50 55 60

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

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Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His

85 90 95

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

100 105 110

Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys

115 120 125

Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn

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Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu

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Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr

165 170 175

Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn

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Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn

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Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

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Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

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Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

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

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Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

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Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

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Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

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Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

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Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

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

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

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

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

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

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Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln

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Lys Ser Leu Ser Leu Ser Leu Gly

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Gly Gly Gly Gly Ser

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Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

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Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

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

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Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

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Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

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Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

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Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

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Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

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Ile Ser Thr Leu Thr

130

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Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

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Ala Val Phe Val Ser Pro Ser His Ala Val Ile His Val Thr Lys Glu

20 25 30

Val Lys Glu Val Ala Thr Leu Ser Cys Gly His Asn Val Ser Val Glu

35 40 45

Glu Leu Ala Gln Thr Arg Ile Tyr Trp Gln Lys Glu Lys Lys Met Val

50 55 60

Leu Thr Met Met Ser Gly Asp Met Asn Ile Trp Pro Glu Tyr Lys Asn

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Arg Thr Ile Phe Asp Ile Thr Asn Asn Leu Ser Ile Val Ile Leu Ala

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Leu Arg Pro Ser Asp Glu Gly Thr Tyr Glu Cys Val Val Leu Lys Tyr

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Glu Lys Asp Ala Phe Lys Arg Glu His Leu Ala Glu Val Thr Leu Ser

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Val Lys Ala Asp Phe Pro Thr Pro Ser Ile Ser Asp Phe Glu Ile Pro

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Thr Ser Asn Ile Arg Arg Ile Ile Cys Ser Thr Ser Gly Gly Phe Pro

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Glu Pro His Leu Ser Trp Leu Glu Asn Gly Glu Glu Leu Asn Ala Ile

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

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Ser Lys Leu Asp Phe Asn Met Thr Thr Asn His Ser Phe Met Cys Leu

195 200 205

Ile Lys Tyr Gly His Leu Arg Val Asn Gln Thr Phe Asn Trp Asn Thr

210 215 220

Thr Lys Gln Glu His Phe Pro Asp Asn Gly Ser Gly Gly Gly Gly Ser

225 230 235 240

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly

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Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

260 265 270

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg

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Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

290 295 300

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

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Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

325 330 335

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

340 345 350

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

355 360 365

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

370 375 380

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

385 390 395 400

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

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Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

420 425 430

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

435 440 445

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala

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Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly

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

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Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile

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

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Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu

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Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

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Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile

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Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala

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Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe

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Cys Gln Ser Ile Ile Ser Thr Leu Thr

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<211> 1857

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<213> Artificial sequence

<220>

<223> nucleotides encoding fusion protein (GI101)

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60

tctccttctc acgctgtgat ccacgtgacc aaagaagtga aagaggtcgc cacactgtcc

120

tgcggccaca acgtttcagt ggaagaactg gcccagacca ggatctactg gcagaaagaa

180

aagaaaatgg tgctgaccat gatgtccggc gacatgaaca tctggcctga gtacaagaac

240

cggaccatct tcgacatcac caacaacctg tccatcgtga ttctggccct gaggccttct

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gatgagggca cctatgagtg cgtggtgctg aagtacgaga aggacgcctt caagcgcgag

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cacctggctg aagtgacact gtccgtgaag gccgactttc cccacaccttc catctccgac

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gagcctcacc tgtcttggct ggaaaacggc gaggaactga acgccatcaa caccaccgtg

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tctcaggacc ccgaaaccga gctgtacgct gtgtcctcca agctggactt caacatgacc

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aactggaaca ccaccaagca agagcacttc cctgacaatg gatctggcgg cggaggttct

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ggcggaggtg gaagcggagg cggaggatct gctgagtcta agtatggccc tccttgtcct

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ccatgtcctg ctccagaagc tgctggcgga ccctctgtgt tcctgtttcc tccaaagcct

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aaggaccagc tcatgatctc tcggacaccc gaagtgacct gcgtggtggt ggatgtgtct

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caagaggacc ctgaggtgca gttcaattgg tacgtggacg gcgtggaagt gcacaacgcc

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aagaccaagc ctagagagga acagttcaac tccacctaca gagtggtgtc cgtgctgacc

1020

gtgctgcacc aggattggct gaacggcaaa gagtacaagt gcaaggtgtc caacaagggc

1080

ctgccttcca gcatcgaaaa gaccatctcc aaggctaagg gccagcctag ggaaccccag

1140

gtttacaccc tgcctccaag ccaagaggaa atgaccaaga accaggtgtc cctgacctgc

1200

ctggtcaagg gcttctaccc ttccgacatt gccgtggaat gggagtccaa tggccagcct

1260

gagaacaact acaagaccac acctcctgtg ctggactccg acggctcctt ctttctgtac

1320

tctcgcctga ccgtggacaa gtctagatgg caagagggca acgtgttctc ctgctctgtg

1380

ctgcacgagg ccctgcacaa tcactacacc cagaagtccc tgtctctgtc tcttggaggt

1440

ggtggcggtt ctgcccctac cagctcctct accaagaaaa cccagctcca gttggagcat

1500

ctgctgctgg acctccagat gattctgaac gggatcaaca actataagaa ccccaagctg

1560

accgccatgc tgaccgctaa gttctacatg cccaagaagg ccaccgagct gaagcacctc

1620

cagtgcctgg aagaagaact gaagcccctg gaagaggtgc tgaatctggc ccagtccaag

1680

aacttccacc tgaggccacg ggacctgatc agcaacatca acgtgatcgt gctggaactg

1740

aagggctccg agacaacctt tatgtgcgag tacgccgacg agacagccac catcgtggaa

1800

tttctgaacc ggtggatcac cttctgccag agcatcatct cccacactgac ctgatga

1857

<210> 9

<211> 592

<212> PRT

<213> Artificial sequence

<220>

<223> fusion protein (GI101)

