IL-2 variant

Abstract

An object of the present invention is to provide a novel IL-2 variant with improved selectivity for IL-2R αβγ and selectively activating Tregs. The present invention relates to an IL-2 variant, a method for producing the IL-2 variant, a composition containing the IL-2 variant and a therapeutic agent for immune diseases, a method for increasing the selectivity of IL-2 for IL-2R αβγ a method for increasing the affinity of IL-2 for the IL-2Rα subunit, a method for reducing the affinity of IL-2 for at least any one of the IL-2Rβ and γ subunits, and a method for selectively activating regulatory T cells.

Description

Technical Field

[0001] The present invention relates to an IL-2 variant, a method for producing the IL-2 variant, a composition and a therapeutic agent containing the IL-2 variant, a method for increasing the affinity of IL-2 for the IL-2Rα subunit, a method for decreasing the affinity of IL-2 for at least any one of the IL-2Rβ and γ subunits, and a method for selectively activating regulatory T cells. Background Art

[0002] Regulatory T cells (Treg) are a subset of CD4-positive T cells characterized by the expression of the transcription factor Foxp3 (forkhead box P3). Treg suppress the activation of effector T cells (Teff) through multiple mechanisms such as the production of inhibitory cytokines such as IL-10 or TGF-β, cytolysis using cytotoxic proteins such as perforin or granzyme, regulation of the function of antigen-presenting cells using CTLA-4, and exhaustion caused by competition for the use of IL-2, and negatively regulate excessive immune responses (Non-Patent Document 1).

[0003] Defects in Treg caused by mutations in Foxp3 lead to IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome presenting with severe systemic autoimmune reactions. In addition, in various autoimmune diseases, the quantity or quality of Treg is reduced, and thus it is considered that the breakdown of immune control by Treg is involved in the pathogenesis of the disease (Non-Patent Documents 2 and 3).

[0004] IL-2 (Interleukin-2) is a cytokine mainly produced by activated T cells and contributes to the proliferation and activation of various immune cells. The molecular weight of the mature form in humans is approximately 15 kDa (133 residues), and it has a four-helix bundle structure composed of four α-helices (Non-Patent Document 4).

[0005] The IL-2 receptor (IL-2R) is composed of three molecules, CD25 (IL-2R α ), CD122 (IL-2R β ), and CD132 (γ c ), and forms a heterodimeric receptor (IL-2R D ≈10 -9 M) showing medium affinity for IL-2, or a high-affinity (K βγ ) receptor (K D ≈10 -11Heterotrimeric receptor (IL-2R αβγ ) transmits signals. Even when CD25 is in its monomeric form, it binds to IL-2 with low affinity (K D ≈ 10 -8 M), but cannot transmit signals (Non-Patent Document 5).

[0006] The expression pattern of IL-2R varies among different immune cells. In CD56 low NK cells or naive T cells, the expression of CD25 is very low, and IL-2R functions in the form of IL-2R βγ . On the other hand, in Treg or CD56 high NK cells, CD25 is expressed, and IL-2R functions in the form of IL-2R αβγ (Non-Patent Document 6).

[0007] In the binding of IL-2 to IL-2R αβγ , IL-2 first binds to CD25, and then sequentially binds to CD122 and CD132, thereby causing the trimerization of IL-2R. The dimerization of CD122 and CD132 by IL-2 promotes the recruitment of JAK1 to the intracellular region of CD122 and the recruitment of JAK3 to the intracellular region of CD132, and then causes the phosphorylation of STAT5. Phosphorylated STAT5 (pSTAT5) forms a dimer and then translocates into the nucleus, promoting the transcription of target genes (Non-Patent Document 7, Non-Patent Document 8).

[0008] IL-2 signaling plays an important role in maintaining the homeostasis of Treg. pSTAT5 generated by IL-2 stimulation directly promotes the expression of Foxp3, thereby enhancing the functions of Treg in promoting proliferation, stabilization, and inhibiting the activation of Teff. In addition to expressing IL-2R αβγ as a high-affinity receptor, Treg has high activities of protein phosphatases 1 (PP1) and 2A that positively regulate IL-2 signaling. Therefore, the phosphorylation of STAT5 in Treg or the gene expression downstream of it caused by IL-2 stimulation occurs in a concentration range that is about 10 to 100 times lower than that in memory T cells (Non-Patent Document 6, Non-Patent Document 9).

[0009] Mice with IL-2 gene or IL-2R gene deficiency show a decrease in Tregs and severe autoimmune reactions. Similarly, in humans, CD25 gene deficiency shows proliferation of self-reactive T cells or symptoms similar to IPEX syndrome. SLE (systemic lupus erythematosus) patients or type 1 diabetes patients have been observed to have a decrease in the IL-2 production capacity of T cells and a concomitant decrease in Tregs (Non-Patent Documents 10, 11, and 12).

[0010] Activation of IL-2 signals enhances Treg function. Administration of IL-2 to MRL / lpr mice showing SLE-like symptoms inhibits inflammatory responses and improves the condition. In addition, administration of IL-2 to patients with GVHD (graft-versus-host disease) or SLE promotes the expansion of Tregs and improves the condition (Non-Patent Documents 13, 14, and 15).

[0011] However, administration of wild-type IL-2 often causes an increase in NK cells or eosinophils, leading to injection site reactions, fever, flu-like symptoms, etc. In addition, the blood half-life of IL-2 is very short, about 1 hour, so when using low-dose IL-2 therapy, IL-2 needs to be administered for several days (Non-Patent Documents 14, 15, 16).

[0012] In order to solve the above-mentioned problems, attempts have been made to create IL-2 variants that selectively activate Tregs and have a prolonged blood half-life.

[0013] IL-2R αβγ Selective attempts. In one approach, attempts were made to target IL-2R βγ A method for introducing mutations into interacting amino acid residues or forming an immune complex with an anti-IL-2 antibody (Patent Document 1, Patent Document 2, Patent Document 3, Patent Document 4, Patent Document 5, Patent Document 6, Non-Patent Document 17).

[0014] However, the introduction of amino acid mutations can lead to increased immunogenicity due to the mutations. Cynomolgus monkeys administered with amino acid-mutated human IL-2 variants produce anti-drug antibodies. αβγ The activity selectively becomes bell-shaped (Patent Documents 2 and 6).

[0015] Attempts have been made to increase the blood half-life of IL-2. In one method, attempts have been made to add a method derived from the Fc sequence of an antibody (Patent Document 2, Patent Document 4, Patent Document 7, Non-Patent Document 18). In other methods, methods of adding a non-toxic water-soluble polymer such as polyethylene glycol (PEG) are known. (Patent Document 8, Patent Document 9, Patent Document 10, Patent Document 11, Non-Patent Document 19, Non-Patent Document 20). In addition, methods of introducing sugar chains have also been attempted (Patent Document 12, Patent Document 13, Patent Document 14).

[0016] However, modification of IL-2 with polyethylene glycol results in a decrease in biological activity (Non-Patent Document 19).

[0017] Prior Art Documents

[0018] Patent Documents

[0019] Patent Document 1: International Publication No. 2010 / 085495

[0020] Patent Document 2: International Publication No. 2014 / 153111

[0021] Patent Document 3: Specification of US Patent Application Publication No. 2015 / 0374788

[0022] Patent Document 4: Specification of US Patent No. 7186804

[0023] Patent Document 5: International Publication No. 2014 / 028748

[0024] Patent Document 6: International Publication No. 2015 / 109212

[0025] Patent Document 7: Specification of US Patent Application Publication No. 2017 / 0051029

[0026] Patent Document 8: International Publication No. 2016 / 025385

[0027] Patent Document 9: Japanese Patent Application Laid-Open No. 2016-202187

[0028] Patent Document 10: Specification of US Patent No. 4902502

[0029] Patent Document 11: Specification of US Patent No. 5206344

[0030] Patent Document 12: Specification of US Patent No. 5153310

[0031] Patent Document 13: Specification of US Patent No. 5312903

[0032] Patent Document 14: Specification of US Patent No. 5,417,970

[0033] Non-Patent Document

[0034] Non-Patent Document 1: Front Immunol, 2013.4(378)

[0035] Non-Patent Document 2: Nat Rev Immunol, 2014.14(5): 343 - 349

[0036] Non-Patent Document 3: Autoimmun Rev, 2015.14(2): 105 - 116

[0037] Non-Patent Document 4: Annu Rev Immunol, 2008.26: 453 - 479

[0038] Non-Patent Document 5: Immunity, 2013.38(1): 13 - 25

[0039] Non-Patent Document 6: Diabetes, 2015.64(6): 2172 - 2183

[0040] Non-Patent Document 7: J Biol Chem, 1997.272(50): 31821 - 31828

[0041] Non-Patent Document 8: Nat Rev Immunol. 2012 Feb 17; 12(3): 180 - 90

[0042] Non-Patent Document 9: Nat Rev Immunol. 2015 May; 15(5): 283 - 94

[0043] Non-Patent Document 10: Proc Natl Acad Sci USA, 1997.94(7): 3168 - 3171

[0044] Non-Patent Document 11: N Engl J Med, 2011.365(22): 2110 - 2121

[0045] Non-Patent Document 12: Curr Diab Rep, 2014.14(12): 553

[0046] Non-Patent Document 13: J Immunol, 2014.193(5): 2168 - 2177

[0047] Non-Patent Document 14: N Engl J Med, 2011.365(22): 2055 - 2066

[0048] Non-patent Document 15: Ann Rheum Dis, 2015. 74(4): 791-792

[0049] Non-patent Document 16: Blood. 2014 Dec 4; 124(24): 3572-6.

[0050] Non-patent Document 17: Curr Pharm Des, 2002. 8(24): 2171-83

[0051] Non-patent Document 18: J Autoimmun, 2015. 56: 66-80

[0052] Non-patent Document 19: American College of Rheumatology Annual Meeting, San Diego, CA, 2017. Poster Abstract 2715: NKTR-358: A Selective, First-in-Class IL-2 Pathway Agonist Which Increases Number and Suppressive Function of Regulatory T Cells for the Treatment of Immune Inflammatory Disorders, Langowski, J., et al. http: / / www.nektar.com / application / files / 6315 / 1001 / 4171 / NKTR-358_2017ACR_ABS2715.pdf

[0053] Non-patent Document 20: Biotechnology (NY), 1990. 8(4): 343-346 Summary of the Invention

[0054] Problems to be Solved by the Invention

[0055] An object of the present invention is to provide a novel IL-2 variant with increased selectivity for IL-2R αβγ and selectively activating Tregs.

[0056] Means for Solving the Problems

[0057] In-depth studies were conducted on the above problems, and as a result, the inventors of the present invention found that an IL-2 variant obtained by modifying IL-2 by binding a sugar chain or PEG thereto can solve the above problems, thereby completing the present invention.

[0058] That is, the present invention is described as follows.

[0059] (1) An interleukin-2 (hereinafter abbreviated as IL-2) variant.

[0060] (2) The IL-2 variant according to (1), which is an IL-2 variant conjugated with a sugar chain and / or an IL-2 variant conjugated with polyethylene glycol (PEG).

[0061] (3) The IL-2 variant according to (1) or (2), which has improved selectivity for the interleukin-2 receptor (hereinafter IL-2R) αβγ and.

[0062] (4) The IL-2 variant according to (2) or (3), wherein a sugar chain is conjugated to at least one amino acid residue selected from the amino acid residues at positions 11, 12, 13, 15, 16, 18, 19, 20, 84, 87, 88, 91, 92, 108, 115, 119, 122, 123 and 130 in the amino acid sequence of IL-2.

[0063] (5) The IL-2 variant according to any one of (2) to (4), wherein the sugar chain is at least one selected from sugar chains having structures represented by the following (Formula 4) to (Formula 8), (Formula Y1), (Formula Y2) or (Formula Y3).

[0064]

[0065]

[0066]

[0067] (6) The IL-2 variant according to any one of (2) to (5), which comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence in which the amino acid residue at position 125 in the amino acid sequence shown in SEQ ID NO: 1 is replaced with a serine residue, and at least one amino acid residue selected from the amino acid residues at positions 11, 12, 13, 15, 16, 18, 19, 20, 84, 87, 88, 91, 92, 108, 115, 119, 122, 123 and 130 is replaced with a group derived from a cysteine residue or an asparagine residue conjugated with a sugar chain.

[0068] (7) The IL-2 variant according to any one of (2) to (6), which comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence in which the amino acid residue at position 125 of the amino acid sequence shown in SEQ ID NO: 1 is substituted with a serine residue, and in which at least one amino acid residue selected from the amino acid residues at positions 12, 15, 16, 19, 88, 91, and 119 is substituted with a group derived from a cysteine residue or an asparagine residue to which a sugar chain is bound.

[0069] (8) The IL-2 variant according to (6) or (7), wherein the group derived from a cysteine residue to which a sugar chain is bound has the structure shown in the following (Formula 1):

[0070]

[0071] [In (Formula 1), Saccharide represents a sugar chain.]

[0072] (9) The IL-2 variant according to (6) or (7), wherein the group derived from an asparagine residue to which a sugar chain is bound has the structure shown in the following (Formula 2):

[0073]

[0074] [In (Formula 2), Saccharide represents a sugar chain.]

[0075] (10) The IL-2 variant according to any one of (2) to (9), wherein the IL-2 variant to which a sugar chain is bound is an IL-2 variant to which PEG is further bound.

[0076] (11) The IL-2 variant according to any one of (2) to (10), which comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence in which the amino acid residue at position 125 of the amino acid sequence shown in SEQ ID NO: 1 is substituted with a serine residue, and in which at least one amino acid residue selected from the amino acid residues at positions 1, 3, 51, and 78 is substituted with an amino acid residue to which PEG is bound.

[0077] (12) The IL-2 variant according to (2) or (3), wherein PEG is bound to at least one amino acid residue selected from the amino acid residues at positions 4, 5, 6, 7, 8, 60, 78, 79, 99, 100, 101, and 129 in the amino acid sequence of IL-2.

[0078] (13) The IL-2 variant according to any one of (2), (3), and (12), which comprises an amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence in which the amino acid residue at position 125 of the amino acid sequence of SEQ ID NO: 1 is substituted with a serine residue, and at least one amino acid residue selected from the amino acid residues at positions 4, 5, 6, 7, 8, 60, 78, 79, 99, 100, 101, and 129 is substituted with an amino acid residue conjugated with PEG.

[0079] (14) The IL-2 variant according to any one of (2), (3), (12), and (13), wherein at least one amino acid residue selected from the amino acid residues at positions 4, 5, 8, 78, and 129 in the amino acid sequence shown in SEQ ID NO: 1, or the amino acid sequence in which the amino acid residue at position 125 of the amino acid sequence shown in SEQ ID NO: 1 is substituted with a serine residue, is substituted with an amino acid residue conjugated with PEG.

[0080] (15) The IL-2 variant according to any one of (2), (3), (12) to (14), wherein at least two amino acid residues selected from the amino acid residues at positions 4, 5, 8, 78, and 129 in the amino acid sequence shown in SEQ ID NO: 1, or the amino acid sequence in which the amino acid residue at position 125 of the amino acid sequence shown in SEQ ID NO: 1 is substituted with a serine residue, are substituted with amino acid residues conjugated with PEG.

[0081] (16) The IL-2 variant according to any one of (2), (3), (12) to (15), wherein at least one amino acid residue selected from the amino acid residues at positions 4, 5, and 8, and the amino acid residue at position 78 or 129 in the amino acid sequence shown in SEQ ID NO: 1, or the amino acid sequence in which the amino acid residue at position 125 of the amino acid sequence shown in SEQ ID NO: 1 is substituted with a serine residue, are substituted with amino acid residues conjugated with PEG.

[0082] (17) The IL-2 variant according to any one of (2), (3), (12) to (16), wherein the amino acid residue conjugated with PEG is a non-natural amino acid residue conjugated with PEG.

[0083] (18) The IL-2 variant according to (17), wherein the non-natural amino acid residue conjugated with PEG is a group conjugated with PEG derived from an amino acid residue having a thiol group (-SH), or a group conjugated with PEG derived from an amino acid residue having an azide group.

[0084] (19) The IL-2 variant according to (17) or (18), wherein the non-natural amino acid residue conjugated with PEG is derived from N 6-[(o-azidobenzyl)oxy]carbonyl-L-lysine (N 6 -[(o-azidobenzyl)oxy]carbonyl-L-lysine) (o-Az-Z-Lys) residue, derived from N 6 -[(m-azidobenzyl)oxy]carbonyl-L-lysine (N 6 -[(m-azidobenzyl)oxy]carbonyl-L-lysine) (m-Az-Z-Lys) residue or a group derived from a cysteine residue.

[0085] (20) The IL-2 variant according to (19), wherein the group derived from the o-Az-Z-Lys residue conjugated with PEG has the structure shown in the following (Formula 11) and / or (Formula 12).

[0086]

[0087]

[0088] (21) The IL-2 variant according to (19), wherein the group derived from the m-Az-Z-Lys residue conjugated with PEG has the structure shown in the following (Formula Y4) and / or (Formula Y5).

[0089]

[0090]

[0091] (22) The IL-2 variant according to (19), wherein the group derived from the cysteine residue conjugated with PEG has the structure shown in the following (Formula X11) and / or (Formula X12) and / or (Formula X13).

[0092]

[0093] (23) The IL-2 variant according to any one of (2), (10) to (22), wherein PEG is linear.

[0094] (24) The IL-2 variant according to any one of (2), (10) to (22), wherein PEG is branched.

[0095] (25) The IL-2 variant according to any one of (2), (10) to (24), wherein PEG is PEG with an average molecular weight of 10 kDa or more.

[0096] (26) The IL-2 variant according to any one of (2), (10) to (25), wherein the conjugated PEG is PEG with an average molecular weight of 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa or 80 kDa.

[0097] (27) The IL-2 variant according to any one of (2), (10) to (26), wherein the conjugated PEG has a structure represented by at least one of the following formulas (13), (14), (15), (16), (X7), (X8), (X9), (X10), (X11), (X13), (X14) or (X15).

[0098]

[0099]

[0100]

[0101] (28) The IL-2 variant according to any one of (1) to (27), wherein a methionine residue is further conjugated to the N-terminus of IL-2.

[0102] (29) The IL-2 variant according to any one of (1) to (28), wherein alanine at the N-terminus of IL-2 is deleted.

[0103] (30) The IL-2 variant according to any one of (1) to (29), wherein alanine at the N-terminus of IL-2 is deleted and methionine is further conjugated.

[0104] (31) A method for producing the IL-2 variant according to any one of (1) to (30).

[0105] (32) A composition comprising the IL-2 variant according to any one of (1) to (30).

[0106] (33) A therapeutic agent for immune diseases comprising the IL-2 variant according to any one of (1) to (30).

[0107] (34) Making IL-2 bind to IL-2R αβγ A method for enhancing selectivity.

[0108] (35) The method according to (34), which comprises a step of binding IL-2 to a sugar chain and / or PEG.

[0109] (36) The method according to (35) includes a step of binding at least one amino acid residue selected from the amino acid residues at positions 11, 12, 13, 15, 16, 18, 19, 20, 84, 87, 88, 91, 92, 108, 115, 119, 122, 123, and 130 in the amino acid sequence of IL-2 to a sugar chain.

[0110] (37) The method according to (35) or (36), wherein the sugar chain is at least one selected from sugar chains having structures represented by the following (Formula 4) to (Formula 8), (Formula Y1), (Formula Y2), or (Formula Y3).

[0111]

[0112]

[0113] (38) The method according to any one of (35) to (37) includes, in IL-2 having the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which the amino acid residue at position 125 of the amino acid sequence shown in SEQ ID NO: 1 is substituted with a serine residue, substituting at least one amino acid residue selected from the amino acid residues at positions 11, 12, 13, 15, 16, 18, 19, 20, 84, 87, 88, 91, 92, 108, 115, 119, 122, 123, and 130 of the amino acid sequence with a group derived from a cysteine residue or an asparagine residue to which a sugar chain is bound.

[0114] (39) The method according to any one of (35) to (38) includes substituting at least one amino acid residue selected from the amino acid residues at positions 12, 15, 16, 19, 88, 91, and 119 in the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which the amino acid residue at position 125 of the amino acid sequence shown in SEQ ID NO: 1 is substituted with a serine residue with a group derived from a cysteine residue or an asparagine residue to which a sugar chain is bound.

[0115] (40) The method according to (38) or (39), wherein the group derived from a cysteine residue to which a sugar chain is bound has a structure represented by the following (Formula 1).

[0116]

[0117] [In (Formula 1), Saccharide represents a sugar chain.]

[0118] (41) The method according to (38) or (39), wherein the group derived from an asparagine residue to which a sugar chain is bound has a structure represented by the following (Formula 2).

[0119]

[0120] [(In formula (2), Saccharide represents a sugar chain.)]

[0121] (42) The method according to any one of (35) to (41), which comprises the step of further binding PEG to the IL-2 variant conjugated with a sugar chain.

[0122] (43) The method according to (42), which comprises an amino acid sequence having at least one amino acid residue selected from the amino acid residues at positions 1, 3, 51, and 78 in the amino acid sequence shown in SEQ ID NO: 1, or in the amino acid sequence in which the amino acid residue at position 125 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with a serine residue, being substituted with an amino acid residue conjugated with PEG.

[0123] (44) The method according to (35), wherein PEG is conjugated to at least one amino acid residue selected from the amino acid residues at positions 4, 5, 6, 7, 8, 60, 78, 79, 99, 100, 101, and 129 in the amino acid sequence of IL-2.

[0124] (45) The method according to (35) or (44), which comprises an amino acid sequence having at least one amino acid residue selected from the amino acid residues at positions 4, 5, 6, 7, 8, 60, 78, 79, 99, 100, 101, and 129 in the amino acid sequence shown in SEQ ID NO: 1, or in the amino acid sequence in which the amino acid residue at position 125 in the amino acid sequence of SEQ ID NO: 1 is substituted with a serine residue, being substituted with an amino acid residue conjugated with PEG.

[0125] (46) The method according to any one of (35), (44), and (45), wherein at least one amino acid residue selected from the amino acid residues at positions 4, 5, 8, 78, and 129 in the amino acid sequence shown in SEQ ID NO: 1, or in the amino acid sequence in which the amino acid residue at position 125 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with a serine residue, is substituted with an amino acid residue conjugated with PEG.

[0126] (47) The method according to any one of (35), (44) to (46), wherein at least two amino acid residues selected from the amino acid residues at positions 4, 5, 8, 78, and 129 in the amino acid sequence shown in SEQ ID NO: 1, or in the amino acid sequence in which the amino acid residue at position 125 in the amino acid sequence shown in SEQ ID NO: 1 is substituted with a serine residue, are substituted with amino acid residues conjugated with PEG.