<400> 9

Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys

1 5 10 15

Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp

20 25 30

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

35 40 45

Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn

50 55 60

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

65 70 75 80

Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His

85 90 95

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

100 105 110

Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys

115 120 125

Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn

130 135 140

Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu

145 150 155 160

Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr

165 170 175

Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn

180 185 190

Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn

195 200 205

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

210 215 220

Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

325 330 335

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

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

355 360 365

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

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser

450 455 460

Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu

465 470 475 480

Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr

485 490 495

Ala Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu

500 505 510

Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val

515 520 525

Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu

530 535 540

Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr

545 550 555 560

Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe

565 570 575

Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr

580 585 590

<210> 10

<211> 133

<212> PRT

<213> Artificial sequence

<220>

<223> hIL-2

<400> 10

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

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

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 11

<211> 288

<212> PRT

<213> Artificial sequence

<220>

<223> CD80

<400> 11

Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys Cys Pro Tyr

1 5 10 15

Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys

20 25 30

Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu

35 40 45

Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile

50 55 60

Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp

65 70 75 80

Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr

85 90 95

Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly

100 105 110

Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg

115 120 125

Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr

130 135 140

Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile

145 150 155 160

Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu

165 170 175

Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp

180 185 190

Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met

195 200 205

Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg

210 215 220

Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro

225 230 235 240

Asp Asn Leu Leu Pro Ser Trp Ala Ile Thr Leu Ile Ser Val Asn Gly

245 250 255

Ile Phe Val Ile Cys Cys Leu Thr Tyr Cys Phe Ala Pro Arg Cys Arg

260 265 270

Glu Arg Arg Arg Asn Glu Arg Leu Arg Arg Glu Ser Val Arg Pro Val

275 280 285

<210> 12

<211> 215

<212> PRT

<213> Artificial sequence

<220>

<223> modified Fc

<400> 12

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

1 5 10 15

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

20 25 30

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

35 40 45

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

50 55 60

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

65 70 75 80

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

85 90 95

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

100 105 110

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

115 120 125

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

130 135 140

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

145 150 155 160

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

165 170 175

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

180 185 190

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

195 200 205

Leu Ser Leu Ser Leu Gly Lys

210 215

<210> 13

<211> 306

<212> PRT

<213> Artificial sequence

<220>

<223>mCD80

<400> 13

Met Ala Cys Asn Cys Gln Leu Met Gln Asp Thr Pro Leu Leu Lys Phe

1 5 10 15

Pro Cys Pro Arg Leu Ile Leu Leu Phe Val Leu Leu Ile Arg Leu Ser

20 25 30

Gln Val Ser Ser Asp Val Asp Glu Gln Leu Ser Lys Ser Val Lys Asp

35 40 45

Lys Val Leu Leu Pro Cys Arg Tyr Asn Ser Pro His Glu Asp Glu Ser

50 55 60

Glu Asp Arg Ile Tyr Trp Gln Lys His Asp Lys Val Val Leu Ser Val

65 70 75 80

Ile Ala Gly Lys Leu Lys Val Trp Pro Glu Tyr Lys Asn Arg Thr Leu

85 90 95

Tyr Asp Asn Thr Thr Tyr Ser Leu Ile Ile Leu Gly Leu Val Leu Ser

100 105 110

Asp Arg Gly Thr Tyr Ser Cys Val Val Gln Lys Lys Glu Arg Gly Thr

115 120 125

Tyr Glu Val Lys His Leu Ala Leu Val Lys Leu Ser Ile Lys Ala Asp

130 135 140

Phe Ser Thr Pro Asn Ile Thr Glu Ser Gly Asn Pro Ser Ala Asp Thr

145 150 155 160

Lys Arg Ile Thr Cys Phe Ala Ser Gly Gly Phe Pro Lys Pro Arg Phe

165 170 175

Ser Trp Leu Glu Asn Gly Arg Glu Leu Pro Gly Ile Asn Thr Thr Ile

180 185 190

Ser Gln Asp Pro Glu Ser Glu Leu Tyr Thr Ile Ser Ser Gln Leu Asp

195 200 205

Phe Asn Thr Thr Arg Asn His Thr Ile Lys Cys Leu Ile Lys Tyr Gly

210 215 220

Asp Ala His Val Ser Glu Asp Phe Thr Trp Glu Lys Pro Pro Glu Asp

225 230 235 240

Pro Pro Asp Ser Lys Asn Thr Leu Val Leu Phe Gly Ala Gly Phe Gly

245 250 255

Ala Val Ile Thr Val Val Val Ile Val Val Ile Ile Lys Cys Phe Cys

260 265 270

Lys His Arg Ser Cys Phe Arg Arg Asn Glu Ala Ser Arg Glu Thr Asn

275 280 285

Asn Ser Leu Thr Phe Gly Pro Glu Glu Ala Leu Ala Glu Gln Thr Val

290 295 300

Phe Leu

305

<210> 14

<211> 1848

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotides encoding fusion protein (mGI101)

<400> 14

atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg

60

tctccttctc acgctgtgga cgagcagctc tccaagtccg tgaaggataa ggtcctgctg

120

ccttgccggt acaactctcc tcacgaggac gagtctgagg accggatcta ctggcagaaa

180

cacgacaagg tggtgctgtc cgtgatcgcc ggaaagctga aagtgtggcc tgagtacaag

240

aacaggaccc tgtacgacaa caccacctac agcctgatca tcctgggcct cgtgctgagc

300

gatagaggca cctattcttg cgtggtgcag aagaaagagc ggggcaccta cgaagtgaag

360

cacctggctc tggtcaagct gtccatcaag gccgacttca gcacccctaa catcaccgag

420

tctggcaacc cttccgccga caccaagaga atcacctgtt tcgcctctgg cggcttccct

480

aagcctcggt tctcttggct ggaaaacggc agagagctgc ccggcatcaa taccaccatt

540

tctcaggacc cagagtccga gctgtacacc atctccagcc agctcgactt taacaccacc

600

agaaaccaca ccatcaagtg cctgattaag tacggcgacg cccacgtgtc cgaggacttt

660

acttgggaga aacctcctga ggaccctcct gactctggat ctggcggcgg aggttctggc

720

ggaggtggaa gcggaggcgg aggatctgct gagtctaagt atggccctcc ttgtcctcca

780

tgtcctgctc cagaagctgc tggcggaccc tctgtgttcc tgtttcctcc aaagcctaag

840

gaccagctca tgatctctcg gacccctgaa gtgacctgcg tggtggtgga tgtgtctcaa

900

gaggaccctg aggtgcagtt caattggtac gtggacggcg tggaagtgca caacgccaag

960

accaagccta gagaggaaca gttcaactcc acctatagag tggtgtccgt gctgaccgtg

1020

ctgcaccagg attggctgaa cggcaaagag tacaagtgca aggtgtccaa caagggcctg

1080

ccttccagca tcgaaaagac catcagcaag gctaagggcc agcctaggga accccaggtt

1140

tacaccctgc ctccaagcca agaggaaatg accaagaacc aggtgtccct gacctgcctg

1200

gtcaagggct tctacccttc cgacattgcc gtggaatggg agtccaatgg ccagcctgag

1260

aacaactaca agaccacacc tcctgtgctg gactccgacg gctccttctt tctgtactct

1320

cgcctgaccg tggacaagtc taggtggcaa gagggcaacg tgttctcctg ctctgtgctg

1380

cacgaggctc tgcacaacca ctacacccag aagtccctgt ctctgtctct tggaggtggt

1440

ggcggttctg cccctacctc cagctctacc aagaaaaccc agctccagtt ggagcatctg

1500

ctgctggacc tccagatgat cctgaatggc atcaacaatt acaagaaccc caagctgacc

1560

gccatgctga ccgctaagtt ctacatgccc aagaaggcca ccgagctgaa gcacttgcag

1620

tgcctggaag aggaactgaa gcccctggaa gaagtgctga atctggccca gtccaagaac

1680

ttccacctga ggcctaggga cctgatctcc aacatcaacg tgatcgtgct ggaactgaaa

1740

ggctccgaga caaccttcat gtgcgagtac gccgacgaga cagccaccat cgtggaattt

1800

ctgaaccggt ggatcacctt ctgccagagc atcatctcca cactgacc

1848

<210> 15

<211> 616

<212> PRT

<213> Artificial sequence

<220>

<223> fusion protein (mGI101)

<400> 15

Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

1 5 10 15

Ala Val Phe Val Ser Pro Ser His Ala Val Asp Glu Gln Leu Ser Lys

20 25 30

Ser Val Lys Asp Lys Val Leu Leu Pro Cys Arg Tyr Asn Ser Pro His

35 40 45

Glu Asp Glu Ser Glu Asp Arg Ile Tyr Trp Gln Lys His Asp Lys Val

50 55 60

Val Leu Ser Val Ile Ala Gly Lys Leu Lys Val Trp Pro Glu Tyr Lys

65 70 75 80

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

85 90 95

Leu Val Leu Ser Asp Arg Gly Thr Tyr Ser Cys Val Val Gln Lys Lys

100 105 110

Glu Arg Gly Thr Tyr Glu Val Lys His Leu Ala Leu Val Lys Leu Ser

115 120 125

Ile Lys Ala Asp Phe Ser Thr Pro Asn Ile Thr Glu Ser Gly Asn Pro

130 135 140

Ser Ala Asp Thr Lys Arg Ile Thr Cys Phe Ala Ser Gly Gly Phe Pro

145 150 155 160

Lys Pro Arg Phe Ser Trp Leu Glu Asn Gly Arg Glu Leu Pro Gly Ile

165 170 175

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

180 185 190

Ser Gln Leu Asp Phe Asn Thr Thr Arg Asn His Thr Ile Lys Cys Leu

195 200 205

Ile Lys Tyr Gly Asp Ala His Val Ser Glu Asp Phe Thr Trp Glu Lys

210 215 220

Pro Pro Glu Asp Pro Pro Asp Ser Gly Ser Gly Gly Gly Gly Ser Gly

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro

245 250 255

Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

260 265 270

Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr

275 280 285

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

290 295 300

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

305 310 315 320

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

325 330 335

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

340 345 350

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

355 360 365

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

370 375 380

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

385 390 395 400

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

405 410 415

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

420 425 430

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

435 440 445

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu

450 455 460

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly

465 470 475 480

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

485 490 495

Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn

500 505 510

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

515 520 525

Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu

530 535 540

Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn

545 550 555 560

Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val

565 570 575

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

580 585 590

Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys

595 600 605

Gln Ser Ile Ile Ser Thr Leu Thr

610 615

<210> 16

<211> 1437

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotides encoding fusion protein (GI101C1)