[0127] (48) The method according to any one of (35), (42) to (47), wherein the amino acid residue conjugated with PEG is a non-natural amino acid residue conjugated with PEG.

[0128] (49) The method according to (48), wherein the non-natural amino acid residue conjugated with PEG is a group derived from an amino acid residue having a thiol group (-SH) conjugated with PEG, or a group derived from an amino acid residue having an azide group conjugated with PEG.

[0129] (50) The method according to (48) or (49), wherein the non-natural amino acid residue conjugated with PEG is derived from N 6 -[(o-azidobenzyl)oxy]carbonyl-L-lysine (o-Az-Z-Lys) residue, a group derived from N 6 -[(m-azidobenzyl)oxy]carbonyl-L-lysine (m-Az-Z-Lys) residue, or a group derived from a cysteine residue.

[0130] (51) The method according to (50), wherein the group derived from the o-Az-Z-Lys residue conjugated with PEG has the structure shown in the following (Formula 11) and / or (Formula 12).

[0131]

[0132]

[0133] (52) The method according to (50), wherein the group derived from the m-Az-Z-Lys residue conjugated with PEG has the structure shown in the following (Formula Y4) and / or (Formula Y5).

[0134]

[0135]

[0136] (53) The method according to (50), wherein the group derived from the cysteine residue conjugated with PEG has the structure shown in the following (Formula X11) and / or (Formula X12) and / or (Formula X13).

[0137]

[0138] (54) The method according to any one of (35), (42) to (53), wherein PEG is linear.

[0139] (55) The method according to any one of (35), (42) to (53), wherein PEG is branched.

[0140] (56) The method according to any one of (35), (42) to (55), wherein the PEG is a PEG having an average molecular weight of 10 kDa or more.

[0141] (57) The method according to any one of (35), (42) to (56), wherein the PEG is a PEG having an average molecular weight of 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa or 80 kDa.

[0142] (58) The method according to any one of (35), (42) to (57), wherein the conjugated PEG has a structure represented by at least one of the following formulas (13), (14), (15), (16), (X7), (X8), (X9), (X10), (X11), (X13), (X14) or (X15).

[0143]

[0144]

[0145]

[0146] (59) The method according to any one of (34) to (58), wherein a methionine residue is further conjugated to the N-terminus of IL-2.

[0147] (60) The method according to any one of (34) to (59), wherein alanine at the N-terminus of IL-2 is deficient.

[0148] (61) The method according to any one of (34) to (60), wherein alanine at the N-terminus of IL-2 is deficient and methionine is conjugated.

[0149] (62) A method for selectively activating regulatory T cells.

[0150] (63) A method for reducing the affinity of IL-2 for at least one of the IL-2Rβ and γ subunits.

[0151] (64) A method for increasing the affinity of IL-2 for the IL-2Rα subunit.

[0152] Effects of the invention

[0153] The IL-2 variant of the present invention and the IL-2R highly expressed in Treg αβγSelectively bind and selectively activate Tregs. According to the present invention, it is possible to provide an IL-2 variant, a method for producing the IL-2 variant, a composition containing the IL-2 variant and a therapeutic agent for immune diseases, a method for increasing the selectivity of IL-2 for IL-2R αβγ and a method for increasing the affinity of IL-2 for the IL-2Rα subunit, a method for reducing the affinity of IL-2 for at least any one of the IL-2Rβ and γ subunits, and a method for selectively activating regulatory T cells. Sequence Listing <110> Kyowa Kirin Co., Ltd. <120> IL-2 Variant <130> PD00976A <150> JP2017-252224 <151> 2017-12-27 <160> 52 <170> PatentIn version 3.5 <210> 1 <211> 133 <212> PRT <213> Human <400> 1 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> 2 <211> 142 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Amino acid sequence of 8His-IL-2 <400> 2 Met His His His His His His His His Ala Pro Thr Ser Ser Ser Thr 1 5 10 15 Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met 20 25 30 Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met 35 40 45 Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His 50 55 60 Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn 65 70 75 80 Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser 85 90 95 Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe 100 105 110 Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn 115 120 125 Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr 130 135 140 <210> 3 <211> 426 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Base sequence of 8His-IL-2 <400> 3 atgcatcatc atcaccatca tcatcacgcc ccgaccagca gcagcaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaactt tcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagcgagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat catcagtacc 420 ctgacc 426 <210> 4 <211> 426 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: Base sequence of S4 (o-Az-Z-Lys) <400> 4 atgcatcatc atcaccatca tcatcacgcc ccgacctaga gcagcaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaactt tcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagcgagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat catcagtacc 420 ctgacc 426 <210> 5 <211> 426 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: Base sequence of S5(o-Az-Z-Lys) <400> 5 atgcatcatc atcaccatca tcatcacgcc ccgaccagct agagcaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaactt tcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagcgagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat catcagtacc 420 ctgacc 426 <210> 6 <211> 426 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: Base sequence of S6(o-Az-Z-Lys) <400> 6 atgcatcatc atcaccatca tcatcacgcc ccgaccagca gctagaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaactt tcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagcgagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat catcagtacc 420 ctgacc 426 <210> 7 <211> 426 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Base sequence of T7(o - Az - Z - Lys) <400> 7 atgcatcatc atcaccatca tcatcacgcc ccgaccagca gcagctagaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaactt tcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagcgagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat catcagtacc 420 ctgacc 426 <210> 8 <211> 426 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Base sequence of K8(o-Az-Z-Lys) <400> 8 atgcatcatc atcaccatca tcatcacgcc ccgaccagca gcagcaccta gaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaactt tcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagcgagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat catcagtacc 420 ctgacc 426 <210> 9 <211> 426 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Base sequence of E60(o-Az-Z-Lys) <400> 9 atgcatcatc atcaccatca tcatcacgcc ccgaccagca gcagcaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctgtaggaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaactt tcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagcgagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat catcagtacc 420 ctgacc 426 <210> 10 <211> 426 <212> DNA <213> Artificial sequence <220> <223> Artificial Sequence Description: Base sequence of F78(o-Az-Z-Lys) <400> 10 atgcatcatc atcaccatca tcatcacgcc ccgaccagca gcagcaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaacta gcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagcgagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat catcagtacc 420 ctgacc 426 <210> 11 <211> 426 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Base sequence of H79(o-Az-Z-Lys) <400> 11 atgcatcatc atcaccatca tcatcacgcc ccgaccagca gcagcaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaactt ttagctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagcgagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat catcagtacc 420 ctgacc 426 <210> 12 <211> 426 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Base sequence of R81(o-Az-Z-Lys) <400> 12 atgcatcatc atcaccatca tcatcacgcc ccgaccagca gcagcaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaactt tcacctgtag ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagcgagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat catcagtacc 420 ctgacc 426 <210> 13 <211> 426 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Base sequence of L94(o-Az-Z-Lys) <400> 13 atgcatcatc atcaccatca tcatcacgcc ccgaccagca gcagcaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaactt tcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgtagg aactgaaggg tagcgagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat catcagtacc 420 ctgacc 426 <210> 14 <211> 426 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: Base sequence of S99(o-Az-Z-Lys) <400> 14 atgcatcatc atcaccatca tcatcacgcc ccgaccagca gcagcaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaactt tcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg ttaggagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat catcagtacc 420 ctgacc 426 <210> 15 <211> 426 <212> DNA <213> Artificial sequence <220> <223> Artificial Sequence Description: Base sequence of E100 (o-Az-Z-Lys) <400> 15 atgcatcatc atcaccatca tcatcacgcc ccgaccagca gcagcaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaactt tcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagctagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat catcagtacc 420 ctgacc 426 <210> 16 <211> 426 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Base sequence of T101 (o-Az-Z-Lys) <400> 16 atgcatcatc atcaccatca tcatcacgcc ccgaccagca gcagcaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaactt tcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagcgagtag accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat catcagtacc 420 ctgacc 426 <210> 17 <211> 426 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Base sequence of Q126 (o-Az-Z-Lys) <400> 17 atgcatcatc atcaccatca tcatcacgcc ccgaccagca gcagcaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaactt tcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagcgagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cctagagcat catcagtacc 420 ctgacc 426 <210> 18 <211> 426 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Base sequence of I129(o-Az-Z-Lys) <400> 18 atgcatcatc atcaccatca tcatcacgcc ccgaccagca gcagcaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaactt tcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagcgagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat ctagagtacc 420 ctgacc 426 <210> 19 <211> 1502 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Amino acid sequence of human IL-2Rαβγ-Azamigreen fusion <400> 19 Met Asp Ser Tyr Leu Leu Met Trp Gly Leu Leu Thr Phe Ile Met Val 1 5 10 15 Pro Gly Cys Gln Ala Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro 20 25 30 His Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn 35 40 45 Cys Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr 50 55 60 Met Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys 65 70 75 80 Gln Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro 85 90 95 Gln Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro 100 105 110 Met Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro 115 120 125 Pro Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val 130 135 140 Gly Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His 145 150 155 160 Arg Gly Pro Ala Glu Ser Val Cys Lys Met Thr His Gly Lys Thr Arg 165 170 175 Trp Thr Gln Pro Gln Leu Ile Cys Thr Gly Glu Met Glu Thr Ser Gln 180 185 190 Phe Pro Gly Glu Glu Lys Pro Gln Ala Ser Pro Glu Gly Arg Pro Glu 195 200 205 Ser Glu Thr Ser Cys Leu Val Thr Thr Thr Asp Phe Gln Ile Gln Thr 210 215 220 Glu Met Ala Ala Thr Met Glu Thr Ser Ile Phe Thr Thr Glu Tyr Gln 225 230 235 240 Val Ala Val Ala Gly Cys Val Phe Leu Leu Ile Ser Val Leu Leu Leu 245 250 255 Ser Gly Leu Thr Trp Gln Arg Arg Gln Arg Lys Ser Arg Arg Thr Ile 260 265 270 Arg Ala Lys Arg Ala Pro Val Lys Gln Thr Leu Asn Phe Asp Leu Leu 275 280 285 Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Met Ala Ala Pro 290 295 300 Ala Leu Ser Trp Arg Leu Pro Leu Leu Ile Leu Leu Leu Pro Leu Ala 305 310 315 320 Thr Ser Trp Ala Ser Ala Ala Val Asn Gly Thr Ser Gln Phe Thr Cys 325 330 335 Phe Tyr Asn Ser Arg Ala Asn Ile Ser Cys Val Trp Ser Gln Asp Gly 340 345 350 Ala Leu Gln Asp Thr Ser Cys Gln Val His Ala Trp Pro Asp Arg Arg 355 360 365 Arg Trp Asn Gln Thr Cys Glu Leu Leu Pro Val Ser Gln Ala Ser Trp 370 375 380 Ala Cys Asn Leu Ile Leu Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr 385 390 395 400 Val Asp Ile Val Thr Leu Arg Val Leu Cys Arg Glu Gly Val Arg Trp 405 410 415 Arg Val Met Ala Ile Gln Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu 420 425 430 Met Ala Pro Ile Ser Leu Gln Val Val His Val Glu Thr His Arg Cys 435 440 445 Asn Ile Ser Trp Glu Ile Ser Gln Ala Ser His Tyr Phe Glu Arg His 450 455 460 Leu Glu Phe Glu Ala Arg Thr Leu Ser Pro Gly His Thr Trp Glu Glu 465 470 475 480 Ala Pro Leu Leu Thr Leu Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu 485 490 495 Thr Leu Thr Pro Asp Thr Gln Tyr Glu Phe Gln Val Arg Val Lys Pro 500 505 510 Leu Gln Gly Glu Phe Thr Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala 515 520 525 Phe Arg Thr Lys Pro Ala Ala Leu Gly Lys Asp Thr Ile Pro Trp Leu 530 535 540 Gly His Leu Leu Val Gly Leu Ser Gly Ala Phe Gly Phe Ile Ile Leu 545 550 555 560 Val Tyr Leu Leu Ile Asn Cys Arg Asn Thr Gly Pro Trp Leu Lys Lys 565 570 575 Val Leu Lys Cys Asn Thr Pro Asp Pro Ser Lys Phe Phe Ser Gln Leu 580 585 590 Ser Ser Glu His Gly Gly Asp Val Gln Lys Trp Leu Ser Ser Pro Phe 595 600 605 Pro Ser Ser Ser Phe Ser Pro Gly Gly Leu Ala Pro Glu Ile Ser Pro 610 615 620 Leu Glu Val Leu Glu Arg Asp Lys Val Thr Gln Leu Leu Leu Gln Gln 625 630 635 640 Asp Lys Val Pro Glu Pro Ala Ser Leu Ser Ser Asn His Ser Leu Thr 645 650 655 Ser Cys Phe Thr Asn Gln Gly Tyr Phe Phe Phe His Leu Pro Asp Ala 660 665 670 Leu Glu Ile Glu Ala Cys Gln Val Tyr Phe Thr Tyr Asp Pro Tyr Ser 675 680 685 Glu Glu Asp Pro Asp Glu Gly Val Ala Gly Ala Pro Thr Gly Ser Ser 690 695 700 Pro Gln Pro Leu Gln Pro Leu Ser Gly Glu Asp Asp Ala Tyr Cys Thr 705 710 715 720 Phe Pro Ser Arg Asp Asp Leu Leu Leu Phe Ser Pro Ser Leu Leu Gly 725 730 735 Gly Pro Ser Pro Pro Ser Thr Ala Pro Gly Gly Ser Gly Ala Gly Glu 740 745 750 Glu Arg Met Pro Pro Ser Leu Gln Glu Arg Val Pro Arg Asp Trp Asp 755 760 765 Pro Gln Pro Leu Gly Pro Pro Thr Pro Gly Val Pro Asp Leu Val Asp 770 775 780 Phe Gln Pro Pro Pro Glu Leu Val Leu Arg Glu Ala Gly Glu Glu Val 785 790 795 800 Pro Asp Ala Gly Pro Arg Glu Gly Val Ser Phe Pro Trp Ser Arg Pro 805 810 815 Pro Gly Gln Gly Glu Phe Arg Ala Leu Asn Ala Arg Leu Pro Leu Asn 820 825 830 Thr Asp Ala Tyr Leu Ser Leu Gln Glu Leu Gln Gly Gln Asp Pro Thr 835 840 845 His Leu Val Arg Ala Lys Arg Ala Pro Val Lys Gln Thr Leu Asn Phe 850 855 860 Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Met 865 870 875 880 Leu Lys Pro Ser Leu Pro Phe Thr Ser Leu Leu Phe Leu Gln Leu Pro 885 890 895 Leu Leu Gly Val Gly Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly Asn 900 905 910 Glu Asp Thr Thr Ala Asp Phe Phe Leu Thr Thr Met Pro Thr Asp Ser 915 920 925 Leu Ser Val Ser Thr Leu Pro Leu Pro Glu Val Gln Cys Phe Val Phe 930 935 940 Asn Val Glu Tyr Met Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro Gln 945 950 955 960 Pro Thr Asn Leu Thr Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn Asp 965 970 975 Lys Val Gln Lys Cys Ser His Tyr Leu Phe Ser Glu Glu Ile Thr Ser 980 985 990 Gly Cys Gln Leu Gln Lys Lys Glu Ile His Leu Tyr Gln Thr Phe Val 995 1000 1005 Val Gln Leu Gln Asp Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln 1010 1015 1020 Met Leu Lys Leu Gln Asn Leu Val Ile Pro Trp Ala Pro Glu Asn 1025 1030 1035 Leu Thr Leu His Lys Leu Ser Glu Ser Gln Leu Glu Leu Asn Trp 1040 1045 1050 Asn Asn Arg Phe Leu Asn His Cys Leu Glu His Leu Val Gln Tyr 1055 1060 1065 Arg Thr Asp Trp Asp His Ser Trp Thr Glu Gln Ser Val Asp Tyr 1070 1075 1080 Arg His Lys Phe Ser Leu Pro Ser Val Asp Gly Gln Lys Arg Tyr 1085 1090 1095 Thr Phe Arg Val Arg Ser Arg Phe Asn Pro Leu Cys Gly Ser Ala 1100 1105 1110 Gln His Trp Ser Glu Trp Ser His Pro Ile His Trp Gly Ser Asn 1115 1120 1125 Thr Ser Lys Glu Asn Pro Phe Leu Phe Ala Leu Glu Ala Val Val 1130 1135 1140 Ile Ser Val Gly Ser Met Gly Leu Ile Ile Ser Leu Leu Cys Val 1145 1150 1155 Tyr Phe Trp Leu Glu Arg Thr Met Pro Arg Ile Pro Thr Leu Lys 1160 1165 1170 Asn Leu Glu Asp Leu Val Thr Glu Tyr His Gly Asn Phe Ser Ala 1175 1180 1185 Trp Ser Gly Val Ser Lys Gly Leu Ala Glu Ser Leu Gln Pro Asp 1190 1195 1200 Tyr Ser Glu Arg Leu Cys Leu Val Ser Glu Ile Pro Pro Lys Gly 1205 1210 1215 Gly Ala Leu Gly Glu Gly Pro Gly Ala Ser Pro Cys Asn Gln His 1220 1225 1230 Ser Pro Tyr Trp Ala Pro Pro Cys Tyr Thr Leu Lys Pro Glu Thr 1235 1240 1245 Arg Ala Lys Arg Ala Pro Val Lys Gln Thr Leu Asn Phe Asp Leu 1250 1255 1260 Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Met Val 1265 1270 1275 Ser Val Ile Lys Pro Glu Met Lys Ile Lys Leu Cys Met Arg Gly 1280 1285 1290 Thr Val Asn Gly His Asn Phe Val Ile Glu Gly Glu Gly Lys Gly 1295 1300 1305 Asn Pro Tyr Glu Gly Thr Gln Ile Leu Asp Leu Asn Val Thr Glu 1310 1315 1320 Gly Ala Pro Leu Pro Phe Ala Tyr Asp Ile Leu Thr Thr Val Phe 1325 1330 1335 Gln Tyr Gly Asn Arg Ala Phe Thr Lys Tyr Pro Ala Asp Ile Gln 1340 1345 1350 Asp Tyr Phe Lys Gln Thr Phe Pro Glu Gly Tyr His Trp Glu Arg 1355 1360 1365 Ser Met Thr Tyr Glu Asp Gln Gly Ile Cys Thr Ala Thr Ser Asn 1370 1375 1380 Ile Ser Met Arg Gly Asp Cys Phe Phe Tyr Asp Ile Arg Phe Asp 1385 1390 1395 Gly Thr Asn Phe Pro Pro Asn Gly Pro Val Met Gln Lys Lys Thr 1400 1405 1410 Leu Lys Trp Glu Pro Ser Thr Glu Lys Met Tyr Val Glu Asp Gly 1415 1420 1425 Val Leu Lys Gly Asp Val Asn Met Arg Leu Leu Leu Glu Gly Gly 1430 1435 1440 Gly His Tyr Arg Cys Asp Phe Lys Thr Thr Tyr Lys Ala Lys Lys 1445 1450 1455 Glu Val Arg Leu Pro Asp Ala His Lys Ile Asp His Arg Ile Glu 1460 1465 1470 Ile Leu Lys His Asp Lys Asp Tyr Asn Lys Val Lys Leu Tyr Glu 1475 1480 1485 Asn Ala Val Ala Arg Tyr Ser Met Leu Pro Ser Gln Ala Lys 1490 1495 1500 <210> 20 <211> 4506 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: Base sequence of human IL-2Rαβγ-Azamigreen fusion <400> 20 atggattcat acctgcttat gtggggtctc ctcaccttca ttatggtccc cggatgtcaa 60 gccgagctct gtgacgatga tccacctgaa atcccccatg caacttttaa ggccatggcc 120 tataaggaag gtaccatgct gaactgtgaa tgcaagcgtg gcttccgcag gattaagagc 180 ggctccctgt acatgctgtg caccgggaac tcttcccatt caagttggga caaccagtgt 240 cagtgtacct cctccgcaac aagaaatacc accaaacagg tgactcccca gccagaggag 300 cagaaggagc ggaagacaac agaaatgcaa agtccaatgc agcccgtgga tcaggccagc 360 ctcccaggcc actgtcggga accaccccct tgggaaaacg aggccacaga acggatatac 420 cacttcgttg tcggtcaaat ggtgtattat cagtgtgtgc agggctacag ggccctgcat 480 cgaggccccg cagaatcagt ctgcaaaatg actcacggca agactcgctg gacccagcct 540 cagctgatat gtaccggaga gatggaaact tctcaattcc ccggggagga gaaaccccag 600 gcatctcctg aagggaggcc tgagtccgaa acctcttgtc tggttaccac aaccgacttc 660 cagatccaga ccgagatggc cgcaaccatg gaaacaagta ttttcactac agagtatcag 720 gtggccgtag caggctgtgt cttcctgttg atttccgtgc tgctgttgtc tgggctgacc 780 tggcaaagaa gacagagaaa gtctaggcgc actatccgag caaagcgggc tccagtgaag 840 cagacattga attttgatct gttgaaactt gccggcgacg ttgagtctaa ccctggccct 900 atggctgcac cagccctgtc ctggcgtttg cctctcctta tattgctgct tcctcttgct 960 acctcttggg cttccgctgc agtgaatggg acctcccaat ttacatgctt ctacaacagt 1020 agagccaaca tttcatgtgt gtggagccag gatggcgctc tgcaagatac atcttgccag 1080 gtccacgcct ggccagatag acggcggtgg aatcagacct gtgagcttct gcccgtgtcc 1140 caggcaagtt gggcttgcaa ccttatactg ggcgctcccg atagtcagaa gctgaccact 1200 gtggacatcg tgaccctgcg ggtgctttgt agggagggcg tccggtggag agtcatggct 1260 atacaggatt ttaaaccctt cgagaacctt cgcctcatgg cccccatctc tctgcaagtc 1320 gtgcatgtag agacacatag gtgtaacatc tcttgggaaa tctctcaggc ttcacactac 1380 tttgagaggc acctggagtt tgaggcccgt actctgagcc caggacatac ttgggaggag 1440 gcacccctgc tcaccctcaa gcagaagcag gagtggatat gtctggagac tttgactccc 1500 gacactcagt atgagttcca ggtgagagtg aaacccctgc agggggagtt tacaacctgg 1560 tcaccatgga gtcagcccct tgcctttcga actaaacctg ccgccctcgg caaggacaca 1620 atcccctggc tcggtcacct gcttgtcggc ctttccggcg cttttggttt tatcatcctg 1680 gtctaccttc tcatcaactg ccgcaatact gggccttggc tgaagaaagt ccttaagtgt 1740 aacaccccag atccaagtaa gttcttctct cagctgtcca gtgagcacgg tggcgatgtc 1800 cagaagtggt tgagcagtcc cttcccctcc tccagtttca gccccggggg cctggctcca 1860 gagattagtc cccttgaggt gctggagcga gacaaggtaa ctcagctgtt gctgcagcaa 1920 gataaggtgc cagagcctgc atctctcagc agtaatcatt ccctgaccag ctgttttact 1980 aaccagggat acttcttttt ccacctgcca gatgctctgg agattgaggc atgtcaagtc 2040 tattttacat acgaccccta cagtgaggaa gacccagatg agggggtcgc aggagctccc 2100 acaggttcat ccccacagcc actccagcct ttgtcaggcg aggatgacgc atactgcaca 2160 tttccaagcc gcgatgatct ccttctgttc tcaccaagcc tgctgggggg acctagtcct 2220 cctagcaccg ctccaggagg gtctggcgca ggagaggaac gaatgcctcc aagtcttcag 2280 gagcgggtcc cccgggattg ggaccctcag cccctgggcc cccccacccc tggagtccct 2340 gacctcgtgg acttccaacc cccacccgag ttggtgctta gagaagccgg agaggaggtg 2400 cctgacgccg gccctagaga gggagtcagt tttccatgga gtcggccacc aggacagggc 2460 gaatttcgag ctctcaacgc tcgtctgcct ttgaacaccg atgcctattt gtctctccag 2520 gaactgcagg ggcaagatcc tacccacctc gtcagagcca aaagagctcc tgtgaagcag 2580 actctgaact ttgatttgct caaactggct ggcgacgtgg aatctaatcc aggcccaatg 2640 ctgaaaccca gtctgccatt cacctctttg ctcttcctgc agctccctct gttgggtgtc 2700 gggctgaaca caactattct gactccaaat gggaacgagg acaccaccgc cgatttcttc 2760 cttactacca tgcccaccga ctccctcagc gtgagtactt tgcccctccc agaagtgcag 2820 tgcttcgtct tcaacgtcga gtacatgaac tgtacttgga acagctcttc agagcctcag 2880 cctaccaacc tcacattgca ctattggtac aagaacagcg ataacgataa agtgcagaag 2940 tgctcccact atctgtttag tgaagagatc accagtgggt gccagctgca gaagaaggaa 3000 attcacctct atcagacttt tgtggtgcag ttgcaagatc cccgggagcc taggaggcag 3060 gccacccaaa tgcttaagct gcaaaatctc gttattcctt gggctcccga gaatctcaca 3120 ttgcacaagc tctccgagtc acagctcgaa ctgaattgga ataataggtt cctgaaccac 3180 tgcctcgagc acctggtgca gtaccggaca gactgggacc acagttggac tgagcaatct 3240 gtggactata gacataaatt ctccctgcct agcgtcgacg ggcagaaacg ttacaccttt 3300 agggtgcggt ctcgttttaa tccactgtgt gggtctgccc agcactggtc agagtggtca 3360 caccctattc attggggtag taatacatct aaggagaatc cattcctctt cgccctcgag 3420 gctgtggtga tcagcgtggg aagtatgggc ctcatcattt ctctcctgtg cgtgtacttt 3480 tggctggaac ggactatgcc tcgtatccct acactcaaaa atctcgagga tctggtgact 3540 gagtatcacg gcaacttttc agcctggtca ggagtgtcaa agggattggc cgaatctctc 3600 cagcccgact atagtgagag gctttgtctg gtttccgaaa tccccccaaa aggcggagca 3660 ttgggtgagg gccccggagc ttcaccctgt aaccaacact ccccttactg ggcaccccct 3720 tgttacactc tgaagccaga aactagagca aagagggctc cagtgaaaca gactttgaat 3780 ttcgacctgc tgaagctggc aggtgacgta gagtcaaacc ccggccccat ggtgtctgtc 3840 attaagcctg aaatgaaaat taagctctgc atgaggggta ctgtgaacgg ccacaatttt 3900 gtgatcgagg gcgaaggaaa aggaaatccc tacgaaggga ctcagattct tgacctgaac 3960 gttactgaag gggctcctct cccctttgct tacgacatcc tgaccaccgt gtttcagtat 4020 ggtaaccgag cttttacaaa gtatcctgct gatatacagg actattttaa gcaaacattc 4080 ccagagggct accactggga gcggtctatg acctatgaag accaaggtat ttgcaccgct 4140 accagcaaca tctcaatgcg aggcgactgt ttcttttacg atatcagatt cgatggaaca 4200 aacttccccc ccaacggacc cgttatgcaa aagaaaacac tgaaatggga acccagcaca 4260 gagaagatgt atgttgagga cggggttctg aagggggacg tgaatatgcg gctcttgctg 4320 gaaggcggag gccactatag atgtgatttc aagacaactt acaaggctaa gaaggaagtg 4380 aggttgcctg acgcccataa gatcgatcat cgaatcgaga ttttgaagca tgacaaggat 4440 tataataagg tgaaactgta cgagaacgcc gtggcccggt actccatgct ccccagtcaa 4500 gctaaa 4506 <210> 21 <211> 1202 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Amino acid sequence of human IL-2Rβγ-Azamigreen fusion <400> 21 Met Ala Ala Pro Ala Leu Ser Trp Arg Leu Pro Leu Leu Ile Leu Leu 1 5 10 15 Leu Pro Leu Ala Thr Ser Trp Ala Ser Ala Ala Val Asn Gly Thr Ser 20 25 30 Gln Phe Thr Cys Phe Tyr Asn Ser Arg Ala Asn Ile Ser Cys Val Trp 35 40 45 Ser Gln Asp Gly Ala Leu Gln Asp Thr Ser Cys Gln Val His Ala Trp 50 55 60 Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys Glu Leu Leu Pro Val Ser 65 70 75 80 Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu Gly Ala Pro Asp Ser Gln 85 90 95 Lys Leu Thr Thr Val Asp Ile Val Thr Leu Arg Val Leu Cys Arg Glu 100 105 110 Gly Val Arg Trp Arg Val Met Ala Ile Gln Asp Phe Lys Pro Phe Glu 115 120 125 Asn Leu Arg Leu Met Ala Pro Ile Ser Leu Gln Val Val His Val Glu 130 135 140 Thr His Arg Cys Asn Ile Ser Trp Glu Ile Ser Gln Ala Ser His Tyr 145 150 155 160 Phe Glu Arg His Leu Glu Phe Glu Ala Arg Thr Leu Ser Pro Gly His 165 170 175 Thr Trp Glu Glu Ala Pro Leu Leu Thr Leu Lys Gln Lys Gln Glu Trp 180 185 190 Ile Cys Leu Glu Thr Leu Thr Pro Asp Thr Gln Tyr Glu Phe Gln Val 195 200 205 Arg Val Lys Pro Leu Gln Gly Glu Phe Thr Thr Trp Ser Pro Trp Ser 210 215 220 Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala Ala Leu Gly Lys Asp Thr 225 230 235 240 Ile Pro Trp Leu Gly His Leu Leu Val Gly Leu Ser Gly Ala Phe Gly 245 250 255 Phe Ile Ile Leu Val Tyr Leu Leu Ile Asn Cys Arg Asn Thr Gly Pro 260 265 270 Trp Leu Lys Lys Val Leu Lys Cys Asn Thr Pro Asp Pro Ser Lys Phe 275 280 285 Phe Ser Gln Leu Ser Ser Glu His Gly Gly Asp Val Gln Lys Trp Leu 290 295 300 Ser Ser Pro Phe Pro Ser Ser Ser Phe Ser Pro Gly Gly Leu Ala Pro 305 310 315 320 Glu Ile Ser Pro Leu Glu Val Leu Glu Arg Asp Lys Val Thr Gln Leu 325 330 335 Leu Leu Gln Gln Asp Lys Val Pro Glu Pro Ala Ser Leu Ser Ser Asn 340 345 350 His Ser Leu Thr Ser Cys Phe Thr Asn Gln Gly Tyr Phe Phe Phe His 355 360 365 Leu Pro Asp Ala Leu Glu Ile Glu Ala Cys Gln Val Tyr Phe Thr Tyr 370 375 380 Asp Pro Tyr Ser Glu Glu Asp Pro Asp Glu Gly Val Ala Gly Ala Pro 385 390 395 400 Thr Gly Ser Ser Pro Gln Pro Leu Gln Pro Leu Ser Gly Glu Asp Asp 405 410 415 Ala Tyr Cys Thr Phe Pro Ser Arg Asp Asp Leu Leu Leu Phe Ser Pro 420 425 430 Ser Leu Leu Gly Gly Pro Ser Pro Pro Ser Thr Ala Pro Gly Gly Ser 435 440 445 Gly Ala Gly Glu Glu Arg Met Pro Pro Ser Leu Gln Glu Arg Val Pro 450 455 460 Arg Asp Trp Asp Pro Gln Pro Leu Gly Pro Pro Thr Pro Gly Val Pro 465 470 475 480 Asp Leu Val Asp Phe Gln Pro Pro Pro Glu Leu Val Leu Arg Glu Ala 485 490 495 Gly Glu Glu Val Pro Asp Ala Gly Pro Arg Glu Gly Val Ser Phe Pro 500 505 510 Trp Ser Arg Pro Pro Gly Gln Gly Glu Phe Arg Ala Leu Asn Ala Arg 515 520 525 Leu Pro Leu Asn Thr Asp Ala Tyr Leu Ser Leu Gln Glu Leu Gln Gly 530 535 540 Gln Asp Pro Thr His Leu Val Arg Ala Lys Arg Ala Pro Val Lys Gln 545 550 555 560 Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn 565 570 575 Pro Gly Pro Met Leu Lys Pro Ser Leu Pro Phe Thr Ser Leu Leu Phe 580 585 590 Leu Gln Leu Pro Leu Leu Gly Val Gly Leu Asn Thr Thr Ile Leu Thr 595 600 605 Pro Asn Gly Asn Glu Asp Thr Thr Ala Asp Phe Phe Leu Thr Thr Met 610 615 620 Pro Thr Asp Ser Leu Ser Val Ser Thr Leu Pro Leu Pro Glu Val Gln 625 630 635 640 Cys Phe Val Phe Asn Val Glu Tyr Met Asn Cys Thr Trp Asn Ser Ser 645 650 655 Ser Glu Pro Gln Pro Thr Asn Leu Thr Leu His Tyr Trp Tyr Lys Asn 660 665 670 Ser Asp Asn Asp Lys Val Gln Lys Cys Ser His Tyr Leu Phe Ser Glu 675 680 685 Glu Ile Thr Ser Gly Cys Gln Leu Gln Lys Lys Glu Ile His Leu Tyr 690 695 700 Gln Thr Phe Val Val Gln Leu Gln Asp Pro Arg Glu Pro Arg Arg Gln 705 710 715 720 Ala Thr Gln Met Leu Lys Leu Gln Asn Leu Val Ile Pro Trp Ala Pro 725 730 735 Glu Asn Leu Thr Leu His Lys Leu Ser Glu Ser Gln Leu Glu Leu Asn 740 745 750 Trp Asn Asn Arg Phe Leu Asn His Cys Leu Glu His Leu Val Gln Tyr 755 760 765 Arg Thr Asp Trp Asp His Ser Trp Thr Glu Gln Ser Val Asp Tyr Arg 770 775 780 His Lys Phe Ser Leu Pro Ser Val Asp Gly Gln Lys Arg Tyr Thr Phe 785 790 795 800 Arg Val Arg Ser Arg Phe Asn Pro Leu Cys Gly Ser Ala Gln His Trp 805 810 815 Ser Glu Trp Ser His Pro Ile His Trp Gly Ser Asn Thr Ser Lys Glu 820 825 830 Asn Pro Phe Leu Phe Ala Leu Glu Ala Val Val Ile Ser Val Gly Ser 835 840 845 Methionine, Glycine, Leucine, Isoleucine, Isoleucine, Serine, Leucine, Leucine, Cysteine, Valine, Tyrosine, Phenylalanine, Tryptophan, Leucine, Glutamic acid, Arginine 850 855 860 Threonine, Methionine, Proline, Arginine, Isoleucine, Proline, Threonine, Leucine, Lysine, Asparagine, Leucine, Glutamic acid, Aspartic acid, Leucine, Valine, Threonine 865 870 875 880 Glutamic acid, Tyrosine, Histidine, Glycine, Asparagine, Phenylalanine, Serine, Alanine, Tryptophan, Serine, Glycine, Valine, Serine, Lysine, Glycine, Leucine 885 890 895 Alanine, Glutamic acid, Serine, Leucine, Glutamine, Proline, Aspartic acid, Tyrosine, Serine, Glutamic acid, Arginine, Leucine, Cysteine, Leucine, Valine, Serine 900 905 910 Glutamic acid, Isoleucine, Proline, Proline, Lysine, Glycine, Glycine, Alanine, Leucine, Glycine, Glutamic acid, Glycine, Proline, Glycine, Alanine, Serine 915 920 925 Proline, Cysteine, Asparagine, Glutamine, Histidine, Serine, Proline, Tyrosine, Tryptophan, Alanine, Proline, Proline, Cysteine, Tyrosine, Threonine, Leucine 930 935 940 Lysine, Proline, Glutamic acid, Threonine, Arginine, Alanine, Lysine, Arginine, Alanine, Proline, Valine, Lysine, Glutamine, Threonine, Leucine, Asparagine 945 950 955 960 Phenylalanine, Aspartic acid, Leucine, Leucine, Lysine, Leucine, Alanine, Glycine, Aspartic acid, Valine, Glutamic acid, Serine, Asparagine, Proline, Glycine, Proline 965 970 975 Methionine, Valine, Serine, Valine, Isoleucine, Lysine, Proline, Glutamic acid, Methionine, Lysine, Isoleucine, Lysine, Leucine, Cysteine, Methionine, Arginine 980 985 990 Glycine, Threonine, Valine, Asparagine, Glycine, Histidine, Asparagine, Phenylalanine, Valine, Isoleucine, Glutamic acid, Glycine, Glutamic acid, Glycine, Lysine, Glycine 995 1000 1005 Asn Pro Tyr Glu Gly Thr Gln Ile Leu Asp Leu Asn Val Thr Glu 1010 1015 1020 Gly Ala Pro Leu Pro Phe Ala Tyr Asp Ile Leu Thr Thr Val Phe 1025 1030 1035 Gln Tyr Gly Asn Arg Ala Phe Thr Lys Tyr Pro Ala Asp Ile Gln 1040 1045 1050 Asp Tyr Phe Lys Gln Thr Phe Pro Glu Gly Tyr His Trp Glu Arg 1055 1060 1065 Ser Met Thr Tyr Glu Asp Gln Gly Ile Cys Thr Ala Thr Ser Asn 1070 1075 1080 Ile Ser Met Arg Gly Asp Cys Phe Phe Tyr Asp Ile Arg Phe Asp 1085 1090 1095 Gly Thr Asn Phe Pro Pro Asn Gly Pro Val Met Gln Lys Lys Thr 1100 1105 1110 Leu Lys Trp Glu Pro Ser Thr Glu Lys Met Tyr Val Glu Asp Gly 1115 1120 1125 Val Leu Lys Gly Asp Val Asn Met Arg Leu Leu Leu Glu Gly Gly 1130 1135 1140 Gly His Tyr Arg Cys Asp Phe Lys Thr Thr Tyr Lys Ala Lys Lys 1145 1150 1155 Glu Val Arg Leu Pro Asp Ala His Lys Ile Asp His Arg Ile Glu 1160 1165 1170 Ile Leu Lys His Asp Lys Asp Tyr Asn Lys Val Lys Leu Tyr Glu 1175 1180 1185 Asn Ala Val Ala Arg Tyr Ser Met Leu Pro Ser Gln Ala Lys 1190 1195 1200 <210> 22 <211> 3606 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Base sequence of human IL-2Rβγ-Azamigreen fusion <400> 22 atggctgcac cagccctgtc ctggcgtttg cctctcctta tattgctgct tcctcttgct 60 acctcttggg cttccgctgc agtgaatggg acctcccaat ttacatgctt ctacaacagt 120 agagccaaca tttcatgtgt gtggagccag gatggcgctc tgcaagatac atcttgccag 180 gtccacgcct ggccagatag acggcggtgg aatcagacct gtgagcttct gcccgtgtcc 240 caggcaagtt gggcttgcaa ccttatactg ggcgctcccg atagtcagaa gctgaccact 300 gtggacatcg tgaccctgcg ggtgctttgt agggagggcg tccggtggag agtcatggct 360 atacaggatt ttaaaccctt cgagaacctt cgcctcatgg cccccatctc tctgcaagtc 420 gtgcatgtag agacacatag gtgtaacatc tcttgggaaa tctctcaggc ttcacactac 480 tttgagaggc acctggagtt tgaggcccgt actctgagcc caggacatac ttgggaggag 540 gcacccctgc tcaccctcaa gcagaagcag gagtggatat gtctggagac tttgactccc 600 gacactcagt atgagttcca ggtgagagtg aaacccctgc agggggagtt tacaacctgg 