<400> 16

atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg

60

tctccttctc acgctgtgat ccacgtgacc aaagaagtga aagaggtcgc cacactgtcc

120

tgcggccaca acgtttcagt ggaagaactg gcccagacca ggatctactg gcagaaagaa

180

aagaaaatgg tgctgaccat gatgtccggc gacatgaaca tctggcctga gtacaagaac

240

cggaccatct tcgacatcac caacaacctg tccatcgtga ttctggccct gaggccttct

300

gatgagggca cctatgagtg cgtggtgctg aagtacgaga aggacgcctt caagcgcgag

360

cacctggctg aagtgacact gtccgtgaag gccgactttc cccacaccttc catctccgac

420

ttcgagatcc ctacctccaa catccggcgg atcatctgtt ctacctctgg cggctttcct

480

gagcctcacc tgtcttggct ggaaaacggc gaggaactga acgccatcaa caccaccgtg

540

tctcaggacc ccgaaaccga gctgtacgct gtgtcctcca agctggactt caacatgacc

600

accaaccaca gcttcatgtg cctgattaag tacggccacc tgagagtgaa ccagaccttc

660

aactggaaca ccaccaagca agagcacttc cctgacaatg gatctggcgg cggaggttct

720

ggcggaggtg gaagcggagg cggaggatct gctgagtcta agtatggccc tccttgtcct

780

ccatgtcctg ctccagaagc tgctggcgga ccctctgtgt tcctgtttcc tccaaagcct

840

aaggaccagc tcatgatctc tcggacaccc gaagtgacct gcgtggtggt ggatgtgtct

900

caagaggacc ctgaggtgca gttcaattgg tacgtggacg gcgtggaagt gcacaacgcc

960

aagaccaagc ctagagagga acagttcaac tccacctaca gagtggtgtc cgtgctgacc

1020

gtgctgcacc aggattggct gaacggcaaa gagtacaagt gcaaggtgtc caacaagggc

1080

ctgccttcca gcatcgaaaa gaccatctcc aaggctaagg gccagcctag ggaaccccag

1140

gtttacaccc tgcctccaag ccaagaggaa atgaccaaga accaggtgtc cctgacctgc

1200

ctggtcaagg gcttctaccc ttccgacatt gccgtggaat gggagtccaa tggccagcct

1260

gagaacaact acaagaccac acctcctgtg ctggactccg acggctcctt ctttctgtac

1320

tctcgcctga ccgtggacaa gtctaggtgg caagagggca acgtgttctc ctgctctgtg

1380

ctgcacgagg ccctgcacaa tcactacacc cagaagtccc tgtctctgtc cctgggc

1437

<210> 17

<211> 454

<212> PRT

<213> Artificial sequence

<220>

<223> fusion protein (GI101C1)

<400> 17

Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys

1 5 10 15

Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp

20 25 30

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

35 40 45

Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn

50 55 60

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

65 70 75 80

Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His

85 90 95

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

100 105 110

Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys

115 120 125

Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn

130 135 140

Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu

145 150 155 160

Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr

165 170 175

Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn

180 185 190

Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn

195 200 205

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

210 215 220

Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

325 330 335

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

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

355 360 365

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

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Leu Gly

450

<210> 18

<211> 1176

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotides encoding fusion protein (GI101C2)

<400> 18

atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg

60

tctccatctc acgccgctga gtctaagtac ggccctcctt gtcctccatg tcctgctcca

120

gaagctgctg gcggaccctc tgtgttcctg tttcctccaa agcctaagga ccagctcatg

180

atctctcgga cccctgaagt gacctgcgtg gtggtggatg tgtctcaaga ggaccctgag

240

gtgcagttca attggtacgt ggacggcgtg gaagtgcaca acgccaagac caagcctaga

300

gaggaacagt tcaactccac ctacagagtg gtgtccgtgc tgaccgtgct gcaccaggat

360

tggctgaacg gcaaagagta caagtgcaag gtgtccaaca agggcctgcc ttccagcatc

420

gaaaagacca tctccaaggc taagggccag cctagggaac cccaggttta caccctgcct

480

ccaagccaag aggaaatgac caagaaccag gtgtccctga cctgcctggt caagggcttc

540

tacccttccg acattgccgt ggaatgggag tccaatggcc agcctgagaa caactacaag

600

accacacctc ctgtgctgga ctccgacggc tccttctttc tgtactctcg cctgaccgtg

660

gacaagtcta ggtggcaaga gggcaacgtg ttctcctgct ctgtgctgca cgaggccctg

720

cacaatcact acacccagaa gtccctgtct ctgtctcttg gcggaggcgg aggatctgct

780

cctacctcca gctccaccaa gaaaacccag ctccagttgg agcatctgct gctggacctc

840

cagatgatcc tgaatggcat caacaattac aagaacccca agctgaccgc catgctgacc

900

gctaagttct acatgcccaa gaaggccacc gagctgaagc acctccagtg cctggaagag

960

gaactgaagc ccctggaaga agtgctgaat ctggcccagt ccaagaactt ccacctgagg

1020

cctagggacc tgatctccaa catcaacgtg atcgtgctgg aactgaaagg ctccgagaca

1080

accttcatgt gcgagtacgc cgacgagaca gccaccatcg tggaatttct gaaccggtgg

1140

atcaccttct gccagtccat catctccaca ctgacc

1176

<210> 19

<211> 367

<212> PRT

<213> Artificial sequence

<220>

<223> fusion protein (GI101C2)

<400> 19

Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu

1 5 10 15

Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

20 25 30

Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

35 40 45

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

50 55 60

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

65 70 75 80

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

85 90 95

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

100 105 110

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

115 120 125

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

130 135 140

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

145 150 155 160

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

165 170 175

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg

180 185 190

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

195 200 205

Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

210 215 220

Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Ala

260 265 270

Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 20

<211> 1434

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotides encoding fusion protein (mGI101C1)

<400> 20

atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg

60

tctccttctc acgctgtgga cgagcagctc tccaagtccg tgaaggataa ggtcctgctg

120

ccttgccggt acaactctcc tcacgaggac gagtctgagg accggatcta ctggcagaaa

180

cacgacaagg tggtgctgtc cgtgatcgcc ggaaagctga aagtgtggcc tgagtacaag

240

aacaggaccc tgtacgacaa caccacctac agcctgatca tcctgggcct cgtgctgagc

300

gatagaggca cctattcttg cgtggtgcag aagaaagagc ggggcaccta cgaagtgaag

360

cacctggctc tggtcaagct gtccatcaag gccgacttca gcacccctaa catcaccgag

420

tctggcaacc cttccgccga caccaagaga atcacctgtt tcgcctctgg cggcttccct

480

aagcctcggt tctcttggct ggaaaacggc agagagctgc ccggcatcaa taccaccatt

540

tctcaggacc cagagtccga gctgtacacc atctccagcc agctcgactt taacaccacc

600

agaaaccaca ccatcaagtg cctgattaag tacggcgacg cccacgtgtc cgaggacttt

660

acttgggaga aacctcctga ggaccctcct gactctggat ctggcggcgg aggttctggc

720

ggaggtggaa gcggaggcgg aggatctgct gagtctaagt atggccctcc ttgtcctcca

780

tgtcctgctc cagaagctgc tggcggaccc tctgtgttcc tgtttcctcc aaagcctaag

840

gaccagctca tgatctctcg gacccctgaa gtgacctgcg tggtggtgga tgtgtctcaa

900

gaggaccctg aggtgcagtt caattggtac gtggacggcg tggaagtgca caacgccaag

960

accaagccta gagaggaaca gttcaactcc acctatagag tggtgtccgt gctgaccgtg

1020

ctgcaccagg attggctgaa cggcaaagag tacaagtgca aggtgtccaa caagggcctg

1080

ccttccagca tcgaaaagac catcagcaag gctaagggcc agcctaggga accccaggtt

1140

tacaccctgc ctccaagcca agaggaaatg accaagaacc aggtgtccct gacctgcctg

1200

gtcaagggct tctacccttc cgacattgcc gtggaatggg agtccaatgg ccagcctgag

1260

aacaactaca agaccacacc tcctgtgctg gactccgacg gctccttctt tctgtactct

1320

cgcctgaccg tggacaagtc taggtggcaa gagggcaacg tgttctcctg ctctgtgctg

1380

cacgaggctc tgcacaacca ctacacccag aagtccctgt ctctgtccct gggc

1434

<210> 21

<211> 478

<212> PRT

<213> Artificial sequence

<220>

<223> fusion protein (mGI101C1)

<400> 21

Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

1 5 10 15

Ala Val Phe Val Ser Pro Ser His Ala Val Asp Glu Gln Leu Ser Lys

20 25 30

Ser Val Lys Asp Lys Val Leu Leu Pro Cys Arg Tyr Asn Ser Pro His

35 40 45

Glu Asp Glu Ser Glu Asp Arg Ile Tyr Trp Gln Lys His Asp Lys Val

50 55 60

Val Leu Ser Val Ile Ala Gly Lys Leu Lys Val Trp Pro Glu Tyr Lys

65 70 75 80

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

85 90 95

Leu Val Leu Ser Asp Arg Gly Thr Tyr Ser Cys Val Val Gln Lys Lys

100 105 110

Glu Arg Gly Thr Tyr Glu Val Lys His Leu Ala Leu Val Lys Leu Ser

115 120 125

Ile Lys Ala Asp Phe Ser Thr Pro Asn Ile Thr Glu Ser Gly Asn Pro

130 135 140

Ser Ala Asp Thr Lys Arg Ile Thr Cys Phe Ala Ser Gly Gly Phe Pro

145 150 155 160

Lys Pro Arg Phe Ser Trp Leu Glu Asn Gly Arg Glu Leu Pro Gly Ile

165 170 175

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

180 185 190

Ser Gln Leu Asp Phe Asn Thr Thr Arg Asn His Thr Ile Lys Cys Leu

195 200 205

Ile Lys Tyr Gly Asp Ala His Val Ser Glu Asp Phe Thr Trp Glu Lys

210 215 220

Pro Pro Glu Asp Pro Pro Asp Ser Gly Ser Gly Gly Gly Gly Ser Gly

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro

245 250 255

Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

260 265 270

Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr

275 280 285

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

290 295 300

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

305 310 315 320

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

325 330 335

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

340 345 350

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

355 360 365

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

370 375 380

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

385 390 395 400

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

405 410 415

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

420 425 430

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

435 440 445

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu

450 455 460

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

465 470 475

<210> 22

<211> 133

<212> PRT

<213> Artificial sequence

<220>

<223> IL-2 variants (3M, M45)