660 tcaccatgga gtcagcccct tgcctttcga actaaacctg ccgccctcgg caaggacaca 720 atcccctggc tcggtcacct gcttgtcggc ctttccggcg cttttggttt tatcatcctg 780 gtctaccttc tcatcaactg ccgcaatact gggccttggc tgaagaaagt ccttaagtgt 840 aacaccccag atccaagtaa gttcttctct cagctgtcca gtgagcacgg tggcgatgtc 900 cagaagtggt tgagcagtcc cttcccctcc tccagtttca gccccggggg cctggctcca 960 gagattagtc cccttgaggt gctggagcga gacaaggtaa ctcagctgtt gctgcagcaa 1020 gataaggtgc cagagcctgc atctctcagc agtaatcatt ccctgaccag ctgttttact 1080 aaccagggat acttcttttt ccacctgcca gatgctctgg agattgaggc atgtcaagtc 1140 tattttacat acgaccccta cagtgaggaa gacccagatg agggggtcgc aggagctccc 1200 acaggttcat ccccacagcc actccagcct ttgtcaggcg aggatgacgc atactgcaca 1260 tttccaagcc gcgatgatct ccttctgttc tcaccaagcc tgctgggggg acctagtcct 1320 cctagcaccg ctccaggagg gtctggcgca ggagaggaac gaatgcctcc aagtcttcag 1380 gagcgggtcc cccgggattg ggaccctcag cccctgggcc cccccacccc tggagtccct 1440 gacctcgtgg acttccaacc cccacccgag ttggtgctta gagaagccgg agaggaggtg 1500 cctgacgccg gccctagaga gggagtcagt tttccatgga gtcggccacc aggacagggc 1560 gaatttcgag ctctcaacgc tcgtctgcct ttgaacaccg atgcctattt gtctctccag 1620 gaactgcagg ggcaagatcc tacccacctc gtcagagcca aaagagctcc tgtgaagcag 1680 actctgaact ttgatttgct caaactggct ggcgacgtgg aatctaatcc aggcccaatg 1740 ctgaaaccca gtctgccatt cacctctttg ctcttcctgc agctccctct gttgggtgtc 1800 gggctgaaca caactattct gactccaaat gggaacgagg acaccaccgc cgatttcttc 1860 cttactacca tgcccaccga ctccctcagc gtgagtactt tgcccctccc agaagtgcag 1920 tgcttcgtct tcaacgtcga gtacatgaac tgtacttgga acagctcttc agagcctcag 1980 cctaccaacc tcacattgca ctattggtac aagaacagcg ataacgataa agtgcagaag 2040 tgctcccact atctgtttag tgaagagatc accagtgggt gccagctgca gaagaaggaa 2100 attcacctct atcagacttt tgtggtgcag ttgcaagatc cccgggagcc taggaggcag 2160 gccacccaaa tgcttaagct gcaaaatctc gttattcctt gggctcccga gaatctcaca 2220 ttgcacaagc tctccgagtc acagctcgaa ctgaattgga ataataggtt cctgaaccac 2280 tgcctcgagc acctggtgca gtaccggaca gactgggacc acagttggac tgagcaatct 2340 gtggactata gacataaatt ctccctgcct agcgtcgacg ggcagaaacg ttacaccttt 2400 agggtgcggt ctcgttttaa tccactgtgt gggtctgccc agcactggtc agagtggtca 2460 caccctattc attggggtag taatacatct aaggagaatc cattcctctt cgccctcgag 2520 gctgtggtga tcagcgtggg aagtatgggc ctcatcattt ctctcctgtg cgtgtacttt 2580 tggctggaac ggactatgcc tcgtatccct acactcaaaa atctcgagga tctggtgact 2640 gagtatcacg gcaacttttc agcctggtca ggagtgtcaa agggattggc cgaatctctc 2700 cagcccgact atagtgagag gctttgtctg gtttccgaaa tccccccaaa aggcggagca 2760 ttgggtgagg gccccggagc ttcaccctgt aaccaacact ccccttactg ggcaccccct 2820 tgttacactc tgaagccaga aactagagca aagagggctc cagtgaaaca gactttgaat 2880 ttcgacctgc tgaagctggc aggtgacgta gagtcaaacc ccggccccat ggtgtctgtc 2940 attaagcctg aaatgaaaat taagctctgc atgaggggta ctgtgaacgg ccacaatttt 3000 gtgatcgagg gcgaaggaaa aggaaatccc tacgaaggga ctcagattct tgacctgaac 3060 gttactgaag gggctcctct cccctttgct tacgacatcc tgaccaccgt gtttcagtat 3120 ggtaaccgag cttttacaaa gtatcctgct gatatacagg actattttaa gcaaacattc 3180 ccagagggct accactggga gcggtctatg acctatgaag accaaggtat ttgcaccgct 3240 accagcaaca tctcaatgcg aggcgactgt ttcttttacg atatcagatt cgatggaaca 3300 aacttccccc ccaacggacc cgttatgcaa aagaaaacac tgaaatggga acccagcaca 3360 gagaagatgt atgttgagga cggggttctg aagggggacg tgaatatgcg gctcttgctg 3420 gaaggcggag gccactatag atgtgatttc aagacaactt acaaggctaa gaaggaagtg 3480 aggttgcctg acgcccataa gatcgatcat cgaatcgaga ttttgaagca tgacaaggat 3540 tataataagg tgaaactgta cgagaacgcc gtggcccggt actccatgct ccccagtcaa 3600 gctaaa 3606 <210> 23 <211> 454 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Amino acid sequence of human CD25ECD-Fc-Avitag <400> 23 Met Arg Val Leu Ile Leu Leu Trp Leu Phe Thr Ala Phe Pro Gly Ile 1 5 10 15 Leu Ser Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr 20 25 30 Phe Lys Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys 35 40 45 Lys Arg Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys 50 55 60 Thr Gly Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr 65 70 75 80 Ser Ser Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu 85 90 95 Glu Gln Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro 100 105 110 Val Asp Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp 115 120 125 Glu Asn Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met 130 135 140 Val Tyr Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro 145 150 155 160 Ala Glu Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln 165 170 175 Pro Gln Leu Ile Cys Thr Gly Glu Met Glu Thr Ser Gln Phe Pro Gly 180 185 190 Glu Glu Lys Pro Gln Ala Ser Pro Glu Gly Arg Pro Glu Ser Glu Thr 195 200 205 Ser Cys Leu Val Thr Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro 210 215 220 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 225 230 235 240 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 245 250 255 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 260 265 270 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 275 280 285 Gln Tyr Gly Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 290 295 300 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 305 310 315 320 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 325 330 335 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 340 345 350 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 355 360 365 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 370 375 380 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 385 390 395 400 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 405 410 415 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 420 425 430 Lys Ser Leu Ser Leu Ser Pro Gly Leu Asn Asp Ile Phe Glu Ala Gln 435 440 445 Lys Ile Glu Trp His Glu 450 <210> 24 <211> 1362 <212> DNA <213> Synthetic sequence <220> <223> Synthetic sequence description: Base sequence of human CD25ECD-Fc-Avitag <400> 24 atgagagtgc ttattttatt gtggctgttc acagcctttc ctggtattct tagtgagctc 60 tgtgacgatg acccgccaga gatcccacac gccacattca aagccatggc ctacaaggaa 120 ggaaccatgt tgaactgtga atgcaagaga ggtttccgca gaataaaaag cgggtcactc 180 tatatgctct gtacaggaaa ctctagccac tcgtcctggg acaaccaatg tcaatgcaca 240 agctctgcca ctcggaacac aacgaaacaa gtgacacctc aacctgaaga acagaaagaa 300 aggaaaacca cagaaatgca aagtccaatg cagccagtgg accaagcgag ccttccaggt 360 cactgcaggg aacctccacc atgggaaaat gaagccacag agagaattta tcatttcgtg 420 gtggggcaga tggtttatta tcagtgcgtc cagggataca gggctctaca cagaggtcct 480 gctgagagcg tctgcaaaat gacccacggg aagacaaggt ggacccagcc ccagctcata 540 tgcacaggtg aaatggagac cagtcagttt ccaggtgaag agaagcctca ggcaagcccc 600 gaaggccgtc ctgagagtga gacttcctgc ctcgtcacaa cagacaaaac tcacacatgc 660 ccaccgtgcc cagcacctga actcctgggg ggaccgtcag tcttcctctt ccccccaaaa 720 cccaaggaca ccctcatgat ctcccggacc cctgaggtca catgcgtggt ggtggacgtg 780 agccacgaag accctgaggt caagttcaac tggtacgtgg acggcgtgga ggtgcataat 840 gccaagacaa agccgcggga ggagcagtac gggagcacgt accgtgtggt cagcgtcctc 900 accgtcctgc accaggactg gctgaatggc aaggagtaca agtgcaaggt ctccaacaaa 960 gccctcccag cccccatcga gaaaaccatc tccaaagcca aagggcagcc ccgagaacca 1020 caggtgtaca ccctgccccc atcccgggaa gagatgacca agaaccaggt cagcctgacc 1080 tgcctggtca aaggcttcta tcccagcgac atcgccgtgg agtgggagag caatgggcag 1140 ccggagaaca actacaagac cacgcctccc gtgctggact ccgacggctc cttcttcctc 1200 tacagcaagc tcaccgtgga caagagcagg tggcagcagg ggaacgtctt ctcatgctcc 1260 gtgatgcatg aggctctgca caaccactac acgcagaaga gcctctccct gtctccggga 1320 cttaacgaca tcttcgaagc acaaaagatc gaatggcacg ag 1362 <210> 25 <211> 1010 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Amino acid sequence of human CD122 ECD-Fc-Avitag-8His_human CD132 ECD-Fc-FLAG <400> 25 Met Arg Val Leu Ile Leu Leu Trp Leu Phe Thr Ala Phe Pro Gly Ile 1 5 10 15 Leu Ser Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser 20 25 30 Arg Ala Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp 35 40 45 Thr Ser Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln 50 55 60 Thr Cys Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu 65 70 75 80 Ile Leu Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val 85 90 95 Thr Leu Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala 100 105 110 Ile Gln Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile 115 120 125 Ser Leu Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp 130 135 140 Glu Ile Ser Gln Ala Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu 145 150 155 160 Ala Arg Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu 165 170 175 Thr Leu Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro 180 185 190 Asp Thr Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu 195 200 205 Phe Thr Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys 210 215 220 Pro Ala Ala Leu Gly Lys Asp Thr Gly Ala Gln Asp Lys Thr His Thr 225 230 235 240 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 245 250 255 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 260 265 270 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 275 280 285 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 290 295 300 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 305 310 315 320 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 325 330 335 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 340 345 350 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro 355 360 365 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val 370 375 380 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 385 390 395 400 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 405 410 415 Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 420 425 430 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 435 440 445 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Leu 450 455 460 Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu His His His 465 470 475 480 His His His His His Arg Ala Lys Arg Ala Pro Val Lys Gln Thr Leu 485 490 495 Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly 500 505 510 Pro Met Asn Leu Gly Leu Ser Leu Ile Phe Leu Ala Leu Ile Leu Lys 515 520 525 Gly Val Gln Cys Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly Asn Glu 530 535 540 Asp Thr Thr Ala Asp Phe Phe Leu Thr Thr Met Pro Thr Asp Ser Leu 545 550 555 560 Ser Val Ser Thr Leu Pro Leu Pro Glu Val Gln Cys Phe Val Phe Asn 565 570 575 Val Glu Tyr Met Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro Gln Pro 580 585 590 Thr Asn Leu Thr Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn Asp Lys 595 600 605 Val Gln Lys Cys Ser His Tyr Leu Phe Ser Glu Glu Ile Thr Ser Gly 610 615 620 Cys Gln Leu Gln Lys Lys Glu Ile His Leu Tyr Gln Thr Phe Val Val 625 630 635 640 Gln Leu Gln Asp Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln Met Leu 645 650 655 Lys Leu Gln Asn Leu Val Ile Pro Trp Ala Pro Glu Asn Leu Thr Leu 660 665 670 His Lys Leu Ser Glu Ser Gln Leu Glu Leu Asn Trp Asn Asn Arg Phe 675 680 685 Leu Asn His Cys Leu Glu His Leu Val Gln Tyr Arg Thr Asp Trp Asp 690 695 700 His Ser Trp Thr Glu Gln Ser Val Asp Tyr Arg His Lys Phe Ser Leu 705 710 715 720 Pro Ser Val Asp Gly Gln Lys Arg Tyr Thr Phe Arg Val Arg Ser Arg 725 730 735 Phe Asn Pro Leu Cys Gly Ser Ala Gln His Trp Ser Glu Trp Ser His 740 745 750 Pro Ile His Trp Gly Ser Asn Thr Ser Lys Glu Asn Pro Phe Leu Phe 755 760 765 Ala Leu Glu Ala Gly Ala Gln Asp Lys Thr His Thr Cys Pro Pro Cys 770 775 780 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 785 790 795 800 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 805 810 815 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 820 825 830 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 835 840 845 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 850 855 860 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 865 870 875 880 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 885 890 895 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu 900 905 910 Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr 915 920 925 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 930 935 940 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 945 950 955 960 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 965 970 975 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 980 985 990 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Asp Tyr Lys Asp Asp Asp 995 1000 1005 Asp Lys 1010 <210> 26 <211> 3030 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: The base sequence of human CD122 ECD-Fc-Avitag-8His_human CD132 ECD-Fc-FLAG <400> 26 atgcgtgtgt tgatactcct ctggcttttc actgcttttc caggaattct ttcagccgta 60 aacggcacca gtcaatttac ctgcttctat aatagtcgcg ccaatatctc atgtgtttgg 120 agtcaagatg gcgctctgca ggacacaagt tgccaagtac acgcctggcc agatcgccgt 180 cggtggaacc agacttgtga gttgcttccc gtttctcagg catcctgggc ttgtaacctt 240 attctggggg ctcccgattc acagaagctg acaacagtgg atatcgttac cctcagagtc 300 ctttgtaggg agggcgtgag gtggcgtgtt atggccatcc aagacttcaa gccatttgaa 360 aacttgcgcc ttatggcccc aatttcattg caagtggttc atgtggagac acacaggtgc 420 aacatcagtt gggaaatttc tcaagcttct cactacttcg agaggcacct tgaatttgaa 480 gcccggaccc tctctcccgg acatacatgg gaagaggctc cattgctgac tctgaagcag 540 aaacaggaat ggatttgcct tgagacattg accccagata cacaatacga attccaggtc 600 cgtgttaagc ctctccaggg ggagtttacc acatggagcc cttggagtca gccccttgct 660 ttccggacca aaccagctgc tctgggtaag gacacagggg ctcaggacaa gacacacaca 720 tgtcctcctt gccccgctcc tgagctcctg ggcgggcctt cagtgtttct cttccctcca 780 aaacctaagg acacccttat gatatctcga acaccagaag tcacttgcgt tgtggtagac 840 gtgtcccatg aagaccctga ggtaaaattc aattggtatg ttgatggagt agaggtacac 900 aatgctaaga caaaacctcg agaggagcag tacaactcca cctatagggt tgtttctgta 960 cttaccgtct tgcatcagga ttggcttaac ggcaaggagt ataaatgtaa ggtgtctaat 1020 aaagcacttc ctgcccctat agaaaaaacc atatccaagg caaagggaca gcctcgtgaa 1080 cctcaggtct gtactctgcc cccatcccgg gacgaattga caaagaatca ggtaagcctc 1140 tcttgcgctg ttaaaggttt ctacccctcc gacatagccg tcgagtggga atccaatggc 1200 cagcccgaga ataattacaa aactactcct cccgtccttg atagcgatgg tagtttcttt 1260 cttgtatcca agttgacagt ggacaagtca agatggcagc agggtaatgt atttagctgc 1320 tccgttatgc atgaggccct tcataaccat tacactcaga aatccctctc actctcccct 1380 ggcaaaggac ttaacgacat cttcgaagca caaaagatcg aatggcacga gcaccaccac 1440 catcaccacc atcacagggc aaagcgggct ccagttaagc agaccttgaa ctttgatctt 1500 ctgaaactgg ccggtgacgt tgagtctaac cccggaccca tgaatttggg gctttccttg 1560 atttttctgg cccttatcct taaaggggtg cagtgtttga acaccaccat tttgactccc 1620 aatgggaacg aagacacaac cgccgatttt tttctcacaa ccatgcctac cgatagcttg 1680 tccgtttcaa ctctgcctct tccagaagtt cagtgtttcg tgtttaatgt cgagtatatg 1740 aattgtacat ggaactcatc ttccgaacca caacctacca accttacttt gcactattgg 1800 tacaagaact ctgacaatga caaggtccag aagtgctctc actatttgtt ctctgaagag 1860 attacatctg gctgtcaatt gcaaaagaaa gagatccacc tttaccaaac cttcgtcgtc 1920 caattgcagg acccacgaga gccccgccgg caagctactc aaatgcttaa gctccagaat 1980 ctcgtcatcc cctgggcccc agagaacctg acacttcata agttgagtga aagtcagctt 2040 gagttgaact ggaacaatag atttctcaac cactgtctgg aacacctcgt ccaataccga 2100 accgactggg atcattcatg gaccgagcaa tctgttgact atcgccataa attctctttg 2160 ccatccgttg atgggcaaaa acgttacacc ttccgtgtcc gctcacgatt taatcctctc 2220 tgtggctccg cacagcattg gagcgagtgg agccacccta tacactgggg ttctaatact 2280 tctaaggaaa accctttcct ctttgcactt gaggccgggg cacaagataa gactcatact 2340 tgtcctccat gtccagcccc cgaattgctg ggtggaccca gcgtcttcct gttcccccca 2400 aagcccaaag acacactcat gataagtagg actcccgagg taacctgtgt cgtagtcgac 2460 gtaagtcatg aagatcctga ggtgaagttt aattggtatg tggatggggt tgaggttcac 2520 aacgctaaaa ccaagccaag agaggagcaa tacaacagta cttatcgcgt cgtgagcgta 2580 ctcacagttc tgcatcaaga ttggctgaat ggcaaagagt acaaatgtaa agtaagcaat 2640 aaggcacttc ctgctcctat cgaaaagact atcagcaaag caaaaggcca accaagggag 2700 cctcaagtat atacactccc accttgtaga gatgagttga ctaagaatca ggtaagtctc 2760 tggtgtcttg tcaagggatt ttatccttca gatatagctg tggagtggga gtctaacggc 2820 caacctgaaa acaactataa gaccaccccc cctgtactgg atagcgatgg tagttttttc 2880 ctctactcca agctcaccgt ggacaagtct cgctggcaac aaggtaacgt gttttcctgc 2940 agcgttatgc acgaggcact tcataatcat tacacacaaa aatcactgtc tttgagtccc 3000 ggtaaagact acaaagacga cgatgacaag 3030 <210> 27 <211> 426 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Base sequence of 8His - S4(oAzZK) / F78(oAzZK) <400> 27 atgcatcatc atcaccatca tcatcacgcc ccgacctaga gcagcaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaacta gcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagcgagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat catcagtacc 420 ctgacc 426 <210> 28 <211> 426 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Base sequence of 8His - S5(oAzZK) / F78(oAzZK) <400> 28 atgcatcatc atcaccatca tcatcacgcc ccgaccagct agagcaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaacta gcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagcgagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat catcagtacc 420 ctgacc 426 <210> 29 <211> 426 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Base sequence of 8His-K8(oAzZK) / F78(oAzZK) <400> 29 atgcatcatc atcaccatca tcatcacgcc ccgaccagca gcagcaccta gaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaacta gcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagcgagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat catcagtacc 420 ctgacc 426 <210> 30 <211> 426 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Base sequence of 8His - F78(oAzZK) / H79(oAzZK) <400> 30 atgcatcatc atcaccatca tcatcacgcc ccgaccagca gcagcaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaacta gtagctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagcgagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat catcagtacc 420 ctgacc 426 <210> 31 <211> 426 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Base sequence of 8His - F78(oAzZK) / S99(oAzZK) <400> 31 atgcatcatc atcaccatca tcatcacgcc ccgaccagca gcagcaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaacta gcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg ttaggagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat catcagtacc 420 ctgacc 426 <210> 32 <211> 426 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Base sequence of 8His-F78(oAzZK) / I129(oAzZK) <400> 32 atgcatcatc atcaccatca tcatcacgcc ccgaccagca gcagcaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaacta gcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagcgagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat ctagagtacc 420 ctgacc 426 <210> 33 <211> 426 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Base sequence of 8His-S4(oAzZK) / I129(oAzZK) <400> 33 atgcatcatc atcaccatca tcatcacgcc ccgacctaga gcagcaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaactt tcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagcgagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat ctagagtacc 420 ctgacc 426 <210> 34 <211> 426 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Base sequence of 8His-S5(oAzZK) / I129(oAzZK) <400> 34 atgcatcatc atcaccatca tcatcacgcc ccgaccagct agagcaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaactt tcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagcgagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat ctagagtacc 420 ctgacc 426 <210> 35 <211> 426 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Base sequence of 8His-K8(oAzZK) / I129(oAzZK) <400> 35 atgcatcatc atcaccatca tcatcacgcc ccgaccagca gcagcaccta gaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaactt tcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagcgagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat ctagagtacc 420 ctgacc 426 <210> 36 <211> 426 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Base sequence of 8His-H79(oAzZK) / I129(oAzZK) <400> 36 atgcatcatc atcaccatca tcatcacgcc ccgaccagca gcagcaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaactt ttagctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg tagcgagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat ctagagtacc 420 ctgacc 426 <210> 37 <211> 426 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Base sequence of 8His - S99(oAzZK) / I129(oAzZK) <400> 37 atgcatcatc atcaccatca tcatcacgcc ccgaccagca gcagcaccaa aaagacccag 60 ctgcagctgg aacatctgct gctggatctg cagatgatcc tgaatggcat taacaactat 120 aaaaacccga agctgacccg catgctgacc tttaaatttt atatgccgaa aaaagccacc 180 gagctgaagc atctgcagtg cctggaagaa gaactgaaac cgctggaaga ggtgctgaac 240 ctggcccaga gcaaaaactt tcacctgcgc ccgcgtgacc tgatcagcaa catcaacgtg 300 atcgtgctgg aactgaaggg ttaggagacc accttcatgt gcgaatatgc cgacgagacc 360 gccaccatcg tggaattcct gaaccgctgg atcacctttt cccagagcat ctagagtacc 420 ctgacc 426 <210> 38 <211> 134 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Amino acid sequence of IL - 2 C125S Nter - Met <400> 38 Met Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu 1 5 10 15 His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr 20 25 30 Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro 35 40 45 Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu 50 55 60 Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His 65 70 75 80 Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu 85 90 95 Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr 100 105 110 Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser 115 120 125 Ile Ile Ser Thr Leu Thr 130 <210> 39 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Base sequence of IL-2 C125S Nter-Met <400> 39 atggcaccta cttcaagttc tacaaagaaa acacagctac aactggagca tttactgctg 60 gatttacaga tgattttgaa tggaattaat aattacaaga atcccaaact caccaggatg 120 ctcacattta agttttacat gcccaagaag gccacagaac tgaaacatct tcagtgtcta 180 gaagaagaac tcaaacctct ggaggaagtg ctaaatttag ctcaaagcaa aaactttcac 240 ttaagaccca gggacttaat cagcaatatc aacgtaatag ttctggaact aaagggatct 300 gaaacaacat tcatgtgtga atatgctgat gagacagcaa ccattgtaga atttctgaac 360 agatggatta ccttttcaca aagcatcatc tcaacactga ct 402 <210> 40 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Amino acid sequence of desAla_IL-2 C125S <400> 40 Met 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 Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 41 <211> 399 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: Base sequence of desAla_IL-2 C125S <400> 41 atgcctactt caagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 60 ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 120 acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 180 gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 240 agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 300 acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 360 tggattacct tttcacaaag catcatctca acactgact 399 <210> 42 <211> 402 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Base sequence of F78(oAzZK) <400> 42 atggcaccta cttcaagttc tacaaagaaa acacagctac aactggagca tttactgctg 60 gatttacaga tgattttgaa tggaattaat aattacaaga atcccaaact caccaggatg 120 ctcacattta agttttacat gcccaagaag gccacagaac tgaaacatct tcagtgtcta 180 gaagaagaac tcaaacctct ggaggaagtg ctaaatttag ctcaaagcaa aaactagcac 240 ttaagaccca gggacttaat cagcaatatc aacgtaatag ttctggaact aaagggatct 300 gaaacaacat tcatgtgtga atatgctgat gagacagcaa ccattgtaga atttctgaac 360 agatggatta ccttttcaca aagcatcatc tcaacactga ct 402 <210> 43 <211> 402 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Base sequence of I129(oAzZK) <400> 43 atggcaccta cttcaagttc tacaaagaaa acacagctac aactggagca tttactgctg 60 gatttacaga tgattttgaa tggaattaat aattacaaga atcccaaact caccaggatg 120 ctcacattta agttttacat gcccaagaag gccacagaac tgaaacatct tcagtgtcta 180 gaagaagaac tcaaacctct ggaggaagtg ctaaatttag ctcaaagcaa aaactttcac 240 ttaagaccca gggacttaat cagcaatatc aacgtaatag ttctggaact aaagggatct 300 gaaacaacat tcatgtgtga atatgctgat gagacagcaa ccattgtaga atttctgaac 360 agatggatta ccttttcaca aagcatctag tcaacactga ct 402 <210> 44 <211> 399 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Base sequence of desAla_I129(oAzZK) <400> 44 atgcctactt caagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 60 ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 120 acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 180 gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 240 agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 300 acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 360 tggattacct tttcacaaag catctagtca acactgact 399 <210> 45 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Base sequence of S4(oAzZK) / F78(oAzZK) <400> 45 atggcaccta cttagagttc tacaaagaaa acacagctac aactggagca tttactgctg 60 gatttacaga tgattttgaa tggaattaat aattacaaga atcccaaact caccaggatg 120 ctcacattta agttttacat gcccaagaag gccacagaac tgaaacatct tcagtgtcta 180 gaagaagaac tcaaacctct ggaggaagtg ctaaatttag ctcaaagcaa aaactagcac 240 ttaagaccca gggacttaat cagcaatatc aacgtaatag ttctggaact aaagggatct 300 gaaacaacat tcatgtgtga atatgctgat gagacagcaa ccattgtaga atttctgaac 360 agatggatta ccttttcaca aagcatcatc tcaacactga ct 402 <210> 46 <211> 402 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Base sequence of S5(oAzZK) / F78(oAzZK) <400> 46 atggcaccta cttcatagtc tacaaagaaa acacagctac aactggagca tttactgctg 60 gatttacaga tgattttgaa tggaattaat aattacaaga atcccaaact caccaggatg 120 ctcacattta agttttacat gcccaagaag gccacagaac tgaaacatct tcagtgtcta 180 gaagaagaac tcaaacctct ggaggaagtg ctaaatttag ctcaaagcaa aaactagcac 240 ttaagaccca gggacttaat cagcaatatc aacgtaatag ttctggaact aaagggatct 300 gaaacaacat tcatgtgtga atatgctgat gagacagcaa ccattgtaga atttctgaac 360 agatggatta ccttttcaca aagcatcatc tcaacactga ct 402 <210> 47 <211> 402 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Base sequence of K8(oAzZK) / F78(oAzZK) <400> 47 atggcaccta cttcaagttc tacatagaaa acacagctac aactggagca tttactgctg 60 gatttacaga tgattttgaa tggaattaat aattacaaga atcccaaact caccaggatg 120 ctcacattta agttttacat gcccaagaag gccacagaac tgaaacatct tcagtgtcta 180 gaagaagaac tcaaacctct ggaggaagtg ctaaatttag ctcaaagcaa aaactagcac 240 ttaagaccca gggacttaat cagcaatatc aacgtaatag ttctggaact aaagggatct 300 gaaacaacat tcatgtgtga atatgctgat gagacagcaa ccattgtaga atttctgaac 360 agatggatta ccttttcaca aagcatcatc tcaacactga ct 402 <210> 48 <211> 402 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Base sequence of S4(oAzZK) / I129(oAzZK) <400> 48 atggcaccta cttagagttc tacaaagaaa acacagctac aactggagca tttactgctg 60 gatttacaga tgattttgaa tggaattaat aattacaaga atcccaaact caccaggatg 120 ctcacattta agttttacat gcccaagaag gccacagaac tgaaacatct tcagtgtcta 180 gaagaagaac tcaaacctct ggaggaagtg ctaaatttag ctcaaagcaa aaactttcac 240 ttaagaccca gggacttaat cagcaatatc aacgtaatag ttctggaact aaagggatct 300 gaaacaacat tcatgtgtga atatgctgat gagacagcaa ccattgtaga atttctgaac 360 agatggatta ccttttcaca aagcatctag tcaacactga ct 402 <210> 49 <211> 402 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Base sequence of S5(oAzZK) / I129(oAzZK) <400> 49 atggcaccta cttcatagtc tacaaagaaa acacagctac aactggagca tttactgctg 60 gatttacaga tgattttgaa tggaattaat aattacaaga atcccaaact caccaggatg 120 ctcacattta agttttacat gcccaagaag gccacagaac tgaaacatct tcagtgtcta 180 gaagaagaac tcaaacctct ggaggaagtg ctaaatttag ctcaaagcaa aaactttcac 240 ttaagaccca gggacttaat cagcaatatc aacgtaatag ttctggaact aaagggatct 300 gaaacaacat tcatgtgtga atatgctgat gagacagcaa ccattgtaga atttctgaac 360 agatggatta ccttttcaca aagcatctag tcaacactga ct 402 <210> 50 <211> 402 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Base sequence of K8(oAzZK) / I129(oAzZK) <400> 50 atggcaccta cttcaagttc tacatagaaa acacagctac aactggagca tttactgctg 60 gatttacaga tgattttgaa tggaattaat aattacaaga atcccaaact caccaggatg 120 ctcacattta agttttacat gcccaagaag gccacagaac tgaaacatct tcagtgtcta 180 gaagaagaac tcaaacctct ggaggaagtg ctaaatttag ctcaaagcaa aaactttcac 240 ttaagaccca gggacttaat cagcaatatc aacgtaatag ttctggaact aaagggatct 300 gaaacaacat tcatgtgtga atatgctgat gagacagcaa ccattgtaga atttctgaac 360 agatggatta ccttttcaca aagcatctag tcaacactga ct 402 <210> 51 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: Amino acid sequence of I129C <400> 51 Met 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 Ser Gln Ser Ile 115 120 125 Cys Ser Thr Leu Thr 130 <210> 52 <211> 399 <212> DNA <213> Artificial sequence <220> <223> Description of artificial sequence: Base sequence of I129C <400> 52 atgcctactt caagttctac aaagaaaaca cagctacaac tggagcattt actgctggat 60 ttacagatga ttttgaatgg aattaataat tacaagaatc ccaaactcac caggatgctc 120 acatttaagt tttacatgcc caagaaggcc acagaactga aacatcttca gtgtctagaa 180 gaagaactca aacctctgga ggaagtgcta aatttagctc aaagcaaaaa ctttcactta 240 agacccaggg acttaatcag caatatcaac gtaatagttc tggaactaaa gggatctgaa 300 acaacattca tgtgtgaata tgctgatgag acagcaacca ttgtagaatt tctgaacaga 360 tggattacct tttcacaaag catctgttca acactgact 399 Description of the drawings