<400> 22

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Ala Met Leu Thr Ala Lys Phe Ala Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 23

<211> 133

<212> PRT

<213> Artificial sequence

<220>

<223> IL-2 variants (3M, M61)

<400> 23

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

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

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 24

<211> 133

<212> PRT

<213> Artificial sequence

<220>

<223> IL-2 variants (3M, M72)

<400> 24

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

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

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Gly Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 25

<211> 1851

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotides encoding fusion protein (GI102-M45)

<400> 25

atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg

60

tctccttctc acgctgtgat ccacgtgacc aaagaagtga aagaggtcgc cacactgtcc

120

tgcggccaca acgtttcagt ggaagaactg gcccagacca ggatctactg gcagaaagaa

180

aagaaaatgg tgctgaccat gatgtccggc gacatgaaca tctggcctga gtacaagaac

240

cggaccatct tcgacatcac caacaacctg tccatcgtga ttctggccct gaggccttct

300

gatgagggca cctatgagtg cgtggtgctg aagtacgaga aggacgcctt caagcgcgag

360

cacctggctg aagtgacact gtccgtgaag gccgactttc cccacaccttc catctccgac

420

ttcgagatcc ctacctccaa catccggcgg atcatctgtt ctacctctgg cggctttcct

480

gagcctcacc tgtcttggct ggaaaacggc gaggaactga acgccatcaa caccaccgtg

540

tctcaggacc ccgaaaccga gctgtacgct gtgtcctcca agctggactt caacatgacc

600

accaaccaca gcttcatgtg cctgattaag tacggccacc tgagagtgaa ccagaccttc

660

aactggaaca ccaccaagca agagcacttc cctgacaatg gatctggcgg cggaggttct

720

ggcggaggtg gaagcggagg cggaggatct gctgagtcta agtatggccc tccttgtcct

780

ccatgtcctg ctccagaagc tgctggcgga ccctctgtgt tcctgtttcc tccaaagcct

840

aaggaccagc tcatgatctc tcggacaccc gaagtgacct gcgtggtggt ggatgtgtct

900

caagaggacc ctgaggtgca gttcaattgg tacgtggacg gcgtggaagt gcacaacgcc

960

aagaccaagc ctagagagga acagttcaac tccacctaca gagtggtgtc cgtgctgacc

1020

gtgctgcacc aggattggct gaacggcaaa gagtacaagt gcaaggtgtc caacaagggc

1080

ctgccttcca gcatcgaaaa gaccatctcc aaggctaagg gccagcctag ggaaccccag

1140

gtttacaccc tgcctccaag ccaagaggaa atgaccaaga accaggtgtc cctgacctgc

1200

ctggtcaagg gcttctaccc ttccgacatt gccgtggaat gggagtccaa tggccagcct

1260

gagaacaact acaagaccac acctcctgtg ctggactccg acggctcctt ctttctgtac

1320

tctcgcctga ccgtggacaa gtctagatgg caagagggca acgtgttctc ctgctctgtg

1380

ctgcacgagg ccctgcacaa tcactacacc cagaagtccc tgtctctgtc tcttggaggt

1440

ggtggcggtt ctgcccctac cagctcctct accaagaaaa cccagctcca gttggagcat

1500

ctgctgctgg acctccagat gattctgaac gggatcaaca actataagaa ccccaagctg

1560

accgccatgc tgaccgctaa gttcgccatg cccaagaagg ccaccgagct gaagcacctc

1620

cagtgcctgg aagaagaact gaagcccctg gaagaggtgc tgaatctggc ccagtccaag

1680

aacttccacc tgaggccacg ggacctgatc agcaacatca acgtgatcgt gctggaactg

1740

aagggctccg agacaacctt tatgtgcgag tacgccgacg agacagccac catcgtggaa

1800

tttctgaacc ggtggatcac cttctgccag agcatcatct ccacactgac c

1851

<210> 26

<211> 592

<212> PRT

<213> Artificial sequence

<220>

<223> fusion protein (GI102-M45)

<400> 26

Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys

1 5 10 15

Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp

20 25 30

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

35 40 45

Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn

50 55 60

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

65 70 75 80

Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His

85 90 95

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

100 105 110

Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys

115 120 125

Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn

130 135 140

Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu

145 150 155 160

Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr

165 170 175

Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn

180 185 190

Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn

195 200 205

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

210 215 220

Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

325 330 335

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

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

355 360 365

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

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser

450 455 460

Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu

465 470 475 480

Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr

485 490 495

Ala Met Leu Thr Ala Lys Phe Ala Met Pro Lys Lys Ala Thr Glu Leu

500 505 510

Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val

515 520 525

Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu

530 535 540

Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr

545 550 555 560

Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe

565 570 575

Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr

580 585 590

<210> 27

<211> 1851

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotides encoding fusion protein (GI102-M61)

<400> 27

atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg

60

tctccttctc acgctgtgat ccacgtgacc aaagaagtga aagaggtcgc cacactgtcc

120

tgcggccaca acgtttcagt ggaagaactg gcccagacca ggatctactg gcagaaagaa

180

aagaaaatgg tgctgaccat gatgtccggc gacatgaaca tctggcctga gtacaagaac

240

cggaccatct tcgacatcac caacaacctg tccatcgtga ttctggccct gaggccttct

300

gatgagggca cctatgagtg cgtggtgctg aagtacgaga aggacgcctt caagcgcgag

360

cacctggctg aagtgacact gtccgtgaag gccgactttc cccacaccttc catctccgac

420

ttcgagatcc ctacctccaa catccggcgg atcatctgtt ctacctctgg cggctttcct

480

gagcctcacc tgtcttggct ggaaaacggc gaggaactga acgccatcaa caccaccgtg

540

tctcaggacc ccgaaaccga gctgtacgct gtgtcctcca agctggactt caacatgacc

600

accaaccaca gcttcatgtg cctgattaag tacggccacc tgagagtgaa ccagaccttc

660

aactggaaca ccaccaagca agagcacttc cctgacaatg gatctggcgg cggaggttct

720

ggcggaggtg gaagcggagg cggaggatct gctgagtcta agtatggccc tccttgtcct

780

ccatgtcctg ctccagaagc tgctggcgga ccctctgtgt tcctgtttcc tccaaagcct

840

aaggaccagc tcatgatctc tcggacaccc gaagtgacct gcgtggtggt ggatgtgtct

900

caagaggacc ctgaggtgca gttcaattgg tacgtggacg gcgtggaagt gcacaacgcc

960

aagaccaagc ctagagagga acagttcaac tccacctaca gagtggtgtc cgtgctgacc

1020

gtgctgcacc aggattggct gaacggcaaa gagtacaagt gcaaggtgtc caacaagggc

1080

ctgccttcca gcatcgaaaa gaccatctcc aaggctaagg gccagcctag ggaaccccag

1140

gtttacaccc tgcctccaag ccaagaggaa atgaccaaga accaggtgtc cctgacctgc

1200

ctggtcaagg gcttctaccc ttccgacatt gccgtggaat gggagtccaa tggccagcct

1260

gagaacaact acaagaccac acctcctgtg ctggactccg acggctcctt ctttctgtac

1320

tctcgcctga ccgtggacaa gtctagatgg caagagggca acgtgttctc ctgctctgtg

1380

ctgcacgagg ccctgcacaa tcactacacc cagaagtccc tgtctctgtc tcttggaggt

1440

ggtggcggtt ctgcccctac cagctcctct accaagaaaa cccagctcca gttggagcat

1500

ctgctgctgg acctccagat gattctgaac gggatcaaca actataagaa ccccaagctg

1560

accgccatgc tgaccgctaa gttctacatg cccaagaagg ccaccgagct gaagcacctc

1620

cagtgcctgg aaagggaact gaagcccctg gaagaggtgc tgaatctggc ccagtccaag

1680

aacttccacc tgaggccacg ggacctgatc agcaacatca acgtgatcgt gctggaactg

1740

aagggctccg agacaacctt tatgtgcgag tacgccgacg agacagccac catcgtggaa

1800

tttctgaacc ggtggatcac cttctgccag agcatcatct ccacactgac c

1851

<210> 28

<211> 592

<212> PRT

<213> Artificial sequence

<220>

<223> fusion protein (GI102-M61)

<400> 28

Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys

1 5 10 15

Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp

20 25 30

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

35 40 45

Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn

50 55 60

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

65 70 75 80

Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His

85 90 95

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

100 105 110

Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys

115 120 125

Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn

130 135 140

Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu

145 150 155 160

Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr

165 170 175

Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn

180 185 190

Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn

195 200 205

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

210 215 220

Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

325 330 335

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

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

355 360 365

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

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser

450 455 460

Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu

465 470 475 480

Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr

485 490 495

Ala Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu

500 505 510

Lys His Leu Gln Cys Leu Glu Arg Glu Leu Lys Pro Leu Glu Glu Val

515 520 525

Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu

530 535 540

Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr

545 550 555 560

Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe

565 570 575

Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr

580 585 590

<210> 29

<211> 1857

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotides encoding fusion protein (GI102-M72)

<400> 29

atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg

60

tctccttctc acgctgtgat ccacgtgacc aaagaagtga aagaggtcgc cacactgtcc

120

tgcggccaca acgtttcagt ggaagaactg gcccagacca ggatctactg gcagaaagaa

180

aagaaaatgg tgctgaccat gatgtccggc gacatgaaca tctggcctga gtacaagaac

240

cggaccatct tcgacatcac caacaacctg tccatcgtga ttctggccct gaggccttct

300

gatgagggca cctatgagtg cgtggtgctg aagtacgaga aggacgcctt caagcgcgag

360

cacctggctg aagtgacact gtccgtgaag gccgactttc cccacaccttc catctccgac

420

ttcgagatcc ctacctccaa catccggcgg atcatctgtt ctacctctgg cggctttcct

480

gagcctcacc tgtcttggct ggaaaacggc gaggaactga acgccatcaa caccaccgtg

540

tctcaggacc ccgaaaccga gctgtacgct gtgtcctcca agctggactt caacatgacc

600

accaaccaca gcttcatgtg cctgattaag tacggccacc tgagagtgaa ccagaccttc

660

aactggaaca ccaccaagca agagcacttc cctgacaatg gatctggcgg cggaggttct

720

ggcggaggtg gaagcggagg cggaggatct gctgagtcta agtatggccc tccttgtcct

780

ccatgtcctg ctccagaagc tgctggcgga ccctctgtgt tcctgtttcc tccaaagcct

840

aaggaccagc tcatgatctc tcggacaccc gaagtgacct gcgtggtggt ggatgtgtct

900

caagaggacc ctgaggtgca gttcaattgg tacgtggacg gcgtggaagt gcacaacgcc

960

aagaccaagc ctagagagga acagttcaac tccacctaca gagtggtgtc cgtgctgacc

1020

gtgctgcacc aggattggct gaacggcaaa gagtacaagt gcaaggtgtc caacaagggc

1080

ctgccttcca gcatcgaaaa gaccatctcc aaggctaagg gccagcctag ggaaccccag

1140

gtttacaccc tgcctccaag ccaagaggaa atgaccaaga accaggtgtc cctgacctgc

1200

ctggtcaagg gcttctaccc ttccgacatt gccgtggaat gggagtccaa tggccagcct

1260

gagaacaact acaagaccac acctcctgtg ctggactccg acggctcctt ctttctgtac

1320

tctcgcctga ccgtggacaa gtctagatgg caagagggca acgtgttctc ctgctctgtg

1380

ctgcacgagg ccctgcacaa tcactacacc cagaagtccc tgtctctgtc tcttggaggt

1440

ggtggcggtt ctgcccctac cagctcctct accaagaaaa cccagctcca gttggagcat

1500

ctgctgctgg acctccagat gattctgaac gggatcaaca actataagaa ccccaagctg

1560

accgccatgc tgaccgctaa gttctacatg cccaagaagg ccaccgagct gaagcacctc

1620

cagtgcctgg aagaagaact gaagcccctg gaagaggtgc tgaatggggc ccagtccaag

1680

aacttccacc tgaggccacg ggacctgatc agcaacatca acgtgatcgt gctggaactg

1740

aagggctccg agacaacctt tatgtgcgag tacgccgacg agacagccac catcgtggaa

1800

tttctgaacc ggtggatcac cttctgccag agcatcatct cccacactgac ctgatga

1857

<210> 30

<211> 592

<212> PRT

<213> Artificial sequence

<220>

<223> fusion protein (GI102-M72)

<400> 30

Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys

1 5 10 15

Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp

20 25 30

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

35 40 45

Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn

50 55 60

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

65 70 75 80

Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His

85 90 95

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

100 105 110

Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys

115 120 125

Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn

130 135 140

Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu

145 150 155 160

Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr

165 170 175

Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn

180 185 190

Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn

195 200 205

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

210 215 220

Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

325 330 335

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

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

355 360 365

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

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser

450 455 460

Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu

465 470 475 480

Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr

485 490 495

Ala Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu

500 505 510

Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val

515 520 525

Leu Asn Gly Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu

530 535 540

Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr

545 550 555 560

Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe

565 570 575

Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr

580 585 590

<210> 31

<211> 1851

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotides encoding fusion protein (GI101w)

<400> 31

atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg

60

tctccttctc acgctgtgat ccacgtgacc aaagaagtga aagaggtcgc cacactgtcc

120

tgcggccaca acgtttcagt ggaagaactg gcccagacca ggatctactg gcagaaagaa

180

aagaaaatgg tgctgaccat gatgtccggc gacatgaaca tctggcctga gtacaagaac

240

cggaccatct tcgacatcac caacaacctg tccatcgtga ttctggccct gaggccttct

300

gatgagggca cctatgagtg cgtggtgctg aagtacgaga aggacgcctt caagcgcgag

360

cacctggctg aagtgacact gtccgtgaag gccgactttc cccacaccttc catctccgac

420

ttcgagatcc ctacctccaa catccggcgg atcatctgtt ctacctctgg cggctttcct

480

gagcctcacc tgtcttggct ggaaaacggc gaggaactga acgccatcaa caccaccgtg

540

tctcaggacc ccgaaaccga gctgtacgct gtgtcctcca agctggactt caacatgacc

600

accaaccaca gcttcatgtg cctgattaag tacggccacc tgagagtgaa ccagaccttc

660

aactggaaca ccaccaagca agagcacttc cctgacaatg gatctggcgg cggaggttct

720

ggcggaggtg gaagcggagg cggaggatct gctgagtcta agtatggccc tccttgtcct

780

ccatgtcctg ctccagaagc tgctggcgga ccctctgtgt tcctgtttcc tccaaagcct

840

aaggaccagc tcatgatctc tcggacaccc gaagtgacct gcgtggtggt ggatgtgtct

900

caagaggacc ctgaggtgca gttcaattgg tacgtggacg gcgtggaagt gcacaacgcc

960

aagaccaagc ctagagagga acagttcaac tccacctaca gagtggtgtc cgtgctgacc

1020

gtgctgcacc aggattggct gaacggcaaa gagtacaagt gcaaggtgtc caacaagggc

1080

ctgccttcca gcatcgaaaa gaccatctcc aaggctaagg gccagcctag ggaaccccag

1140

gtttacaccc tgcctccaag ccaagaggaa atgaccaaga accaggtgtc cctgacctgc

1200

ctggtcaagg gcttctaccc ttccgacatt gccgtggaat gggagtccaa tggccagcct

1260

gagaacaact acaagaccac acctcctgtg ctggactccg acggctcctt ctttctgtac

1320

tctcgcctga ccgtggacaa gtctagatgg caagagggca acgtgttctc ctgctctgtg

1380

ctgcacgagg ccctgcacaa tcactacacc cagaagtccc tgtctctgtc tcttggaggt

1440

ggtggcggtt ctgcccctac cagctcctct accaagaaaa cccagctcca gttggagcat

1500

ctgctgctgg acctccagat gattctgaac gggatcaaca actataagaa ccccaagctg

1560

acccgcatgc tgacctttaa gttctacatg cccaagaagg ccaccgagct gaagcacctc

1620

cagtgcctgg aagaagaact gaagcccctg gaagaggtgc tgaatctggc ccagtccaag

1680

aacttccacc tgaggccacg ggacctgatc agcaacatca acgtgatcgt gctggaactg

1740

aagggctccg agacaacctt tatgtgcgag tacgccgacg agacagccac catcgtggaa

1800

tttctgaacc ggtggatcac cttctgccag agcatcatct ccacactgac c

1851

<210> 32

<211> 592

<212> PRT

<213> Artificial sequence

<220>

<223> fusion protein (GI101w)