[0154] Figure 1A It is a graph showing the Treg proliferation promoting activity of various sugar chain - conjugated IL - 2 variants. The black dots represent the activity of IL - 2 manufactured by Peprotech [hereinafter referred to as IL - 2(P)], the black triangles represent the activity of H16C - 2, the black quadrilaterals represent the activity of L19C - 9, and the black horizontal bars represent the activity of N88C - 2. The horizontal axis represents the IL - 2 concentration (pM), and the vertical axis represents the IL - 2 - dependent cell proliferation rate (%).

[0155] Figure 1BIt is a graph showing the Treg proliferation-promoting activities of various sugar chain-conjugated IL-2 variants. The black dots represent the activity of E15C-11, the black triangles represent the activity of L19C-11*, the black quadrilaterals represent the activity of L12C-11 / V91C-11, the black diamonds represent the activity of V91C-11 / V115C-11, the black bars represent the activity of V91C-11 / N119C-11, and the white dots represent the activity of A1C-11 / T3C-11 / S5C-11 / L12C-11 / V91C-11. The horizontal axis represents the IL-2 concentration (pM), and the vertical axis represents the IL-2-dependent cell proliferation rate (%).

[0156] Figure 1C It is a graph showing the Treg proliferation-promoting activities of various Cys-PEGylated, sugar chain-conjugated IL-2 variants. The black dots represent the activity of A1C-Y50(IAc) / L12C-11 / V91C-11, the black triangles represent the activity of T3C-Li20(IAc) / L12C-11 / V91C-11, the black quadrilaterals represent the activity of T3C-Y50(IAc) / L12C-11 / V91C-11, the black diamonds represent the activity of T3C-Y50(IAc) / E15C-11, the black bars represent the activity of T3C-V40(IAc) / E15C-11, the white dots represent the activity of T3C-V80(Mal) / E15C-11, and the white triangles represent the activity of F78C-V40(IAc) / L12C-11. The horizontal axis represents the IL-2 concentration (pM), and the vertical axis represents the IL-2-dependent cell proliferation rate (%).

[0157] Figure 1D It is a graph showing the Treg proliferation-promoting activities of various PEG-conjugated IL-2 variants. The black dots represent the activity of 8His-IL-2, the black triangles represent the activity of 8His-F78(oAzZK)-Li20, and the black quadrilaterals represent the activity of 8His-I129(oAzZK)-Li20. The horizontal axis represents the IL-2 concentration (pM), and the vertical axis represents the IL-2-dependent cell proliferation rate (%).

[0158] Figure 1E It is a graph showing the Treg proliferation-promoting activities of various PEG-conjugated IL-2 variants. The black dots represent the activity of 8His-S4(oAzZK)-Li20, the black triangles represent the activity of 8His-S5(oAzZK)-Li20, the black quadrilaterals represent the activity of 8His-S6(oAzZK)-Li20, the black diamonds represent the activity of 8His-T7(oAzZK)-Li20, and the black bars represent the activity of 8His-K8(oAzZK)-Li20. The horizontal axis represents the IL-2 concentration (pM), and the vertical axis represents the IL-2-dependent cell proliferation rate (%).

[0159] Figure 1F It is a graph showing the Treg proliferation-promoting activity of various PEG-conjugated IL-2 variants. The black dots represent the activity of 8His-E60(oAzZK)-Li20, the black triangles represent the activity of 8His-H79(oAzZK)-Li20, the black quadrilaterals represent the activity of 8His-R81(oAzZK)-Li20, and the black diamonds represent the activity of 8His-L94(oAzZK)-Li20. The horizontal axis represents the IL-2 concentration (pM), and the vertical axis represents the IL-2-dependent cell proliferation rate (%).

[0160] Figure 1G It is a graph showing the Treg proliferation-promoting activity of various PEG-conjugated IL-2 variants. The black dots represent the activity of 8His-S99(oAzZK)-Li20, the black triangles represent the activity of 8His-E100(oAzZK)-Li20, the black quadrilaterals represent the activity of 8His-T101(oAzZK)-Li20, and the black diamonds represent the activity of 8His-Q126(oAzZK)-Li20. The horizontal axis represents the IL-2 concentration (pM), and the vertical axis represents the IL-2-dependent cell proliferation rate (%).

[0161] Figure 1H It is a graph showing the Treg proliferation-promoting activity of various PEG-conjugated IL-2 variants. The black dots represent the activity of 8His-F78(oAzZK)-V40, the black triangles represent the activity of 8His-F78(oAzZK)-W40, the black quadrilaterals represent the activity of 8His-I129(oAzZK)-Li40, the black diamonds represent the activity of 8His-I129(oAzZK)-V40, the black bars represent the activity of 8His-I129(oAzZK)-W40, the white dots represent the activity of 8His-I129(oAzZK)-Y50, the white triangles represent the activity of I129(oAzZK)-V40, the white quadrilaterals represent the activity of I129(oAzZK)-W80, and the white diamonds represent the activity of I129C-V40(Mal). The horizontal axis represents the IL-2 concentration (pM), and the vertical axis represents the IL-2-dependent cell proliferation rate (%).

[0162] Figure 1IIt is a graph showing the Treg proliferation-promoting activity of various PEG-conjugated IL-2 variants. The black dots represent the activity of 8His-S4(oAzZK)-Li30 / I129(oAzZK)-Li30, the black triangles represent the activity of S4(oAzZK)-Y50 / I129(oAzZK)-Y50, the black quadrilaterals represent the activity of 8His-S5(oAzZK)-Li30 / I129(oAzZK)-Li30, the black diamonds represent the activity of S5(oAzZK)-Y50 / I129(oAzZK)-Y50, the black bars represent the activity of 8His-K8(oAzZK)-Li30 / I129(oAzZK)-Li30, the white dots represent the activity of K8(oAzZK)-Y50 / I129(oAzZK)-Y50, the white triangles represent the activity of 8His-F78(oAzZK)-Li30 / I129(oAzZK)-Li30, the white quadrilaterals represent the activity of 8His-H79(oAzZK)-Li30 / I129(oAzZK)-Li30, and the white diamonds represent the activity of 8His-S99(oAzZK)-Li30 / I129(oAzZK)-Li30. The horizontal axis represents the IL-2 concentration (pM), and the vertical axis represents the IL-2-dependent cell proliferation rate (%).

[0163] Figure 1J It is a graph showing the Treg proliferation-promoting activity of various PEG-conjugated IL-2 variants. The black dots represent the activity of 8His-S4(oAzZK)-Li30 / F78(oAzZK)-Li30, the black triangles represent the activity of 8His-S5(oAzZK)-Li30 / F78(oAzZK)-Li30, the black quadrilaterals represent the activity of 8His-K8(oAzZK)-Li30 / F78(oAzZK)-Li30, the black diamonds represent the activity of 8His-F78(oAzZK)-Li30 / H79(oAzZK)-Li30, and the black bars represent the activity of 8His-F78(oAzZK)-Li30 / S99(oAzZK)-Li30. The horizontal axis represents the IL-2 concentration (pM), and the vertical axis represents the IL-2-dependent cell proliferation rate (%).

[0164] Figure 2A It is a graph showing the NK cell proliferation-promoting activity of various sugar chain-conjugated IL-2 variants. The black diamonds represent the activity of IL-2(P), the black quadrilaterals represent the activity of H16C-2, the black triangles represent the activity of L19C-9, and the black dots represent the activity of N88C-2. The horizontal axis represents the IL-2 concentration (pM), and the vertical axis represents the IL-2-dependent cell proliferation rate (%).

[0165] Figure 2BIt is a graph showing the NK cell proliferation promoting activities of various PEG-conjugated IL-2 variants. The black dots represent the activity of 8His-IL-2, the black triangles represent the activity of 8His-F78(oAzZK)-Li20, and the black quadrilaterals represent the activity of 8His-I129(oAzZK)-Li20. The horizontal axis represents the IL-2 concentration (pM), and the vertical axis represents the IL-2-dependent cell proliferation rate (%).

[0166] Figure 2C It is a graph showing the NK cell proliferation promoting activities of various PEG-conjugated IL-2 variants. The black dots represent the activity of 8His-S4(oAzZK)-Li20, the black triangles represent the activity of 8His-S5(oAzZK)-Li20, the black quadrilaterals represent the activity of 8His-S6(oAzZK)-Li20, the black diamonds represent the activity of 8His-T7(oAzZK)-Li20, and the black horizontal bars represent the activity of 8His-K8(oAzZK)-Li20. The horizontal axis represents the IL-2 concentration (pM), and the vertical axis represents the IL-2-dependent cell proliferation rate (%).

[0167] Figure 2D It is a graph showing the NK cell proliferation promoting activities of various PEG-conjugated IL-2 variants. The black dots represent the activity of 8His-S99(oAzZK)-Li20, the black triangles represent the activity of 8His-E100(oAzZK)-Li20, the black quadrilaterals represent the activity of 8His-T101(oAzZK)-Li20, and the black diamonds represent the activity of 8His-Q126(oAzZK)-Li20. The horizontal axis represents the IL-2 concentration (pM), and the vertical axis represents the IL-2-dependent cell proliferation rate (%).

[0168] Figure 2E It is a graph showing the NK cell proliferation promoting activities of various PEG-conjugated IL-2 variants. The black dots represent the activity of 8His-E60(oAzZK)-Li20, the black triangles represent the activity of 8His-H79(oAzZK)-Li20, the black quadrilaterals represent the activity of 8His-R81(oAzZK)-Li20, and the black diamonds represent the activity of 8His-L94(oAzZK)-Li20. The horizontal axis represents the IL-2 concentration (pM), and the vertical axis represents the IL-2-dependent cell proliferation rate (%).

[0169] Figure 2FIt is a graph showing the NK cell proliferation-promoting activities of various PEG-conjugated IL-2 variants. The black dots represent the activity of 8His-F78(oAzZK)-V40, the black triangles represent the activity of 8His-F78(oAzZK)-W40, the black quadrilaterals represent the activity of 8His-I129(oAzZK)-Li40, the black diamonds represent the activity of 8His-I129(oAzZK)-V40, the black horizontal bars represent the activity of 8His-I129(oAzZK)-W40, the white dots represent the activity of 8His-I129(oAzZK)-Y50, the white triangles represent the activity of I129(oAzZK)-W80, and the white quadrilaterals represent the activity of I129C-V40(Mal). The horizontal axis represents the IL-2 concentration (pM), and the vertical axis represents the IL-2-dependent cell proliferation rate (%).

[0170] Figure 2G It is a graph showing the NK cell proliferation-promoting activities of various PEG-conjugated IL-2 variants. The black dots represent the activity of 8His-S4(oAzZK)-Li30 / I129(oAzZK)-Li30, the black triangles represent the activity of S4(oAzZK)-Y50 / I129(oAzZK)-Y50, the black quadrilaterals represent the activity of 8His-S5(oAzZK)-Li30 / I129(oAzZK)-Li30, the black diamonds represent the activity of S5(oAzZK)-Y50 / I129(oAzZK)-Y50, the black horizontal bars represent the activity of 8His-K8(oAzZK)-Li30 / I129(oAzZK)-Li30, and the white dots represent the activity of K8(oAzZK)-Y50 / I129(oAzZK)-Y50. The horizontal axis represents the IL-2 concentration (pM), and the vertical axis represents the IL-2-dependent cell proliferation rate (%).

[0171] Figure 2HIt is a graph showing the NK cell proliferation-promoting activities of various glycan-conjugated IL-2 variants and various Cys-PEGylated, glycan-conjugated IL-2 variants. The black dots represent the activity of L12C-11 / V91C-11, the black triangles represent the activity of A1C-Y50(IAc) / L12C-11 / V91C-11, the black quadrilaterals represent the activity of T3C-Li20(IAc) / L12C-11 / V91C-11, the black diamonds represent the activity of T3C-Y50(IAc) / L12C-11 / V91C-11, the black bars represent the activity of T3C-Y50(IAc) / E15C-11, the white dots represent the activity of T3C-V40(IAc) / E15C-11, the white triangles represent the activity of T3C-V80(Mal) / E15C-11, and the white quadrilaterals represent the activity of F78C-V40(IAc) / L12C-11. The horizontal axis represents the IL-2 concentration (pM), and the vertical axis represents the IL-2-dependent cell proliferation rate (%).

[0172] Figure 2I It is a graph showing the NK cell proliferation-promoting activities of various PEG-conjugated IL-2 variants. The black dots represent the activity of IL-2(P), the black triangles represent the activity of I129(oAzZK)-V40, the black quadrilaterals represent the activity of 8His-F78(oAzZK)-Li30 / I129(oAzZK)-Li30, the black diamonds represent the activity of 8His-H79(oAzZK)-Li30 / I129(oAzZK)-Li30, and the black bars represent the activity of 8His-S99(oAzZK)-Li30 / I129(oAzZK)-Li30. The horizontal axis represents the IL-2 concentration (pM), and the vertical axis represents the IL-2-dependent cell proliferation rate (%).

[0173] Figure 2J It is a graph showing the NK cell proliferation-promoting activities of various PEG-conjugated IL-2 variants. The black dots represent the activity of 8His-S4(oAzZK)-Li30 / F78(oAzZK)-Li30, the black triangles represent the activity of 8His-S5(oAzZK)-Li30 / F78(oAzZK)-Li30, the black quadrilaterals represent the activity of 8His-K8(oAzZK)-Li30 / F78(oAzZK)-Li30, the black diamonds represent the activity of 8His-F78(oAzZK)-Li30 / H79(oAzZK)-Li30, and the black bars represent the activity of 8His-F78(oAzZK)-Li30 / S99(oAzZK)-Li30. The horizontal axis represents the IL-2 concentration (pM), and the vertical axis represents the IL-2-dependent cell proliferation rate (%).

[0174] Figure 2KIt is a graph showing the NK cell proliferation-promoting activity of various sugar chain-conjugated IL-2 variants. The black dots represent the activity of E15C-11, the black triangles represent the activity of L19C-11*, the black quadrilaterals represent the activity of V91C-11 / V115C-11, the black diamonds represent the activity of V91C-11 / N119C-11, and the black bars represent the activity of A1C-11 / T3C-11 / S5C-11 / L12C-11 / V91C-11. The horizontal axis represents the IL-2 concentration (pM), and the vertical axis represents the IL-2-dependent cell proliferation rate (%).

[0175] Figure 3 is a graph showing the proliferation rate of effector T cells (Tresp) in the coexistence of unstimulated Tregs or various IL-2 variant-stimulated Tregs. Figure 3(A) shows the proliferation rate of CD4-positive Tresp, and Figure 3(B) shows the proliferation rate of CD8-positive Tresp. In either graph, the horizontal axis represents the presence ratio of Tresp to Treg, and the vertical axis represents the proliferation rate (%) of Tresp. The white diamonds represent unstimulated, the black diamonds represent IL-2(P), the black quadrilaterals represent H16C-2, the black triangles represent L19C-9, and the black dots represent N88C-2.

[0176] Figures 4(A)-4(E) are graphs showing the cytokine concentrations in the culture supernatant when various IL-2 variants are added to human PBMC reconstructed with autologous plasma and cultured. Figure 4(A) shows the IL-4 concentration, Figure 4(B) shows the IL-6 concentration, Figure 4(C) shows the IL-10 concentration, Figure 4(D) shows the IFNγ concentration, and Figure 4(E) shows the TNFα concentration. In either graph, the horizontal axis represents the added IL-2 concentration (pM), and the vertical axis represents the cytokine production amount (pg / mL). Figure 4(F) is a graph showing the results of evaluating the Treg-selective proliferation activity. The vertical axis represents the ratio [Treg(%) / Teff(%)] when the CD4-positive fraction is Treg with CD25 + Foxp3 high as the Treg component and CD25 + Foxp3 low as the effector T cell (Teff) component. The horizontal axis represents the added IL-2 concentration. In either graph, the white diamonds represent unstimulated, the black diamonds represent IL-2(P), the black quadrilaterals represent H16C-2, the black triangles represent L19C-9, and the black dots represent N88C-2.

[0177] Detailed implementation manners of the invention

[0178] Hereinafter, the present invention will be described in detail.

[0179] "Treg" or "Treg cell" refers to regulatory T cells. Regulatory T cells are a type of T cells that inhibit the activity of other immune cells, and are defined using a flow cytometer by CD4 + CD25 + FOXP3 + as cell markers of phenotype.

[0180] FOXP3 is an intracellular protein, and for staining, cell fixation and permeabilization are required. Therefore, to define viable Tregs, CD4 can be used as a cell surface phenotype + CD25 + CD127 low .

[0181] Tregs also include various Treg subclasses such as tTregs (derived from the thymus) and pTregs (derived from the periphery and differentiated from naive T cells in the periphery). All Tregs express IL-2R αβγ , and proliferate in an IL-2-dependent manner. However, the IL-2 variants of the present invention can selectively activate at least one Treg subclass, preferably can selectively activate all subclasses.

[0182] "IL-2" can be either wild-type IL-2 or an IL-2 variant.

[0183] "Wild-type IL-2" also includes any of the following IL-2s 1) to 3).

[0184] 1) Human wild-type mature IL-2 consisting of the amino acid sequence shown in SEQ ID NO: 1.

[0185] 2) IL-2 having an amino acid modification that can be applied when preparing the gene recombinant of 1) above.

[0186] 3) IL-2 with the amino acid residues at the N-terminus of the IL-2s of 1) and 2) above deleted.

[0187] Examples of the amino acid changes in 2) above include modifications such as binding a methionine residue encoded by the start codon to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 in order to express IL-2 in E. coli; binding the amino acid sequence shown by MHHHHHHHH (polyhistidine bound to methionine) to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 in order to express IL-2 in E. coli and facilitate purification; and modifying the 125th amino acid residue of human wild-type mature IL-2 to an alanine residue or a serine residue in order to improve the physical properties of IL-2, etc.

[0188] The IL-2 with the N-terminal amino acid residue deletion of the above 3) can include, for example, IL-2 with an amino acid sequence in which the alanine residue at the N-terminal of the amino acid sequence shown in SEQ ID NO: 1 is deleted, or IL-2 with an amino acid sequence in which the alanine residue and the proline residue are deleted, etc.

[0189] Specific examples of wild-type IL-2 can include IL-2 with the following amino acid sequences: the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence in which a methionine residue is bound to the N-terminal of the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence in which the amino acid sequence shown as MHHHHHHHH is bound to the N-terminal of the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence in which the alanine residue at the N-terminal of the amino acid sequence shown in SEQ ID NO: 1 is deleted, the amino acid sequence in which the alanine residue at the N-terminal of the amino acid sequence shown in SEQ ID NO: 1 is deleted and methionine is bound, the amino acid sequence in which the alanine residue and the proline residue at the N-terminal of the amino acid sequence shown in SEQ ID NO: 1 are deleted, or the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence in which a methionine residue is bound to the N-terminal of the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence in which the amino acid sequence shown as MHHHHHHHH is bound to the N-terminal of the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence in which the alanine residue at the N-terminal of the amino acid sequence shown in SEQ ID NO: 1 is deleted, the amino acid sequence in which the alanine residue at the N-terminal of the amino acid sequence shown in SEQ ID NO: 1 is deleted and methionine is bound, or the amino acid sequence in which the amino acid residue at the 125th position in the amino acid sequence in which the alanine residue and the proline residue at the N-terminal of the amino acid sequence shown in SEQ ID NO: 1 are deleted is substituted with a serine residue or an alanine residue. The amino acid sequences on the N-terminal side and the serine residue at the 125th position of the amino acid sequence shown in SEQ ID NO: 1 do not affect the activity of IL-2 and are acceptable amino acid sequence variations from the viewpoints of protein expression or protein stability. Such amino acid sequence variations are also included in the IL-2 variants of the present invention.