<400> 32

Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys

1 5 10 15

Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp

20 25 30

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

35 40 45

Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn

50 55 60

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

65 70 75 80

Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His

85 90 95

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

100 105 110

Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys

115 120 125

Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn

130 135 140

Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu

145 150 155 160

Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr

165 170 175

Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn

180 185 190

Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn

195 200 205

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

210 215 220

Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

325 330 335

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

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

355 360 365

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

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser

450 455 460

Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu

465 470 475 480

Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr

485 490 495

Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu

500 505 510

Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val

515 520 525

Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu

530 535 540

Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr

545 550 555 560

Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe

565 570 575

Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr

580 585 590

<210> 33

<211> 1848

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotides encoding fusion protein (mGI102-M61)

<400> 33

atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg

60

tctccttctc acgctgtgga cgagcagctc tccaagtccg tgaaggataa ggtcctgctg

120

ccttgccggt acaactctcc tcacgaggac gagtctgagg accggatcta ctggcagaaa

180

cacgacaagg tggtgctgtc cgtgatcgcc ggaaagctga aagtgtggcc tgagtacaag

240

aacaggaccc tgtacgacaa caccacctac agcctgatca tcctgggcct cgtgctgagc

300

gatagaggca cctattcttg cgtggtgcag aagaaagagc ggggcaccta cgaagtgaag

360

cacctggctc tggtcaagct gtccatcaag gccgacttca gcacccctaa catcaccgag

420

tctggcaacc cttccgccga caccaagaga atcacctgtt tcgcctctgg cggcttccct

480

aagcctcggt tctcttggct ggaaaacggc agagagctgc ccggcatcaa taccaccatt

540

tctcaggacc cagagtccga gctgtacacc atctccagcc agctcgactt taacaccacc

600

agaaaccaca ccatcaagtg cctgattaag tacggcgacg cccacgtgtc cgaggacttt

660

acttgggaga aacctcctga ggaccctcct gactctggat ctggcggcgg aggttctggc

720

ggaggtggaa gcggaggcgg aggatctgct gagtctaagt atggccctcc ttgtcctcca

780

tgtcctgctc cagaagctgc tggcggaccc tctgtgttcc tgtttcctcc aaagcctaag

840

gaccagctca tgatctctcg gacccctgaa gtgacctgcg tggtggtgga tgtgtctcaa

900

gaggaccctg aggtgcagtt caattggtac gtggacggcg tggaagtgca caacgccaag

960

accaagccta gagaggaaca gttcaactcc acctatagag tggtgtccgt gctgaccgtg

1020

ctgcaccagg attggctgaa cggcaaagag tacaagtgca aggtgtccaa caagggcctg

1080

ccttccagca tcgaaaagac catcagcaag gctaagggcc agcctaggga accccaggtt

1140

tacaccctgc ctccaagcca agaggaaatg accaagaacc aggtgtccct gacctgcctg

1200

gtcaagggct tctacccttc cgacattgcc gtggaatggg agtccaatgg ccagcctgag

1260

aacaactaca agaccacacc tcctgtgctg gactccgacg gctccttctt tctgtactct

1320

cgcctgaccg tggacaagtc taggtggcaa gagggcaacg tgttctcctg ctctgtgctg

1380

cacgaggctc tgcacaacca ctacacccag aagtccctgt ctctgtctct tggaggtggt

1440

ggcggttctg cccctacctc cagctctacc aagaaaaccc agctccagtt ggagcatctg

1500

ctgctggacc tccagatgat cctgaatggc atcaacaatt acaagaaccc caagctgacc

1560

gccatgctga ccgctaagtt ctacatgccc aagaaggcca ccgagctgaa gcacttgcag

1620

tgcctggaaa gggaactgaa gcccctggaa gaagtgctga atctggccca gtccaagaac

1680

ttccacctga ggcctaggga cctgatctcc aacatcaacg tgatcgtgct ggaactgaaa

1740

ggctccgaga caaccttcat gtgcgagtac gccgacgaga cagccaccat cgtggaattt

1800

ctgaaccggt ggatcacctt ctgccagagc atcatctcca cactgacc

1848

<210> 34

<211> 616

<212> PRT

<213> Artificial sequence

<220>

<223> fusion protein (mGI102-M61)

<400> 34

Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

1 5 10 15

Ala Val Phe Val Ser Pro Ser His Ala Val Asp Glu Gln Leu Ser Lys

20 25 30

Ser Val Lys Asp Lys Val Leu Leu Pro Cys Arg Tyr Asn Ser Pro His

35 40 45

Glu Asp Glu Ser Glu Asp Arg Ile Tyr Trp Gln Lys His Asp Lys Val

50 55 60

Val Leu Ser Val Ile Ala Gly Lys Leu Lys Val Trp Pro Glu Tyr Lys

65 70 75 80

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

85 90 95

Leu Val Leu Ser Asp Arg Gly Thr Tyr Ser Cys Val Val Gln Lys Lys

100 105 110

Glu Arg Gly Thr Tyr Glu Val Lys His Leu Ala Leu Val Lys Leu Ser

115 120 125

Ile Lys Ala Asp Phe Ser Thr Pro Asn Ile Thr Glu Ser Gly Asn Pro

130 135 140

Ser Ala Asp Thr Lys Arg Ile Thr Cys Phe Ala Ser Gly Gly Phe Pro

145 150 155 160

Lys Pro Arg Phe Ser Trp Leu Glu Asn Gly Arg Glu Leu Pro Gly Ile

165 170 175

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

180 185 190

Ser Gln Leu Asp Phe Asn Thr Thr Arg Asn His Thr Ile Lys Cys Leu

195 200 205

Ile Lys Tyr Gly Asp Ala His Val Ser Glu Asp Phe Thr Trp Glu Lys

210 215 220

Pro Pro Glu Asp Pro Pro Asp Ser Gly Ser Gly Gly Gly Gly Ser Gly

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly Pro

245 250 255

Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

260 265 270

Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr

275 280 285

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

290 295 300

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

305 310 315 320

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

325 330 335

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

340 345 350

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

355 360 365

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

370 375 380

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

385 390 395 400

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

405 410 415

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

420 425 430

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

435 440 445

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu

450 455 460

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly

465 470 475 480

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

485 490 495

Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn

500 505 510

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

515 520 525

Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Arg

530 535 540

Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn

545 550 555 560

Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val

565 570 575

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

580 585 590

Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys

595 600 605

Gln Ser Ile Ile Ser Thr Leu Thr

610 615

<210> 35

<211> 153

<212> PRT

<213> Artificial sequence

<220>

<223> wild type hIL-2

<400> 35

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu

20 25 30

Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile

35 40 45

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

50 55 60

Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu

65 70 75 80

Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

85 90 95

Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile

100 105 110

Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala

115 120 125

Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe

130 135 140

Cys Gln Ser Ile Ile Ser Thr Leu Thr

145 150

<210> 36

<211> 158

<212> PRT

<213> Artificial sequence

<220>

<223> IL-2 with signal sequence

<400> 36

Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

1 5 10 15

Ala Val Phe Val Ser Pro Ser His Ala Ala Pro Thr Ser Ser Ser Thr

20 25 30

Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met

35 40 45

Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met

50 55 60

Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His

65 70 75 80

Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn

85 90 95

Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser

100 105 110

Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe

115 120 125

Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn

130 135 140

Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr

145 150 155

<210> 37

<211> 474

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotide sequence encoding IL-2 with signal

sequence

<400> 37

atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg

60

tctccttctc acgctgcccc taccagctcc tctaccaaga aaacccagct ccagttggag

120

catctgctgc tggacctcca gatgattctg aacgggatca acaactataa gaaccccaag

180

ctgacccgca tgctgacctt taagttctac atgcccaaga aggccaccga gctgaagcac

240

ctccagtgcc tggaagaaga actgaagccc ctggaagagg tgctgaatct ggcccagtcc

300

aagaacttcc acctgaggcc acgggacctg atcagcaaca tcaacgtgat cgtgctggaa

360

ctgaagggct ccgagacaac ctttatgtgc gagtacgccg acgagacagc caccatcgtg

420

gaatttctga accggtggat caccttctgc cagagcatca tctccacact gacc

474

<210> 38

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> hinge

<400> 38

Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro

1 5 10

<210> 39

<211> 1461

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotides encoding CD80-Fc protein

<400> 39

ggatccgcca ccatggatgc tatgctgaga ggcctgtgtt gcgtgctgct gctgtgtggc

60

gctgtgttcg tgtctccttc tcacgctgtg atccacgtga ccaaagaagt gaaagaggtc

120

gccacactgt cctgcggcca caacgtttca gtggaagaac tggcccagac caggatctac

180

tggcagaaag aaaagaaaat ggtgctgacc atgatgtccg gcgacatgaa catctggcct

240

gagtacaaga accggaccat cttcgacatc accaacaacc tgtccatcgt gattctggcc

300

ctgaggcctt ctgatgaggg cacctatgag tgcgtggtgc tgaagtacga gaaggacgcc

360

ttcaagcgcg agcacctggc tgaagtgaca ctgtccgtga aggccgactt tcccacacct

420

tccatctccg acttcgagat ccctacctcc aacatccggc ggatcatctg ttctacctct

480

ggcggctttc ctgagcctca cctgtcttgg ctggaaaacg gcgaggaact gaacgccatc

540

aacaccaccg tgtctcagga ccccgaaacc gagctgtacg ctgtgtcctc caagctggac

600

ttcaacatga ccaccaacca cagcttcatg tgcctgatta agtacggcca cctgagagtg

660

aaccagacct tcaactggaa caccaccaag caagagcact tccctgacaa tggatctggc

720

ggcggaggtt ctggcggagg tggaagcgga ggcggaggat ctgctgagtc taagtatggc

780

cctccttgtc ctccatgtcc tgctccagaa gctgctggcg gaccctctgt gttcctgttt

840

cctccaaagc ctaaggacca gctcatgatc tctcggacac ccgaagtgac ctgcgtggtg

900

gtggatgtgt ctcaagagga ccctgaggtg cagttcaatt ggtacgtgga cggcgtggaa

960

gtgcacaacg ccaagaccaa gcctagagag gaacagttca actccaccta cagagtggtg

1020

tccgtgctga ccgtgctgca ccaggattgg ctgaacggca aagagtacaa gtgcaaggtg

1080

tccaacaagg gcctgccttc cagcatcgaa aagaccatct ccaaggctaa gggccagcct

1140

agggaacccc aggtttacac cctgcctcca agccaagagg aaatgaccaa gaaccaggtg

1200

tccctgacct gcctggtcaa gggcttctac ccttccgaca ttgccgtgga atgggagtcc

1260

aatggccagc ctgagaacaa ctacaagacc acacctcctg tgctggactc cgacggctcc

1320

ttctttctgt actctcgcct gaccgtggac aagtctaggt ggcaagaggg caacgtgttc

1380

tcctgctctg tgctgcacga ggccctgcac aatcactaca cccagaagtc cctgtctctg

1440

tccctgggct gatgactcga g

1461

<210> 40

<211> 479

<212> PRT

<213> Artificial sequence

<220>

<223>CD80-Fc protein

<400> 40

Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

1 5 10 15

Ala Val Phe Val Ser Pro Ser His Ala Val Ile His Val Thr Lys Glu

20 25 30

Val Lys Glu Val Ala Thr Leu Ser Cys Gly His Asn Val Ser Val Glu

35 40 45

Glu Leu Ala Gln Thr Arg Ile Tyr Trp Gln Lys Glu Lys Lys Met Val

50 55 60

Leu Thr Met Met Ser Gly Asp Met Asn Ile Trp Pro Glu Tyr Lys Asn

65 70 75 80

Arg Thr Ile Phe Asp Ile Thr Asn Asn Leu Ser Ile Val Ile Leu Ala

85 90 95

Leu Arg Pro Ser Asp Glu Gly Thr Tyr Glu Cys Val Val Leu Lys Tyr

100 105 110

Glu Lys Asp Ala Phe Lys Arg Glu His Leu Ala Glu Val Thr Leu Ser

115 120 125

Val Lys Ala Asp Phe Pro Thr Pro Ser Ile Ser Asp Phe Glu Ile Pro

130 135 140

Thr Ser Asn Ile Arg Arg Ile Ile Cys Ser Thr Ser Gly Gly Phe Pro

145 150 155 160

Glu Pro His Leu Ser Trp Leu Glu Asn Gly Glu Glu Leu Asn Ala Ile

165 170 175

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

180 185 190

Ser Lys Leu Asp Phe Asn Met Thr Thr Asn His Ser Phe Met Cys Leu

195 200 205

Ile Lys Tyr Gly His Leu Arg Val Asn Gln Thr Phe Asn Trp Asn Thr

210 215 220

Thr Lys Gln Glu His Phe Pro Asp Asn Gly Ser Gly Gly Gly Gly Ser

225 230 235 240

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys Tyr Gly

245 250 255

Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser

260 265 270

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg

275 280 285

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro

290 295 300

Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

305 310 315 320

Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val

325 330 335

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

340 345 350

Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

420 425 430

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

435 440 445

Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala

450 455 460

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly

465 470 475

<210> 41

<211> 1851

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotides encoding fusion protein (hCD80-Fc-IL2wt)