[0190] In addition, the numbering of the amino acid residues of IL-2 described in the present invention all represents the numbering (position) of the amino acid residues based on the amino acid sequence of IL-2 shown in SEQ ID NO: 1. Therefore, the alanine residue at the N-terminal of the amino acid sequence shown in SEQ ID NO: 1 is defined as the 1st position, the proline residue is defined as the 2nd position, and the methionine residue bound to the N-terminal is defined as the -1st position.

[0191] "IL-2 variant" includes all proteins with the functions of wild-type IL-2 produced by imposing certain modifications on wild-type IL-2. As variants, for example, IL-2 variants modified from wild-type IL-2 by amino acid modifications (such as substitution, deletion, addition, etc.), IL-2 variants modified from wild-type IL-2 by sugar chain modification, IL-2 variants modified from wild-type IL-2 by chemical modification, etc. can be listed. The above modifications include any modifications of naturally occurring modifications and artificial modifications.

[0192] "Function of wild-type IL-2" refers to at least one function selected from the following functions: binding to IL-2R αβγ ; binding to IL-2R βγ ; activation of the intracellular signal transduction pathway via the intracellular regions of CD122 and CD132, phosphorylation of JAK1, phosphorylation of JAK3, phosphorylation of STAT5, phosphorylation of STAT3, phosphorylation of PI3K, phosphorylation of MEK, promotion of Foxp3 expression, promotion of the expression of the gene group controlled by Foxp3 through transcription, promotion of DNA demethylation in the TSDR (Treg-specific demethylation region) region of the Foxp3 gene, promotion of the proliferation and survival of immune cells expressing IL-2R βγ ; promotion of the cytokine production of immune cells expressing IL-2R βγ ; promotion of the proliferation and survival of immune cells expressing IL-2R αβγ ; promotion of the cytokine production of immune cells expressing IL-2R αβγ ; promotion of Treg proliferation and survival, and improvement of the ability of Treg to inhibit Teff activation.

[0193] As a mode of the IL-2 variant of the present invention, IL-2 variants with sugar chains bound to a specified region of IL-2, IL-2 variants with PEG bound to a specified region of IL-2, and IL-2 variants with both sugar chains and PEG bound to a specified region of IL-2 can be listed. As the binding, for example, covalent binding, non-covalent binding, etc. can be listed, and the binding mode is not limited.

[0194] "Amino acid residue" includes any case of natural amino acid residues and unnatural amino acid residues.

[0195] "Natural amino acid residue" may include selenocysteine residue and the following 20 kinds of α - amino acid residues: alanine residue, asparagine residue, aspartic acid residue, glutamine residue, glutamic acid residue, glycine residue, histidine residue, isoleucine residue, leucine residue, lysine residue, methionine residue, phenylalanine residue, proline residue, serine residue, threonine residue, tryptophan residue, tyrosine residue, valine residue or cysteine residue. Natural amino acid residues include both L - form and D - form, and the L - form is preferred for humans.

[0196] "Unnatural amino acid residue" refers to all amino acid residues other than natural amino acid residues. As unnatural amino acid residues, for example, amino acid residues modified from natural amino acid residues and amino acid residues obtained by artificial design can be cited.

[0197] "Modification" includes all modifications such as chemical modification and post - translational modification.

[0198] As an embodiment of the IL - 2 variant of the present invention, an IL - 2 variant with improved selectivity for IL - 2R αβγ can be cited. By the IL - 2 variant with improved selectivity for IL - 2R αβγ , Tregs expressing IL - 2R αβγ can be selectively activated.

[0199] "Selectivity for IL - 2R αβγ " refers to the property that IL - 2 selectively binds to IL - 2R βγ compared with IL - 2R αβγ . In addition, "improved selectivity for IL - 2R αβγ " means that the IL - 2 variant has improved selectivity for IL - 2R αβγ compared with wild - type IL - 2.

[0200] The selectivity for IL - 2R αβγ or the improved selectivity for IL - 2R αβγ can be judged, for example, by the methods described below.

[0201] (1) For various IL - 2s, measure the EC αβγ value of the binding activity to IL - 2R 50 and the EC βγ value of the binding activity to IL - 2R 50 . If the EC αβγ of IL - 2R 50 is less than the EC βγ of IL - 2R 50 or the EC 50 ratio value (IL - 2R βγEC 50 / IL-2R αβγ EC 50 ) greater than 1, it can be determined that the IL-2 is selective for IL-2R αβγ Have selectivity.

[0202] In addition, in the EC of the IL-2 variant 50 The ratio value is greater than the EC of wild-type IL-2 50 Ratio value or normalized EC 50 Ratio value (EC of IL-2 variant 50 Ratio value / EC of wild-type IL-2 50 Ratio value) greater than 1, it can be determined that the IL-2 variant is more selective for IL-2R αβγ . The normalized EC 50 Ratio values are preferably greater than 1, 5 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more. Instead of wild-type IL-2, an IL-2 variant having an EC 50 Ratio value equivalent to that of wild-type IL-2 can also be used.

[0203] As a method for measuring the EC 50 Value, specifically, for example, a method using the procedures shown in the following (A) to (C) can be cited. As a more specific method, the method described in the following examples can be cited.

[0204] (A) Express human IL-2R αβγ Or human IL-2R βγ In mammalian cells to produce a human IL-2-dependent survival cell line, and inoculate each cell line into a 96-well plate.

[0205] (B) Taking the relative fluorescence unit (relative fluorescence units, RLU) value of the well supplemented with the control IL-2 at 1000 ng / ml as 100%, and the RLU value of the well supplemented with the medium without IL-2 as 0%, calculate the IL-2-dependent cell proliferation rate of the IL-2 variant as the test substance.

[0206] (C) According to the data obtained in (B), use statistical analysis software (for example, XLfit5 version 5.3.1.3 manufactured by IBDS Co., Ltd.) to calculate the EC 50 Value.

[0207] (2) In wild-type IL-2 and IL-2 variants, the affinity of CD25ECD-Fc and IL-2R βγ ECD-Fc, which are IL-2R extracellular domain (ECD)-Fc fusion proteins, was measured by Biacore, respectively. Compared with the wild type, in the IL-2 variant, when the K D value of CD25ECD-Fc is small and / or the K βγ value of IL-2R D ECD-Fc is large, it can be judged that the selectivity of this IL-2 variant for IL-2R αβγ is improved. In addition, when the relative value of the K βγ value of IL-2R D to the K D value of CD25ECD-Fc increases compared with the wild type in the IL-2 variant, it can also be judged that the selectivity of this IL-2 variant for IL-2R αβγ is improved.

[0208] "Selectively activating Treg" means at least any one of the following (a) to (c).

[0209] (a) Compared with wild-type IL-2, the Treg proliferation activity of the IL-2 variant is high and / or the NK cell proliferation activity is low.

[0210] (b) Compared with wild-type IL-2, in the IL-2 variant, the ratio of the proportion of Treg to the proportion of effector T cells (Teff) in the cell population [Treg(%) / Teff(%)] is high.

[0211] (c) Compared with wild-type IL-2, in the IL-2 variant, the production amount of inflammatory cytokines is reduced and / or the production amount of anti-inflammatory cytokines is increased.

[0212] In any of the above cases (a) to (c), an IL-2 variant having the same activity as wild-type IL-2 can be used to replace wild-type IL-2.

[0213] The Treg proliferation activity and NK cell proliferation activity can be measured by the methods described below, for example. Treg or NK cells are inoculated into a 96-well plate. Taking the RLU value of the well with control IL-2 added as 100% and the RLU value of the well with medium without IL-2 added as 0%, the Treg or NK cell proliferation rate of the IL-2 variant as the test substance is calculated. As a more specific method, the methods described in the following examples can be cited.

[0214] The Treg(%) / Teff(%) can be measured, for example, by the method described below. Human peripheral blood mononuclear cells (hereinafter also simply referred to as PBMC) are suspended in autologous plasma, anti-CD3 antibody OKT3 is added thereto, and after inoculation into a 96-well plate, various IL-2s are added for culture. After reacting the obtained human PBMC with a fluorescently labeled anti-human CD4 antibody, a fluorescently labeled CD25 antibody, and a fluorescently labeled anti-Foxp3 antibody, the fluorescence intensities are measured using a flow cytometer (for example, LSR Fortessa manufactured by BD Biosciences).

[0215] The obtained data is analyzed using data analysis software (for example, FlowJo, version 7.6.5 manufactured by TreeStar). Among the CD4-positive fractions, the CD25 + Foxp3 high fraction is regarded as Treg, and the CD25 + Foxp3 low fraction is regarded as effector T cells (Teff), and their abundance ratio [Treg(%) / Teff(%)] is calculated. As a more specific method, the method described in the following examples can be cited.

[0216] The production amount of various cytokines can be measured, for example, by the method described below. Human PBMC are suspended in autologous plasma, anti-CD3 antibody OKT3 is added thereto, and after inoculation into a 96-well plate, various IL-2s are added for culture, and the cytokine production amount in the supernatant is quantified. As a more specific method, the method described in the following examples can be cited.

[0217] As one mode of the IL-2 variant of the present invention, an IL-2 variant modified by binding a sugar chain to IL-2 (hereinafter also simply referred to as a sugar chain-binding IL-2 variant), and an IL-2 variant modified by binding IL-2 to PEG (hereinafter also simply referred to as a PEG-binding IL-2 variant) can be cited. Hereinafter, each variant will be described.

[0218] [Sugar chain-binding IL-2 variant]

[0219] As one embodiment of the IL-2 variant of the present invention, an IL-2 variant in which at least one amino acid residue selected from the amino acid residues at positions 11, 12, 13, 15, 16, 18, 19, 20, 84, 87, 88, 91, 92, 108, 115, 119, 122, 123, and 130 in the amino acid sequence of IL-2 is bound with a sugar chain is preferred.

[0220] In the present specification, "sugar chain" means a monosaccharide or two or more monosaccharides bound via a bond called a glycosidic bond, and any sugar chain can be used.

[0221] As the sugar chain that binds to IL-2, specifically, for example, at least one selected from sugar chains having the structures represented by the following (Formula 4) to (Formula 8) and (Formula Y1) to (Formula Y3) can be exemplified. By binding the above amino acid residues in the amino acid sequence of IL-2 to this sugar chain, the selectivity for IL-2R αβγ can be improved. In addition, a sugar chain in which one N-acetylglucosamine (GlcNAc) is bound to each of the mannose (Man) of the α1-6 arm and the α1-3 arm in (Formula 6), a sugar chain (G1) obtained by removing one galactose (Gal) from various Man-GlcNAc of the α1-6 arm and the α1-3 arm in (Formula 7), a sugar chain in which one sialic acid (Sial) at the non-reducing end in (Formula 8) is removed, and a sugar chain in which 1 to 4 sials at the non-reducing end in (Formula Y3) are removed can also be used as the sugar chain of the IL-2 variant of the present invention.

[0222]

[0223]

[0224]

[0225] As an embodiment of the IL-2 variant of the present invention, an IL-2 variant preferably includes an amino acid sequence in which at least one amino acid residue selected from the amino acid residues at positions 11, 12, 13, 15, 16, 18, 19, 20, 84, 87, 88, 91, 92, 108, 115, 119, 122, 123, and 130 in the amino acid sequence of wild-type IL-2 is replaced with a group derived from a cysteine residue or an asparagine residue to which a sugar chain is bound, and more preferably an IL-2 variant includes an amino acid sequence in which at least one amino acid residue selected from the amino acid residues at positions 12, 13, 15, 16, 19, 88, 91, and 119 in the amino acid sequence of wild-type IL-2 is replaced with a group derived from a cysteine residue or an asparagine residue to which a sugar chain is bound.

[0226] In this embodiment, the amino acid sequence of wild-type IL-2 is more preferably the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence in which the amino acid residue at position 125 in the amino acid sequence shown in SEQ ID NO: 1 is replaced with a serine residue or an alanine residue.

[0227] The group derived from a cysteine residue or an asparagine residue respectively refers to a group in which the side-chain thiol of the cysteine residue or the side-chain amide of the asparagine residue is modified.

[0228] A group derived from a cysteine residue or an asparagine residue to which a sugar chain is bound refers to a group in which a sugar chain is bound by chemical modification to the side-chain thiol of a cysteine residue or the side-chain amide of an asparagine residue. The group derived from a cysteine residue or an asparagine residue may be modified by a linker or the like, or a cysteine residue or an asparagine residue may be bound to a sugar chain via a linker.

[0229] As an example of a group derived from a cysteine residue to which a sugar chain is bound, an amino acid residue having a structure in which a sugar chain is bound to the side-chain thiol of a cysteine residue via a CH2CONH linker, as shown in the following (Formula 1), can be cited. The side-chain thiol of a cysteine residue and a sugar chain may be bound without a linker.

[0230]

[0231] In the above (Formula 1), Saccharide represents a sugar chain.

[0232] As an example of a group derived from an asparagine residue to which a sugar chain is bound, a structure in which a sugar chain is bound by chemical modification to the side-chain amide of an asparagine residue, as shown in the following (Formula 2), can be cited. The side-chain amide of an asparagine residue and a sugar chain may be bound via a linker.

[0233]

[0234] In the above (Formula 2), Saccharide represents a sugar chain.

[0235] As an embodiment of the IL-2 variant of the present invention, for example, an IL-2 variant in which at least one amino acid residue selected from the amino acid residues at positions 11, 12, 13, 15, 16, 18, 19, 20, 84, 87, 88, 91, 92, 108, 115, 119, 122, 123, and 130 in the amino acid sequence of wild-type IL-2 is substituted with an amino acid residue to which a sugar chain is bound can be cited.

[0236] As an example of an IL-2 variant having one sugar chain bound to wild-type IL-2, the IL-2 variants described below can be cited.

[0237] · An IL-2 variant in which the amino acid residue at position 11 in the amino acid sequence of wild-type IL-2 is substituted with an amino acid residue to which a sugar chain is bound.

[0238] · An IL-2 variant in which the amino acid residue at position 12 in the amino acid sequence of wild-type IL-2 is substituted with an amino acid residue to which a sugar chain is bound.

[0239] · An IL-2 variant in which the amino acid residue at position 13 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with a sugar chain.

[0240] · An IL-2 variant in which the amino acid residue at position 15 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with a sugar chain.

[0241] · An IL-2 variant in which the amino acid residue at position 16 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with a sugar chain.

[0242] · An IL-2 variant in which the amino acid residue at position 18 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with a sugar chain.

[0243] · An IL-2 variant in which the amino acid residue at position 19 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with a sugar chain.

[0244] · An IL-2 variant in which the amino acid residue at position 20 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with a sugar chain.

[0245] · An IL-2 variant in which the amino acid residue at position 84 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with a sugar chain.

[0246] · An IL-2 variant in which the amino acid residue at position 87 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with a sugar chain.

[0247] · An IL-2 variant in which the amino acid residue at position 88 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with a sugar chain.

[0248] · An IL-2 variant in which the amino acid residue at position 91 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with a sugar chain.

[0249] · An IL-2 variant in which the amino acid residue at position 92 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with a sugar chain.

[0250] · An IL-2 variant in which the amino acid residue at position 108 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with a sugar chain.

[0251] · An IL-2 variant in which the amino acid residue at position 115 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with a sugar chain.

[0252] · An IL-2 variant in which the amino acid residue at position 119 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with a sugar chain.

[0253] · An IL-2 variant in which the amino acid residue at position 122 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with a sugar chain.

[0254] · An IL-2 variant in which the amino acid residue at position 123 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with a sugar chain.

[0255] · An IL-2 variant in which the amino acid residue at position 130 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with a sugar chain.

[0256] In the above IL-2 variants, the conjugated sugar chain can be any sugar chain, for example, sugar chains having structures represented by (Formula 4), (Formula 5), (Formula 6), (Formula 7), (Formula 8), or (Formula Y3) can be cited.

[0257] · An IL-2 variant in which the amino acid residue at position 11 in the amino acid sequence of wild-type IL-2 is replaced with a sugar chain-conjugated group derived from a cysteine residue represented by (Formula 1), and the structure of Saccharide in (Formula 1) is the structure represented by (Formula 8).

[0258] · An IL-2 variant in which the amino acid residue at position 12 in the amino acid sequence of wild-type IL-2 is replaced with a sugar chain-conjugated group derived from a cysteine residue represented by (Formula 1), and the structure of Saccharide in (Formula 1) is the structure represented by (Formula 7) or (Formula 8).

[0259] · An IL-2 variant in which the amino acid residue at position 13 in the amino acid sequence of wild-type IL-2 is replaced with a sugar chain-conjugated group derived from a cysteine residue represented by (Formula 1), and the structure of Saccharide in (Formula 1) is the structure represented by (Formula 4) or (Formula 8).

[0260] · An IL-2 variant in which the amino acid residue at position 15 in the amino acid sequence of wild-type IL-2 is replaced with a sugar chain-conjugated group derived from a cysteine residue represented by (Formula 1), and the structure of Saccharide in (Formula 1) is the structure represented by (Formula 4), (Formula 8), or (Formula Y3).

[0261] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 16 is replaced with a sugar chain-linked cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is a structure represented by (Formula 4), (Formula 5), (Formula 6), or (Formula 7), an IL-2 variant.

[0262] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 18 is replaced with a sugar chain-linked cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is a structure represented by (Formula 4) or (Formula 8), an IL-2 variant.

[0263] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 19 is replaced with a sugar chain-linked cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is a structure represented by (Formula 4), (Formula 7), (Formula 8), or (Formula Y3), an IL-2 variant.

[0264] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 20 is replaced with a sugar chain-linked cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is a structure represented by (Formula 4) or (Formula 8), an IL-2 variant.

[0265] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 84 is replaced with a sugar chain-linked cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is a structure represented by (Formula 4), an IL-2 variant.

[0266] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 87 is replaced with a sugar chain-linked cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is a structure represented by (Formula 4) or (Formula 8), an IL-2 variant.

[0267] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 88 is replaced with a sugar chain-linked cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is a structure represented by (Formula 4), (Formula 7), or (Formula 8), an IL-2 variant.

[0268] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 91 is replaced with a sugar chain-linked cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is a structure represented by (Formula 4), (Formula 7), or (Formula 8), an IL-2 variant.

[0269] · An IL-2 variant in which the amino acid residue at position 92 in the amino acid sequence of wild-type IL-2 is replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is the structure represented by (Formula 4).

[0270] · An IL-2 variant in which the amino acid residue at position 108 in the amino acid sequence of wild-type IL-2 is replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is the structure represented by (Formula 4) or (Formula 7).

[0271] · An IL-2 variant in which the amino acid residue at position 115 in the amino acid sequence of wild-type IL-2 is replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is the structure represented by (Formula 4).

[0272] · An IL-2 variant in which the amino acid residue at position 119 in the amino acid sequence of wild-type IL-2 is replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is the structure represented by (Formula 4) or (Formula 7).

[0273] · An IL-2 variant in which the amino acid residue at position 122 in the amino acid sequence of wild-type IL-2 is replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is the structure represented by (Formula 4).

[0274] · An IL-2 variant in which the amino acid residue at position 123 in the amino acid sequence of wild-type IL-2 is replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is the structure represented by (Formula 8).

[0275] · An IL-2 variant in which the amino acid residue at position 130 in the amino acid sequence of wild-type IL-2 is replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is the structure represented by (Formula 4) or (Formula 7).

[0276] As an embodiment of the present invention, an IL-2 variant having at least 2 sugar chains bound to wild-type IL-2 can also be cited. As an example of an IL-2 variant having 2 sugar chains bound to wild-type IL-2, an IL-2 variant can be cited in which at least 2 amino acid residues selected from the amino acid residues at positions 1, 3, 4, 5, 8, 11, 12, 13, 15, 16, 18, 19, 20, 23, 32, 38, 51, 76, 84, 87, 88, 91, 92, 100, 102, 104, 108, 115, 119, 122, 123, 127, and 130 in the amino acid sequence of wild-type IL-2 are substituted with a group derived from a cysteine residue or an asparagine residue having a sugar chain bound thereto.

[0277] As an example of an IL-2 variant having 2 sugar chains bound to wild-type IL-2, an IL-2 variant is preferably one in which 1 amino acid residue selected from the amino acid residues at positions 1, 3, 4, 5, 8, 11, 12, 13, 15, 16, 18, 19, 20, 23, 32, 38, 51, 76, 84, 87, 88, 91, 92, 100, 102, 104, 108, 115, 119, 122, 123, 127, and 130 in the amino acid sequence of wild-type IL-2, and 1 amino acid residue selected from the amino acid residues at positions 11, 12, 18, 20, 84, 87, 88, 91, 108, 115, 119, 122, and 123 are substituted with a group derived from a cysteine residue or an asparagine residue having a sugar chain bound thereto.

[0278] As an embodiment of a specific IL-2 variant of the present invention, the following IL-2 variants can be cited.

[0279] · An IL-2 variant in which the amino acid residues at positions 8 and 19 in the amino acid sequence of wild-type IL-2 are respectively substituted with amino acid residues having a sugar chain bound thereto.

[0280] · An IL-2 variant in which the amino acid residues at positions 12 and 16 in the amino acid sequence of wild-type IL-2 are respectively substituted with amino acid residues having a sugar chain bound thereto.

[0281] · An IL-2 variant in which the amino acid residues at positions 15 and 119 in the amino acid sequence of wild-type IL-2 are respectively substituted with amino acid residues having a sugar chain bound thereto.

[0282] · An IL-2 variant in which the amino acid residues at positions 19 and 23 in the amino acid sequence of wild-type IL-2 are respectively substituted with amino acid residues having a sugar chain bound thereto.

[0283] · An IL-2 variant in which the amino acid residues at positions 12 and 91 in the amino acid sequence of wild-type IL-2 are each replaced with an amino acid residue conjugated with a sugar chain.

[0284] · An IL-2 variant in which the amino acid residues at positions 12 and 115 in the amino acid sequence of wild-type IL-2 are each replaced with an amino acid residue conjugated with a sugar chain.

[0285] · An IL-2 variant in which the amino acid residues at positions 12 and 119 in the amino acid sequence of wild-type IL-2 are each replaced with an amino acid residue conjugated with a sugar chain.

[0286] · An IL-2 variant in which the amino acid residues at positions 13 and 91 in the amino acid sequence of wild-type IL-2 are each replaced with an amino acid residue conjugated with a sugar chain.

[0287] · An IL-2 variant in which the amino acid residues at positions 13 and 115 in the amino acid sequence of wild-type IL-2 are each replaced with an amino acid residue conjugated with a sugar chain.

[0288] · An IL-2 variant in which the amino acid residues at positions 13 and 119 in the amino acid sequence of wild-type IL-2 are each replaced with an amino acid residue conjugated with a sugar chain.

[0289] · An IL-2 variant in which the amino acid residues at positions 19 and 115 in the amino acid sequence of wild-type IL-2 are each replaced with an amino acid residue conjugated with a sugar chain.

[0290] · An IL-2 variant in which the amino acid residues at positions 91 and 115 in the amino acid sequence of wild-type IL-2 are each replaced with an amino acid residue conjugated with a sugar chain.

[0291] · An IL-2 variant in which the amino acid residues at positions 91 and 119 in the amino acid sequence of wild-type IL-2 are each replaced with an amino acid residue conjugated with a sugar chain.

[0292] In the above IL-2 variants, the conjugated sugar chain can be any sugar chain, for example, a sugar chain having the structure shown in (Formula 4) or (Formula 8) can be cited.

[0293] Further, as an embodiment of a specific IL-2 variant of the present invention, the following IL-2 variants can also be cited.

[0294] · An IL-2 variant in which the amino acid residues at positions 8 and 19 in the amino acid sequence of wild-type IL-2 are replaced with a group derived from a cysteine residue conjugated with a sugar chain shown in (Formula 1), and the structure of Saccharide in (Formula 1) is (Formula 8).

[0295] · An IL-2 variant in which the amino acid residues at positions 12 and 16 in the amino acid sequence of wild-type IL-2 are replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), the structure of the Saccharide of (Formula 1) bound to position 12 is (Formula 8), and the structure of the Saccharide of (Formula 1) bound to position 16 is (Formula 4).

[0296] · An IL-2 variant in which the amino acid residues at positions 15 and 119 in the amino acid sequence of wild-type IL-2 are replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide of (Formula 1) is (Formula 8).

[0297] · An IL-2 variant in which the amino acid residues at positions 19 and 23 in the amino acid sequence of wild-type IL-2 are replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide of (Formula 1) is (Formula 8).

[0298] · An IL-2 variant in which the amino acid residues at positions 12 and 91 in the amino acid sequence of wild-type IL-2 are replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide of (Formula 1) is (Formula 8).

[0299] · An IL-2 variant in which the amino acid residues at positions 12 and 115 in the amino acid sequence of wild-type IL-2 are replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide of (Formula 1) is (Formula 8).

[0300] · An IL-2 variant in which the amino acid residues at positions 12 and 119 in the amino acid sequence of wild-type IL-2 are replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide of (Formula 1) is (Formula 8).

[0301] · An IL-2 variant in which the amino acid residues at positions 13 and 91 in the amino acid sequence of wild-type IL-2 are replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide of (Formula 1) is (Formula 8).

[0302] · An IL-2 variant in which the amino acid residues at positions 13 and 115 in the amino acid sequence of wild-type IL-2 are replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide of (Formula 1) is (Formula 8).

[0303] · An IL-2 variant in which the amino acid residues at positions 13 and 119 in the amino acid sequence of wild-type IL-2 are replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of Saccharide in (Formula 1) is (Formula 8).

[0304] · An IL-2 variant in which the amino acid residues at positions 19 and 115 in the amino acid sequence of wild-type IL-2 are replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of Saccharide in (Formula 1) is (Formula 8).

[0305] · An IL-2 variant in which the amino acid residues at positions 91 and 115 in the amino acid sequence of wild-type IL-2 are replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of Saccharide in (Formula 1) is (Formula 8).

[0306] · An IL-2 variant in which the amino acid residues at positions 91 and 119 in the amino acid sequence of wild-type IL-2 are replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of Saccharide in (Formula 1) is (Formula 8).

[0307] As an embodiment of the present invention, an IL-2 variant having at least 3 sugar chains bound to wild-type IL-2 can also be cited. As an example of an IL-2 having 3 sugar chains bound to wild-type IL-2, an IL-2 variant having an amino acid sequence in which at least 3 amino acid residues selected from the amino acid residues at positions 1, 3, 4, 5, 8, 11, 12, 13, 15, 16, 18, 19, 20, 23, 32, 38, 51, 76, 84, 87, 88, 91, 92, 100, 102, 104, 108, 115, 119, 122, 123, 127, and 130 in the amino acid sequence of wild-type IL-2 are replaced with a sugar chain-bound cysteine residue-derived group or asparagine residue-derived group can be cited.

[0308] In addition, as an example of wild-type IL-2 with three sugar chains attached, it is more preferable to include at least one amino acid residue selected from the amino acid residues at positions 1, 3, 4, 5, 8, 11, 12, 13, 15, 16, 18, 19, 20, 23, 32, 38, 51, 76, 84, 87, 88, 91, 92, 100, 102, 104, 108, 115, 119, 122, 123, 127, and 130 in the amino acid sequence of wild-type IL-2, and at least one amino acid residue selected from the amino acid residues at positions 11, 12, 18, 20, 84, 87, 88, 91, 108, 115, 119, 122, and 123, which are replaced by a group derived from a cysteine residue or an asparagine residue with a sugar chain attached, in the amino acid sequence of the IL-2 variant.

[0309] As an embodiment of the present invention, an IL-2 variant with at least four sugar chains attached to wild-type IL-2 can also be cited. As an example of wild-type IL-2 with four sugar chains attached, it can be cited that at least four amino acid residues selected from the amino acid residues at positions 1, 3, 4, 5, 8, 11, 12, 13, 15, 16, 18, 19, 20, 23, 32, 38, 51, 76, 84, 87, 88, 91, 92, 100, 102, 104, 108, 115, 119, 122, 123, 127, and 130 in the amino acid sequence of wild-type IL-2 are replaced by a group derived from a cysteine residue or an asparagine residue with a sugar chain attached, in the amino acid sequence of the IL-2 variant.

[0310] In addition, as an example of wild-type IL-2 with four sugar chains attached, it is more preferable to include at least one amino acid residue selected from the amino acid residues at positions 1, 3, 4, 5, 8, 11, 12, 13, 15, 16, 18, 19, 20, 23, 32, 38, 51, 76, 84, 87, 88, 91, 92, 100, 102, 104, 108, 115, 119, 122, 123, 127, and 130 in the amino acid sequence of wild-type IL-2, and at least one amino acid residue selected from the amino acid residues at positions 11, 12, 18, 20, 84, 87, 88, 91, 108, 115, 119, 122, and 123, which are replaced by a group derived from a cysteine residue or an asparagine residue with a sugar chain attached, in the amino acid sequence of the IL-2 variant.

[0311] As an embodiment of the present invention, an IL-2 variant having at least 5 sugar chains bound to wild-type IL-2 can also be cited. As an example of an IL-2 having 5 sugar chains bound to wild-type IL-2, an IL-2 variant can be cited that has the amino acid residues selected from positions 1, 3, 4, 5, 8, 11, 12, 13, 15, 16, 18, 19, 20, 23, 32, 38, 51, 76, 84, 87, 88, 91, 92, 100, 102, 104, 108, 115, 119, 122, 123, 127, and 130 in the amino acid sequence of wild-type IL-2 replaced by 5 amino acid residues that are groups derived from cysteine residues or asparagine residues to which sugar chains are bound.

[0312] In addition, as an example of an IL-2 having 5 sugar chains bound to wild-type IL-2, an IL-2 variant is more preferably cited that has at least 1 amino acid residue selected from the amino acid residues at positions 1, 3, 4, 5, 8, 11, 12, 13, 15, 16, 18, 19, 20, 23, 32, 38, 51, 76, 84, 87, 88, 91, 92, 100, 102, 104, 108, 115, 119, 122, 123, 127, and 130 in the amino acid sequence of wild-type IL-2 and at least 1 amino acid residue selected from the amino acid residues at positions 1, 3, 5, 12, 32, 51, 76, 91, 100, 102, and 104 replaced by groups derived from cysteine residues or asparagine residues to which sugar chains are bound.

[0313] As an embodiment of the IL-2 variant of the present invention, the following IL-2 variants can be cited.

[0314] · An IL-2 variant in which the amino acid residues at positions 3, 12, 32, 76, and 91 in the amino acid sequence of wild-type IL-2 are each replaced by an amino acid residue to which a sugar chain is bound.

[0315] · An IL-2 variant in which the amino acid residues at positions 1, 3, 5, 12, and 91 in the amino acid sequence of wild-type IL-2 are each replaced by an amino acid residue to which a sugar chain is bound.

[0316] · An IL-2 variant in which the amino acid residues at positions 3, 12, 51, 91, and 100 in the amino acid sequence of wild-type IL-2 are each replaced by an amino acid residue to which a sugar chain is bound.

[0317] · An IL-2 variant in which the amino acid residues at positions 3, 12, 76, 91, and 100 in the amino acid sequence of wild-type IL-2 are each replaced by an amino acid residue to which a sugar chain is bound.

[0318] · An IL-2 variant in which the amino acid residues at positions 12, 91, 100, 102, and 104 in the amino acid sequence of wild-type IL-2 are each replaced with an amino acid residue conjugated with a sugar chain.

[0319] In the above IL-2 variant, the conjugated sugar chain can be any sugar chain, for example, a sugar chain having the structure shown in (Formula 8) can be cited.

[0320] In addition, as an embodiment of the IL-2 variant of the present invention, the following IL-2 variants can also be cited.

[0321] · An IL-2 variant in which the amino acid residues at positions 3, 12, 32, 76, and 91 in the amino acid sequence of wild-type IL-2 are replaced with a cysteine-residue-derived group conjugated with a sugar chain shown in (Formula 1) and the structure of the Saccharide in (Formula 1) is (Formula 8).

[0322] · An IL-2 variant in which the amino acid residues at positions 1, 3, 5, 12, and 91 in the amino acid sequence of wild-type IL-2 are replaced with a cysteine-residue-derived group conjugated with a sugar chain shown in (Formula 1) and the structure of the Saccharide in (Formula 1) is (Formula 8).

[0323] · An IL-2 variant in which the amino acid residues at positions 3, 12, 51, 91, and 100 in the amino acid sequence of wild-type IL-2 are replaced with a cysteine-residue-derived group conjugated with a sugar chain shown in (Formula 1) and the structure of the Saccharide in (Formula 1) is (Formula 8).

[0324] · An IL-2 variant in which the amino acid residues at positions 3, 12, 76, 91, and 100 in the amino acid sequence of wild-type IL-2 are replaced with a cysteine-residue-derived group conjugated with a sugar chain shown in (Formula 1) and the structure of the Saccharide in (Formula 1) is (Formula 8).

[0325] · An IL-2 variant in which the amino acid residues at positions 12, 91, 100, 102, and 104 in the amino acid sequence of wild-type IL-2 are replaced with a cysteine-residue-derived group conjugated with a sugar chain shown in (Formula 1) and the structure of the Saccharide in (Formula 1) is (Formula 8).

[0326] In the present embodiment, the amino acid sequence of wild-type IL-2 is more preferably the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence in which the amino acid residue at position 125 of the amino acid sequence shown in SEQ ID NO: 1 is substituted with a serine residue or an alanine residue.

[0327] [Method for producing a sugar chain-binding IL-2 variant]

[0328] As a method for producing a sugar chain-binding IL-2 variant, a method of chemically synthesizing a sugar chain-binding peptide and then folding it (chemical synthesis method) can be mentioned; or a method of expressing an IL-2 variant in which the amino acid residue at the sugar chain-introducing position in the amino acid sequence of wild-type IL-2 is substituted with an amino acid residue capable of binding a sugar chain in a host cell such as Escherichia coli, and then binding the amino acid residue capable of binding a sugar chain to a sugar chain (expression method).

[0329] In this specification, "peptide" means a chain formed by connecting multiple amino acid residues via peptide bonds. Unless otherwise specified, the side chains of each amino acid residue may contain a protecting group, and the amino group at the N-terminus and the carboxyl group at the C-terminus may also be modified.

[0330] A sugar chain-binding IL-2 variant can be produced by a combination of the chemical synthesis method and the expression method. Each method will be described below.

[0331] (Production of a sugar chain-binding IL-2 variant by the chemical synthesis method)

[0332] In the chemical synthesis method, it is preferable to sequentially link at least one or more sugar chain-binding peptide fragments and peptide fragments and then fold them to produce a sugar chain-binding IL-2 variant.

[0333] As the total number of the linked peptide fragments and sugar chain-binding peptide fragments, it is preferable to use 2 to 15 fragments, and more preferably 2 to 5 fragments. The sugar chain-binding peptide fragments and peptide fragments can also be thioesterified and linked in the form of sugar chain-binding peptide thioesters and peptide thioesters.

[0334] As a method for synthesizing peptide fragments and peptide thioesters, for example, methods commonly used in peptide synthesis [for example, the methods described in the 5th Edition of Experimental Chemistry Course 16: Synthesis of Organic Compounds IV: Carboxylic Acids, Amino Acids, and Peptides (edited by the Chemical Society of Japan, Maruzen Co., Ltd., 2005), Chemical Ligation: Tools for Biomolecule Synthesis and Modification (written by Luca D. DiAndrea et al., Wiley, 2017), etc.] can be mentioned.

[0335] At this time, in order to improve the solubility of the peptide, etc., pseudoproline (J. Am. Chem. Soc., 1996, 118, 9218 - 9227) or isopeptide (Angew. Chem. Int. Ed., 2015, 54, 8226 - 8230) can also be used instead of 2 - amino acids.

[0336] In addition, as methods for synthesizing peptide fragments and peptide thioesters, for example, liquid - phase synthesis methods such as the Ajiphase technology developed by Takahashi et al. (Tetrahedron Lett., 2012, 53, 1936.) and the Molecular Hiving technology developed by Okada et al. (J. Org. Chem., 2013, 78, 320 - 327) can be used instead of the peptide solid - phase synthesis method.

[0337] In addition, as a method for synthesizing peptide fragments, for example, a method known in the art can be used, such as a method of manufacturing by recombinant DNA methods including polymerase chain reaction (PCR), preparation of plasmid DNA, cleavage of DNA using endonucleases, preparation of oligonucleotides, ligation of DNA, isolation of mRNA, obtaining of transformants by introducing DNA into appropriate host cells, and culturing of transformants, or a cell - free protein expression method (Current Opinion in Biotechnology 2002, 13:297 - 303) and other methods described therein. As a method for synthesizing peptide thioesters, for example, a method described in (Proc Natl Acad Sci USA 1998, 95:6705 - 6710) and other methods can be used.

[0338] As a method for binding a sugar chain to a peptide fragment, peptide thioester, etc., for example, when binding a sugar chain to the side - chain thiol of a cysteine residue of a peptide fragment, a method described in Japanese Patent No. 4607017 and other publications can be cited. In addition, for example, when binding a sugar chain to the side - chain amide of an asparagine residue of a peptide fragment, a method described in Japanese Patent No. 4119428 and other publications can be cited. In addition, as a method for manufacturing a sugar chain, for example, a method described in International Publication No. 03 / 008431 and other methods can be cited.

[0339] As a method for linking peptide fragments and / or glycan-binding peptide fragments, methods commonly used in peptide synthesis can be cited [for example, the methods described in Experimental Chemistry Course, 5th Edition, Vol. 16, Synthesis of Organic Compounds IV: Carboxylic Acids, Amino Acids, Peptides (edited by The Chemical Society of Japan, Maruzen Co., Ltd., 2005), Chemical Ligation: Tools for Biomolecule Synthesis and Modification (by Luca D. DiAndrea et al., Wiley, 2017), Chemoselective and Bioorthogonal Ligation Reactions Volume 1, 2 (by W. Russ Algar et al., Wiley, 2017), etc.]. Preferably, the native chemical ligation (NCL) method of a peptide fragment having a C-terminal thioester and another peptide fragment having a cysteine residue at the N-terminus is used.

[0340] The linking of peptide fragments and / or glycan-binding peptide fragments can be carried out at any site. In the case of using the NCL method, as the N-terminal amino acid residue of the C-terminal side peptide fragment, cysteine residue and alanine residue are preferred, and cysteine residue is more preferred.

[0341] As a method for linking peptide fragments and / or glycan-binding peptide fragments, specifically, for example, when an alanine residue is used as the N-terminal amino acid residue of the C-terminal side fragment, a method can be cited in which, according to a conventional method [the method described in Chemical Ligation: Tools for Biomolecule Synthesis and Modification (by Luca D. DiAndrea et al., Wiley, 2017), etc.], after linking a C-terminal side peptide fragment in which the alanine residue is replaced with a cysteine residue and an N-terminal side peptide thioester fragment by the NCL method, the cysteine residue is converted to an alanine residue by a desulfurization reaction.

[0342] As a method for folding glycan-binding peptides, for example, methods commonly used in peptide folding can be cited [for example, the methods described in Experimental Chemistry Course, 5th Edition, Vol. 16, Synthesis of Organic Compounds IV: Carboxylic Acids, Amino Acids, Peptides (edited by The Chemical Society of Japan, Maruzen Co., Ltd., 2005), Chemical Ligation: Tools for Biomolecule Synthesis and Modification (by Luca D. DiAndrea et al., Wiley, 2017), etc.].

[0343] (Production of glycan-binding IL-2 variant using an expression method)

[0344] In the expression method, the sugar chain-conjugated IL-2 variant can be produced by known methods, for example, by recombinant DNA methods including polymerase chain reaction (PCR), preparation of plasmid DNA, cleavage of DNA using restriction enzymes, preparation of oligonucleotides, ligation of DNA, isolation of mRNA, obtaining a transformant by introducing DNA into an appropriate host cell, culturing the transformant, and introduction of sugar chains by chemical modification.

[0345] For example, the sugar chain-conjugated IL-2 variant can be obtained by recombining an expression cassette containing a base sequence encoding an amino acid sequence in which a mutation has been introduced in a manner that contains an amino acid residue capable of binding a sugar chain into an appropriate expression vector in the amino acid sequence of wild-type IL-2, introducing the expression vector into a host cell, and binding a sugar chain to the resulting protein by chemical modification.

[0346] As the base sequence of wild-type IL-2 used in the production of the above-mentioned expression vector, examples include the base sequence obtained by removing the base sequence encoding the signal sequence from the base sequence shown in NCBI accession No. NM_000586, the base sequence encoding the amino acid sequence described in Sequence Listing No. 1, etc.

[0347] The base sequence encoding IL-2 can be obtained by artificial gene synthesis or by designing appropriate primers from the sequences registered in gene banks such as the DNA Data Bank of Japan (DDBJ) and performing RT-PCR on mRNA extracted from cells or tissues of the animal.

[0348] In addition, the sugar chain-conjugated IL-2 variant can also be obtained by introducing the above-mentioned expression vector into a host cell capable of biosynthesizing the target sugar chain.

[0349] Specifically, for example, the above-mentioned expression vector can be obtained by ligating the base sequence encoding IL-2 into which the above-mentioned mutation has been introduced downstream of a promoter in a vector suitable for expression recombined at a desired position (for example, the 5' end). The expression vector may have a secretion signal depending on the host.

[0350] As a method for site-specifically introducing mutations into the amino acid sequence of wild-type IL-2 in such a way that it contains amino acid residues capable of binding to sugar chains, known methods can be used (U.S. Patent Application Publication No. 2004 / 0171154; Storici et al, 2001, Nature Biotechnology, 19, p. 773-776; Kren et al, 1998, Nat. Med., vol. 4, p. 285-290; Calissano and Macino, 1996, Fungal Genet. News lett., Vol. 43, p. 15-16). In addition, commercially available kits can also be used for introducing site-specific mutations.

[0351] Specifically, for example, when a cysteine residue is used as the amino acid residue capable of binding to sugar chains, an IL-2 variant in which the amino acid residue of wild-type IL-2 is replaced with a cysteine residue can be prepared according to the methods described in U.S. Patent No. 5206344, International Publication No. 2016 / 025385, etc., and the binding of sugar chains to this IL-2 variant can be carried out according to the method described in Japanese Patent No. 4607017 Gazette.

[0352] The region containing the base sequence encoding IL-2 has a translation start codon at the 5'-end, and in addition, it may have a translation stop codon at the 3'-end. In addition, in order to express the base sequence encoding IL-2, it is preferred to ligate a promoter upstream thereof.

[0353] As the promoter, there is no particular limitation as long as it is a promoter corresponding to the host used in the expression of the gene. When the transformed host is Bacillus subtilis, for example, SP01, SP02, and PenP promoters can be mentioned. When the host is yeast, for example, PH05, PGK, GAP, and ADH promoters can be mentioned. When the host is Escherichia coli, the trp promoter (Ptrp), lac promoter, etc. can be mentioned. When the host is an animal cell, for example, promoters derived from SV40 and retroviruses can be mentioned.

[0354] The IL-2 protein can also be expressed in E. coli without an accompanying signal sequence, and this protein can be recovered from inclusion bodies and refolded into an active form. Such an expression system is described in U.S. Patent No. 7105653.

[0355] A signal sequence can also be used to facilitate the expression of IL-2 protein. As signal sequences for mammalian cells, for example, a natural human IL-2 signal sequence, a signal sequence homologous to the TCR coding sequence, and a signal sequence homologous to the mouse IL-2 coding sequence can be cited. In addition, as other suitable signal sequence / host cell pairs, for example, it includes the Bacillus subtilis sacB signal sequence for secretion in B. subtilis, the Saccharomyces cerevisiae α mating factor signal sequence for secretion using P. pastoris, or the P. pastoris acid phosphatase phoI signal sequence. The signal sequence can also be directly linked to the protein coding sequence via a sequence encoding a signal peptidase cleavage site, or can be linked via a short nucleotide linker.

[0356] Elements for enhancing transcription and translation in eukaryotic protein expression systems can be used. For example, by configuring the cauliflower mosaic virus (CaMV) promoter at positions 1000 bp on both sides of a heterologous promoter, the transcription level in plant cells can be increased by 10 to 400 times.

[0357] The host cell is not particularly limited, and prokaryotic cells and eukaryotic cells can be cited. As preferred host cells, for example, prokaryotic cells such as E. coli and Bacillus subtillus, and animal cells such as HEK, J558, NSO, SP2-O, CHO, COS, KB, NIH3T3, BALB / c3T3, umbilical vein endothelial cells, and yeast strains such as S. cerevisiae and Pichia pastoris, and insect cells such as Sf9 or Tn can be cited.

[0358] The host cell can also be a cell modified in a manner capable of biosynthesizing the target sugar chain.

[0359] The transformation of the host can be any method commonly used for each host or a method adaptable thereto. For example, when the host is Escherichia coli or yeast, an expression vector containing recombinant DNA is introduced into competent cells prepared by methods such as the lithium method using the heat shock method or electroporation. If the host is an animal cell, an expression vector containing recombinant DNA can be introduced into cells in the proliferation phase or the like by the calcium phosphate method, liposome transfection method, or electroporation method.