<400> 41

atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg

60

tctccttctc acgctgtgat ccacgtgacc aaagaagtga aagaggtcgc cacactgtcc

120

tgcggccaca acgtttcagt ggaagaactg gcccagacca ggatctactg gcagaaagaa

180

aagaaaatgg tgctgaccat gatgtccggc gacatgaaca tctggcctga gtacaagaac

240

cggaccatct tcgacatcac caacaacctg tccatcgtga ttctggccct gaggccttct

300

gatgagggca cctatgagtg cgtggtgctg aagtacgaga aggacgcctt caagcgcgag

360

cacctggctg aagtgacact gtccgtgaag gccgactttc cccacaccttc catctccgac

420

ttcgagatcc ctacctccaa catccggcgg atcatctgtt ctacctctgg cggctttcct

480

gagcctcacc tgtcttggct ggaaaacggc gaggaactga acgccatcaa caccaccgtg

540

tctcaggacc ccgaaaccga gctgtacgct gtgtcctcca agctggactt caacatgacc

600

accaaccaca gcttcatgtg cctgattaag tacggccacc tgagagtgaa ccagaccttc

660

aactggaaca ccaccaagca agagcacttc cctgacaatg gatctggcgg cggaggttct

720

ggcggaggtg gaagcggagg cggaggatct gctgagtcta agtatggccc tccttgtcct

780

ccatgtcctg ctccagaagc tgctggcgga ccctctgtgt tcctgtttcc tccaaagcct

840

aaggaccagc tcatgatctc tcggacaccc gaagtgacct gcgtggtggt ggatgtgtct

900

caagaggacc ctgaggtgca gttcaattgg tacgtggacg gcgtggaagt gcacaacgcc

960

aagaccaagc ctagagagga acagttcaac tccacctaca gagtggtgtc cgtgctgacc

1020

gtgctgcacc aggattggct gaacggcaaa gagtacaagt gcaaggtgtc caacaagggc

1080

ctgccttcca gcatcgaaaa gaccatctcc aaggctaagg gccagcctag ggaaccccag

1140

gtttacaccc tgcctccaag ccaagaggaa atgaccaaga accaggtgtc cctgacctgc

1200

ctggtcaagg gcttctaccc ttccgacatt gccgtggaat gggagtccaa tggccagcct

1260

gagaacaact acaagaccac acctcctgtg ctggactccg acggctcctt ctttctgtac

1320

tctcgcctga ccgtggacaa gtctagatgg caagagggca acgtgttctc ctgctctgtg

1380

ctgcacgagg ccctgcacaa tcactacacc cagaagtccc tgtctctgtc tcttggaggt

1440

ggtggcggtt ctgcccctac cagctcctct accaagaaaa cccagctcca gttggagcat

1500

ctgctgctgg acctccagat gattctgaac gggatcaaca actataagaa ccccaagctg

1560

acccgcatgc tgacctttaa gttctacatg cccaagaagg ccaccgagct gaagcacctc

1620

cagtgcctgg aagaagaact gaagcccctg gaagaggtgc tgaatctggc ccagtccaag

1680

aacttccacc tgaggccacg ggacctgatc agcaacatca acgtgatcgt gctggaactg

1740

aagggctccg agacaacctt tatgtgcgag tacgccgacg agacagccac catcgtggaa

1800

tttctgaacc ggtggatcac cttctgccag agcatcatct ccacactgac c

1851

<210> 42

<211> 367

<212> PRT

<213> Artificial sequence

<220>

<223> Fc-IL2wt

<400> 42

Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu

1 5 10 15

Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

20 25 30

Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

35 40 45

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

50 55 60

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

65 70 75 80

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

85 90 95

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

100 105 110

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

115 120 125

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

130 135 140

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

145 150 155 160

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

165 170 175

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg

180 185 190

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

195 200 205

Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

210 215 220

Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 43

<211> 1176

<212> DNA

<213> Artificial sequence

<220>

<223> Fc-IL2wt

<400> 43

atggatgcta tgctgagagg cctgtgttgc gtgctgctgc tgtgtggcgc tgtgttcgtg

60

tctccttctc acgctgctga gtctaagtat ggccctcctt gtcctccatg tcctgctcca

120

gaagctgctg gcggaccctc tgtgttcctg tttcctccaa agcctaagga ccagctcatg

180

atctctcgga cacccgaagt gacctgcgtg gtggtggatg tgtctcaaga ggaccctgag

240

gtgcagttca attggtacgt ggacggcgtg gaagtgcaca acgccaagac caagcctaga

300

gaggaacagt tcaactccac ctacagagtg gtgtccgtgc tgaccgtgct gcaccaggat

360

tggctgaacg gcaaagagta caagtgcaag gtgtccaaca agggcctgcc ttccagcatc

420

gaaaagacca tctccaaggc taagggccag cctagggaac cccaggttta caccctgcct

480

ccaagccaag aggaaatgac caagaaccag gtgtccctga cctgcctggt caagggcttc

540

tacccttccg acattgccgt ggaatgggag tccaatggcc agcctgagaa caactacaag

600

accacacctc ctgtgctgga ctccgacggc tccttctttc tgtactctcg cctgaccgtg

660

gacaagtcta gatggcaaga gggcaacgtg ttctcctgct ctgtgctgca cgaggccctg

720

cacaatcact acacccagaa gtccctgtct ctgtctcttg gaggtggtgg cggttctgcc

780

cctaccagct cctctaccaa gaaaacccag ctccagttgg agcatctgct gctggacctc

840

cagatgattc tgaacgggat caacaactat aagaacccca agctgacccg catgctgacc

900

tttaagttct acatgcccaa gaaggccacc gagctgaagc acctccagtg cctggaagaa

960

gaactgaagc ccctggaaga ggtgctgaat ctggcccagt ccaagaactt ccacctgagg

1020

ccacgggacc tgatcagcaa catcaacgtg atcgtgctgg aactgaaggg ctccgagaca

1080

acctttatgt gcgagtacgc cgacgagaca gccaccatcg tggaatttct gaaccggtgg

1140

atcaccttct gccagagcat catctccaca ctgacc

1176

<210> 44

<211> 392

<212> PRT

<213> Artificial sequence

<220>

<223> Fc-IL2v2

<400> 44

Met Asp Ala Met Leu Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly

1 5 10 15

Ala Val Phe Val Ser Pro Ser His Ala Ala Glu Ser Lys Tyr Gly Pro

20 25 30

Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val

35 40 45

Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr

50 55 60

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

65 70 75 80

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

85 90 95

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

100 105 110

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

115 120 125

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

130 135 140

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

145 150 155 160

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

165 170 175

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

180 185 190

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

195 200 205

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

210 215 220

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu

225 230 235 240

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly

245 250 255

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

260 265 270

Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn

275 280 285

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

290 295 300

Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu

305 310 315 320

Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn

325 330 335

Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val

340 345 350

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

355 360 365

Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys

370 375 380

Gln Ser Ile Ile Ser Thr Leu Thr

385 390

<210> 45

<211> 1200

<212> DNA

<213> Artificial sequence

<220>

<223> nucleotides encoding Fc-IL2v2

<400> 45

ggatccgcca ccatggatgc tatgctgaga ggcctgtgtt gcgtgctgct gctgtgtggc

60

gctgtgttcg tgtctccatc tcacgccgct gagtctaagt acggccctcc ttgtcctcca

120

tgtcctgctc cagaagctgc tggcggaccc tctgtgttcc tgtttcctcc aaagcctaag

180

gaccagctca tgatctctcg gacccctgaa gtgacctgcg tggtggtgga tgtgtctcaa

240

gaggaccctg aggtgcagtt caattggtac gtggacggcg tggaagtgca caacgccaag

300

accaagccta gagaggaaca gttcaactcc acctacagag tggtgtccgt gctgaccgtg

360

ctgcaccagg attggctgaa cggcaaagag tacaagtgca aggtgtccaa caagggcctg

420

ccttccagca tcgaaaagac catctccaag gctaagggcc agcctaggga accccaggtt

480

tacaccctgc ctccaagcca agaggaaatg accaagaacc aggtgtccct gacctgcctg

540

gtcaagggct tctacccttc cgacattgcc gtggaatggg agtccaatgg ccagcctgag

600

aacaactaca agaccacacc tcctgtgctg gactccgacg gctccttctt tctgtactct

660

cgcctgaccg tggacaagtc taggtggcaa gagggcaacg tgttctcctg ctctgtgctg

720

cacgaggccc tgcacaatca ctacacccag aagtccctgt ctctgtctct tggcggaggc

780

ggaggatctg ctcctacctc cagctccacc aagaaaaccc agctccagtt ggagcatctg

840

ctgctggacc tccagatgat cctgaatggc atcaacaatt acaagaaccc caagctgacc

900

gccatgctga ccgctaagtt ctacatgccc aagaaggcca ccgagctgaa gcacctccag

960

tgcctggaag aggaactgaa gcccctggaa gaagtgctga atctggccca gtccaagaac

1020

ttccacctga ggcctaggga cctgatctcc aacatcaacg tgatcgtgct ggaactgaaa

1080

ggctccgaga caaccttcat gtgcgagtac gccgacgaga cagccaccat cgtggaattt

1140

ctgaaccggt ggatcacctt ctgccagtcc atcatctcca cactgacctg atgactcgag

1200

1200

<210> 46

<211> 592

<212> PRT

<213> Artificial sequence

<220>

<223>CD80-Fc-IL2wt

<400> 46

Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys

1 5 10 15

Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp

20 25 30

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

35 40 45

Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn

50 55 60

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

65 70 75 80

Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His

85 90 95

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

100 105 110

Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys

115 120 125

Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn

130 135 140

Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu

145 150 155 160

Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr

165 170 175

Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn

180 185 190

Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Asn

195 200 205

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

210 215 220

Ser Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Gln Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

325 330 335

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

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

355 360 365

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

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Leu Gly Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser

450 455 460

Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu

465 470 475 480

Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr

485 490 495

Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu

500 505 510

Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val

515 520 525

Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu

530 535 540

Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr

545 550 555 560

Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe

565 570 575

Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr

580 585 590

<---

Claims (24)

1. A method for culturing regulatory T cells, comprising: culturing isolated regulatory T cells in a medium containing a fusion protein dimer comprising IL-2 protein or a variant thereof and CD80 protein or a fragment thereof, wherein the fusion protein is characterized by the following structural formulas (I) or (II): N'-X-[linker (1)]n-domain Fc-[linker (2)]m-Y-C' – formula (I), N'-Y-[linker (1)]n-domain Fc-[linker (2)]m-X-C' – formula (II), where N' represents the N-terminus of the fusion protein, C' represents the C-terminus of the fusion protein, X is CD80 protein or a fragment thereof, Y is IL-2 protein or a variant thereof, linkers (1) and (2) are peptide linkers and n and m are each independently equal to 0 or 1, wherein the IL-2 protein variant contains a substitution of the 38th and 42nd amino acids in the amino acid sequence of SEQ ID NO: 10, and the CD80 protein fragment contains the CD80 extracellular domain. 2. The method for culturing regulatory T cells according to claim 1, wherein the regulatory T cells are CD4+CD25+CD127- T cells. 3. A method for cultivating regulatory T cells according to claim 1, in which the cultivation is carried out for 1-20 days. 4. The method for culturing regulatory T cells according to claim 2, in which CD4+CD25+CD127- T cells are obtained by culturing CD4+ T cells or culturing CD25+ T cells. 5. A method for cultivating regulatory T cells according to claim 1, wherein the IL-2 protein has the amino acid sequence SEQ ID NO: 10. 6. A method for cultivating regulatory T cells according to claim 1, wherein the IL-2 protein variant has the amino acid sequence SEQ ID NO: 6. 7. The method of culturing regulatory T cells according to claim 1, wherein CD80 has the amino acid sequence SEQ ID NO: 11. 8. The method for cultivating regulatory T cells according to claim 1, wherein the IL-2 protein variant contains any substitution selected from the following combinations of substitutions (a)-(d) in the amino acid sequence SEQ ID NO: 10: (a) R38A/F42A, (b) R38A/F42A/Y45A, (c) R38A/F42A/E61R, (d) R38A/F42A/L72G. 9. The method of culturing regulatory T cells according to claim 1, wherein the fusion protein has the amino acid sequence SEQ ID NO: 9, 26, 28 or 30.