[0360] The resulting transformant can be cultured using a medium commonly used for each host or a suitable culture solution to express the protein, and if necessary, a sugar chain can be chemically modified and bound thereto to produce a sugar chain-bound IL-2 protein. As the culture solution, for example, when the host is Escherichia coli, culture solutions such as LB medium can be cited; when the host is yeast, culture solutions such as YPD medium can be cited; and when the host is an animal cell, a culture solution obtained by adding fetal bovine serum to Dulbecco's MEM can be cited.

[0361] The culture may be carried out under conditions commonly used for each host or conditions that can be applied. For example, if the host is yeast, it is cultured at about 25 to 37°C for about 12 hours to 2 weeks, and aeration or stirring can be applied as needed. When the host is an animal cell, it is cultured at 37°C under conditions of 5% carbon dioxide and 100% humidity for about 24 hours to 2 weeks, and the gas phase conditions can be changed or stirring can be carried out as needed.

[0362] [PEG-conjugated IL-2 variant]

[0363] As an embodiment of the IL-2 variant of the present invention, a variant in which at least one amino acid residue selected from the amino acid residues at positions 4, 5, 6, 7, 8, 60, 78, 79, 99, 100, 101, and 129 in the amino acid sequence of IL-2 is conjugated with PEG is preferred, and an IL-2 variant in which at least one of the amino acid residues at positions 78 and 129 is conjugated with PEG is more preferred.

[0364] As an embodiment of the IL-2 variant of the present invention, an IL-2 variant comprising an amino acid sequence in which at least one amino acid residue selected from the amino acid residues at positions 4, 5, 6, 7, 8, 60, 78, 79, 99, 100, 101, and 129 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with PEG is more preferred, and an IL-2 variant comprising an amino acid sequence in which at least one of the amino acid residues at positions 78 and 129 is replaced with an amino acid residue conjugated with PEG is more preferred.

[0365] In the present embodiment, the amino acid sequence of wild-type IL-2 is more preferably the amino acid sequence shown in SEQ ID NO: 1, an amino acid sequence in which a methionine residue is bound to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1, an amino acid sequence in which the N-terminal alanine residue of the amino acid sequence shown in SEQ ID NO: 1 is deleted, an amino acid sequence in which the N-terminal alanine residue of the amino acid sequence shown in SEQ ID NO: 1 is deleted and a methionine residue is bound, or an amino acid sequence in which the amino acid residue at the 125th position of the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1 with a methionine residue bound to the N-terminus, the amino acid sequence in which the N-terminal alanine residue of the amino acid sequence shown in SEQ ID NO: 1 is deleted, and the amino acid sequence in which the N-terminal alanine residue of the amino acid sequence shown in SEQ ID NO: 1 is deleted and a methionine residue is bound, is substituted with a serine residue or an alanine residue.

[0366] "PEG" refers to a water-soluble polymer containing a structure formed by polymerization of ethylene glycol represented by 「-(CH2CH2O) n -」(n is 2 or more), that is, a poly(ethylene glycol) molecule. As PEG, PEG4 and PEG having an average molecular weight of 10 kDa or more are preferred. For example, those having an average molecular weight of 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 200 kDa, etc. can be mentioned, but there is no particular limitation. In addition, as the shape of PEG, it can be linear or branched, and is not limited to these. By chemically binding PEG to the above amino acid residue in the amino acid sequence of IL-2, the selectivity for IL-2R αβγ can be improved. By the IL-2 variant with improved selectivity for IL-2R αβγ Treg can be selectively activated.

[0367] As the amino acid residue bound to PEG, for example, a cysteine residue bound to PEG and a non-natural amino acid residue can be mentioned.

[0368] As the non-natural amino acid residue bound to PEG, for example, a group derived from an amino acid residue having a thiol group with PEG bound thereto and a group derived from an amino acid residue having an azide group with PEG bound thereto can be mentioned. As the amino acid residue having a thiol group, specifically, for example, acetylcysteine, homocysteine, etc. can be mentioned, but are not limited to these. As the amino acid residue having an azide group, specifically, for example, o-Az-Z-Lys residue, m-Az-Z-Lys residue, N 6- Overlapping lysine, para-azidophenylalanine, but not limited to these. As non-natural amino acid residues, they can also be those described in International Publication No. 2017 / 030156, [Nature. 2017 Nov 29; 551(7682):644 - 647.], International Publication No. 2013 / 068874, US Patent Application Publication No. 2014 / 0046030, [Bioconj. Chem., 2014, 25(2), pp 351 - 361], International Publication No. 2014 / 044872, [Bioconj. Chem. 2015 Nov 18; 26(11):2249 - 60], International Publication No. 2014 / 124258, [Proc Natl Acad Sci U S A. 2011 Jun 28; 108(26):10437 - 42], etc. PEG and the above non-natural amino acid residues can be bound via a linker. The linker can be appropriately changed according to the types of PEG or non-natural amino acid residues.

[0369] The above o-Az-Z-Lys residue is an amino acid residue having the structure shown in the following (Formula 10).

[0370]

[0371] The above m-Az-Z-Lys residue is an amino acid residue having the structure shown in the following (Formula XX1).

[0372]

[0373] Examples of the group derived from the o-Az-Z-Lys residue bound to PEG or the group derived from the m-Az-Z-Lys residue bound to PEG include structures in which PEG is bound via a linker obtained by reacting acetylene with the azide group of the o-Az-Z-Lys residue or m-Az-Z-Lys. As acetylene, for example, dibenzylcyclooctyne (DBCO), bicyclo[6.1.0]nonyne (BCN), etc. can be mentioned. In addition, instead of acetylene, structures in which PEG is bound via an amide bond according to the methods described in [J. Am. Chem. Soc. 2006, 128, pp 8820], [Org. Lett. 2000, 2, pp2141], [Org. Lett. 2000, 2, pp1939.], etc. can also be mentioned, but not limited to these.

[0374] Groups derived from o-Az-Z-Lys residues or m-Az-Z-Lys residues conjugated with PEG can specifically include, for example, the structures shown in the following (Formula 11) and / or (Formula 12), or the following (Formula Y4) and / or (Formula Y5).

[0375]

[0376]

[0377] As the PEG in the formula, various PEGs can be used according to the average molecular weight or structure of PEG. Specifically, for example, the following (Formula 13), the following formula (14) when the average molecular weight is 20 kDa, the following formula (14) when the average molecular weight is 30 kDa, the following (Formula X0) when the average molecular weight is 40 kDa, the following (Formula 15) when the average molecular weight is 50 kDa, the following (Formula 16) when the average molecular weight is 40 kDa, the following (Formula 16) when the average molecular weight is 80 kDa, the following (Formula X1) when the average molecular weight is 40 kDa, the following (Formula X2) when the average molecular weight is 80 kDa, or the following (Formula X3) when the average molecular weight is 40 kDa can be listed, but are not limited to these. In addition, when the PEG in the formula is the structure shown in the following (Formula X3), it is not limited to O(CH2CH2O) n When there are 4 branches, 2 branches or 3 branches of CH3, they can also be used in the same way.

[0378]

[0379]

[0380] Groups derived from cysteine residues conjugated with PEG can specifically include, for example, the structures shown in the following (Formula Y6) and / or (Formula Y7) and / or (Formula Y8) conjugated with PEG via a linker obtained by reacting maleimide with the thiol group of a cysteine residue, the structure shown in the following (Formula Y9) conjugated with PEG via a linker obtained by reacting haloacetyl with the thiol group of a cysteine residue, etc., but are not limited to these.

[0381]

[0382] Specifically, for example, the PEG in the formula can include the structures shown in (Formula X7) when the average molecular weight is 40 kDa, or (Formula X7) when the average molecular weight is 80 kDa, or (Formula X8) when the average molecular weight is 80 kDa, but is not limited to these.

[0383]

[0384] As an embodiment of the IL-2 variant of the present invention, the IL-2 variants described below are preferred.

[0385] As an embodiment of the IL-2 variant of the present invention, for example, the following described IL-2 variants in which one PEG is conjugated to IL-2 can be mentioned.

[0386] · An IL-2 variant in which the amino acid residue at position 4 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with PEG.

[0387] · An IL-2 variant in which the amino acid residue at position 5 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with PEG.

[0388] · An IL-2 variant in which the amino acid residue at position 6 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with PEG.

[0389] · An IL-2 variant in which the amino acid residue at position 7 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with PEG.

[0390] · An IL-2 variant in which the amino acid residue at position 8 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with PEG.

[0391] · An IL-2 variant in which the amino acid residue at position 60 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with PEG.

[0392] · An IL-2 variant in which the amino acid residue at position 78 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with PEG.

[0393] · An IL-2 variant in which the amino acid residue at position 79 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with PEG.

[0394] · An IL-2 variant in which the amino acid residue at position 99 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with PEG.

[0395] · An IL-2 variant in which the amino acid residue at position 100 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with PEG.

[0396] · An IL-2 variant in which the amino acid residue at position 101 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with PEG.

[0397] · An IL-2 variant in which the amino acid residue at position 129 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with PEG.

[0398] In the above IL-2 variant, as the size of the conjugated PEG, PEG with an average molecular weight of 20 kDa or more is preferred, and examples thereof include PEG with an average molecular weight of 20, 30, 40, 50, 60, 70, or 80 kDa.

[0399] As one embodiment of the IL-2 variant of the present invention, for example, the following-described IL-2 variant conjugated with 1 PEG on IL-2 can also be cited.

[0400] · An IL-2 variant in which the amino acid residue at position 4 in the amino acid sequence of wild-type IL-2 is replaced with a group derived from an o-Az-Z-Lys residue conjugated with PEG as shown in (Formula 14) when the average molecular weight is 20 kDa or the average molecular weight is 30 kDa.

[0401] · An IL-2 variant in which the amino acid residue at position 5 in the amino acid sequence of wild-type IL-2 is replaced with a group derived from an o-Az-Z-Lys residue conjugated with PEG as shown in (Formula 14) when the average molecular weight is 20 kDa or the average molecular weight is 30 kDa.

[0402] · An IL-2 variant in which the amino acid residue at position 6 in the amino acid sequence of wild-type IL-2 is replaced with a group derived from an o-Az-Z-Lys residue conjugated with PEG as shown in (Formula 14) when the average molecular weight is 20 kDa or the average molecular weight is 30 kDa.

[0403] · An IL-2 variant in which the amino acid residue at position 7 in the amino acid sequence of wild-type IL-2 is replaced with a group derived from an o-Az-Z-Lys residue conjugated with PEG as shown in (Formula 14) when the average molecular weight is 30 kDa.

[0404] · An IL-2 variant in which the amino acid residue at position 8 in the amino acid sequence of wild-type IL-2 is replaced with a group derived from an o-Az-Z-Lys residue conjugated with PEG as shown in (Formula 14) when the average molecular weight is 20 kDa or the average molecular weight is 30 kDa.

[0405] · An IL-2 variant in which the amino acid residue at position 60 in the amino acid sequence of wild-type IL-2 is replaced with a group derived from an o-Az-Z-Lys residue conjugated with PEG as shown in (Formula 14) when the average molecular weight is 30 kDa.

[0406] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 78 is replaced with a PEG-conjugated group derived from an o-Az-Z-Lys residue as shown in (Formula 14) when the average molecular weight is 20 kDa or 30 kDa, (Formula X0) when the average molecular weight is 40 kDa, (Formula 15) when the average molecular weight is 50 kDa, (Formula 16) when the average molecular weight is 40 kDa or 80 kDa, (Formula X1) when the average molecular weight is 40 kDa, (Formula X2) when the average molecular weight is 80 kDa, or (Formula X3) when the average molecular weight is 40 kDa, resulting in an IL-2 variant.

[0407] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 79 is replaced with a PEG-conjugated group derived from an o-Az-Z-Lys residue as shown in (Formula 14) when the average molecular weight is 20 kDa or 30 kDa, resulting in an IL-2 variant.

[0408] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 99 is replaced with a PEG-conjugated group derived from an o-Az-Z-Lys residue as shown in (Formula 14) when the average molecular weight is 30 kDa, resulting in an IL-2 variant.

[0409] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 100 is replaced with a PEG-conjugated group derived from an o-Az-Z-Lys residue as shown in (Formula 14) when the average molecular weight is 20 kDa, resulting in an IL-2 variant.

[0410] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 101 is replaced with a PEG-conjugated group derived from an o-Az-Z-Lys residue as shown in (Formula 14) when the average molecular weight is 20 kDa or 30 kDa, resulting in an IL-2 variant.

[0411] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 129 is replaced with (Formula 13), or a PEG-conjugated group derived from an o-Az-Z-Lys residue as shown in (Formula 14) when the average molecular weight is 20 kDa or 30 kDa, (Formula X0) when the average molecular weight is 40 kDa, (Formula 16) when the average molecular weight is 40 kDa or 80 kDa, (Formula X1) when the average molecular weight is 40 kDa, (Formula X2) when the average molecular weight is 80 kDa, (Formula 15) when the average molecular weight is 50 kDa, or (Formula X3) when the average molecular weight is 40 kDa, resulting in an IL-2 variant.

[0412] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 129 is replaced with an IL-2 variant having a PEG-conjugated group derived from an o-Az-Z-Lys residue as shown in (Formula 16) when it is 40 kDa.

[0413] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 129 is replaced with an IL-2 variant having a PEG-conjugated group derived from a cysteine residue as shown in (Formula X7) and / or (Formula X8) when the average molecular weight is 40 kDa or 80 kDa.

[0414] As an embodiment of the IL-2 variant of the present invention, preferably the following-described IL-2 variant in which at least 2 PEGs are conjugated to IL-2.

[0415] · In the amino acid sequence of wild-type IL-2, the amino acid residues at positions 4 and 78 are respectively replaced with PEG-conjugated amino acid residues.

[0416] · In the amino acid sequence of wild-type IL-2, the amino acid residues at positions 5 and 78 are respectively replaced with PEG-conjugated amino acid residues.

[0417] · In the amino acid sequence of wild-type IL-2, the amino acid residues at positions 8 and 78 are respectively replaced with PEG-conjugated amino acid residues.

[0418] · In the amino acid sequence of wild-type IL-2, the amino acid residues at positions 78 and 79 are respectively replaced with PEG-conjugated amino acid residues.

[0419] · In the amino acid sequence of wild-type IL-2, the amino acid residues at positions 78 and 99 are respectively replaced with PEG-conjugated amino acid residues.

[0420] · In the amino acid sequence of wild-type IL-2, the amino acid residues at positions 78 and 129 are respectively replaced with PEG-conjugated amino acid residues.

[0421] · In the amino acid sequence of wild-type IL-2, the amino acid residues at positions 4 and 129 are respectively replaced with PEG-conjugated amino acid residues.

[0422] · In the amino acid sequence of wild-type IL-2, the amino acid residues at positions 5 and 129 are respectively replaced with PEG-conjugated amino acid residues.

[0423] · An IL-2 variant in which the amino acid residues at positions 8 and 129 in the amino acid sequence of wild-type IL-2 are each replaced with an amino acid residue conjugated with PEG.

[0424] · An IL-2 variant in which the amino acid residues at positions 79 and 129 in the amino acid sequence of wild-type IL-2 are each replaced with an amino acid residue conjugated with PEG.

[0425] · An IL-2 variant in which the amino acid residues at positions 99 and 129 in the amino acid sequence of wild-type IL-2 are each replaced with an amino acid residue conjugated with PEG.

[0426] As the size of the PEG conjugated in the above IL-2 variant, PEG with an average molecular weight of 20 kDa or more is preferred, and examples thereof include PEG with an average molecular weight of 20, 30, 40, 50, 60, 70, or 80 kDa.

[0427] As one embodiment of the IL-2 variant of the present invention, the following-described IL-2 variant in which at least two PEGs are conjugated to IL-2 is also preferred.

[0428] · An IL-2 variant in which the amino acid residues at positions 4 and 78 in the amino acid sequence of wild-type IL-2 are each replaced with a group derived from an o-Az-Z-Lys residue conjugated with PEG as shown in (Formula 14) when the average molecular weight is 30 kDa.

[0429] · An IL-2 variant in which the amino acid residues at positions 4 and 78 in the amino acid sequence of wild-type IL-2 are each replaced with a group derived from an o-Az-Z-Lys residue conjugated with PEG as shown in (Formula X0) when the average molecular weight is 40 kDa.

[0430] · An IL-2 variant in which the amino acid residues at positions 4 and 78 in the amino acid sequence of wild-type IL-2 are each replaced with a group derived from an o-Az-Z-Lys residue conjugated with PEG as shown in (Formula 15) when the average molecular weight is 50 kDa.

[0431] · An IL-2 variant in which the amino acid residues at positions 5 and 78 in the amino acid sequence of wild-type IL-2 are each replaced with a group derived from an o-Az-Z-Lys residue conjugated with PEG as shown in (Formula 14) when the average molecular weight is 30 kDa.

[0432] · An IL-2 variant in which the amino acid residues at positions 5 and 78 in the amino acid sequence of wild-type IL-2 are each replaced with a group derived from an o-Az-Z-Lys residue conjugated with PEG as shown in (Formula X0) when the average molecular weight is 40 kDa.

[0433] · In the amino acid sequence of wild-type IL-2, the amino acid residues at positions 8 and 78 are respectively replaced with the groups of PEG-conjugated o-Az-Z-Lys residues shown in (Formula 14) when the average molecular weight is 30 kDa and (Formula X0) when the average molecular weight is 40 kDa, to form an IL-2 variant.

[0434] · In the amino acid sequence of wild-type IL-2, the amino acid residues at positions 78 and 79 are respectively replaced with the groups of PEG-conjugated o-Az-Z-Lys residues shown in (Formula 14) when the average molecular weight is 30 kDa, to form an IL-2 variant.

[0435] · In the amino acid sequence of wild-type IL-2, the amino acid residues at positions 78 and 99 are respectively replaced with the groups of PEG-conjugated o-Az-Z-Lys residues shown in (Formula 14) when the average molecular weight is 30 kDa, to form an IL-2 variant.

[0436] · In the amino acid sequence of wild-type IL-2, the amino acid residues at positions 78 and 129 are respectively replaced with the groups of PEG-conjugated o-Az-Z-Lys residues shown in (Formula 14) when the average molecular weight is 30 kDa, to form an IL-2 variant.

[0437] · In the amino acid sequence of wild-type IL-2, the amino acid residues at positions 4 and 129 are respectively replaced with the groups of PEG-conjugated o-Az-Z-Lys residues shown in (Formula 14) when the average molecular weight is 30 kDa, (Formula X0) when the average molecular weight is 40 kDa, or (Formula 15) when the average molecular weight is 50 kDa, to form an IL-2 variant.

[0438] · In the amino acid sequence of wild-type IL-2, the amino acid residues at positions 5 and 129 are respectively replaced with the groups of PEG-conjugated o-Az-Z-Lys residues shown in (Formula 14) when the average molecular weight is 30 kDa, (Formula X0) when the average molecular weight is 40 kDa, or (Formula 15) when the average molecular weight is 50 kDa, to form an IL-2 variant.

[0439] · In the amino acid sequence of wild-type IL-2, the amino acid residues at positions 8 and 129 are respectively replaced with the groups of PEG-conjugated o-Az-Z-Lys residues shown in (Formula 14) when the average molecular weight is 30 kDa, (Formula X0) when the average molecular weight is 40 kDa, or (Formula 15) when the average molecular weight is 50 kDa, to form an IL-2 variant.

[0440] · In the amino acid sequence of wild-type IL-2, the amino acid residues at positions 79 and 129 are respectively replaced with an IL-2 variant having a PEG-bound group derived from an o-Az-Z-Lys residue as shown in (Formula 14) when the average molecular weight is 30 kDa.

[0441] · In the amino acid sequence of wild-type IL-2, the amino acid residues at positions 99 and 129 are respectively replaced with an IL-2 variant having a PEG-bound group derived from an o-Az-Z-Lys residue as shown in (Formula 14) when the average molecular weight is 30 kDa.

[0442] In the present embodiment, the amino acid sequence of wild-type IL-2 is preferably the amino acid sequence shown in SEQ ID NO: 1, an amino acid sequence having a methionine residue bound to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1, an amino acid sequence lacking the N-terminal alanine residue of the amino acid sequence shown in SEQ ID NO: 1, an amino acid sequence lacking the N-terminal alanine residue of the amino acid sequence shown in SEQ ID NO: 1 and having a methionine residue bound thereto, or an amino acid sequence in which the amino acid residue at position 125 in the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence having a methionine residue bound to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence lacking the N-terminal alanine residue of the amino acid sequence shown in SEQ ID NO: 1, or the amino acid sequence lacking the N-terminal alanine residue of the amino acid sequence shown in SEQ ID NO: 1 and having a methionine residue bound thereto is replaced with a serine residue or an alanine residue.

[0443] [Method for producing PEG-bound IL-2 variant]

[0444] As methods for producing a PEG-bound IL-2 variant, a chemical synthesis method and an expression method can be cited. A PEG-bound IL-2 variant can be produced by a combination of a chemical synthesis method and an expression method. Each method will be described below.

[0445] (Production of PEG-bound IL-2 variant by chemical synthesis method)

[0446] As a method for producing a PEG-bound IL-2 variant by chemical synthesis method, for example, the following methods can be cited: a method of chemically synthesizing a peptide in which the amino acid residue at the PEG-introducing position in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue having a chemical reactivity for site-specific PEGylation of a protein, then folding it to obtain an IL-2 variant, and binding PEG to the obtained IL-2 variant to produce a PEG-bound IL-2 variant; and a method of chemically synthesizing a PEG-bound peptide fragment and then folding it to produce a PEG-bound IL-2 variant.

[0447] The PEG-binding peptide fragment can be produced by introducing PEG into an amino acid residue having a chemical reactivity for site-specific PEGylation of a protein in the peptide fragment.

[0448] As a method for folding the PEG-binding peptide fragment after chemical synthesis, examples include a method of sequentially linking at least one or more PEG-binding peptide fragments and a peptide fragment followed by folding, or a method of introducing PEG into the full-length IL-2 peptide fragment obtained by chemical synthesis and then folding it.

[0449] As a method for synthesizing a peptide fragment and a method for folding the peptide fragment after sequential linking, for example, the same methods as those described in the section (Production of a sugar chain-conjugated IL-2 variant by chemical synthesis) can be cited.

[0450] As a method for introducing PEG by folding the IL-2 variant obtained by substituting the amino acid residue at the PEG-introducing position in the amino acid sequence of wild-type IL-2 with an amino acid residue having a chemical reactivity for site-specific PEGylation of a protein, for example, the methods described in U.S. Patent No. 5,206,344 and International Publication No. 2012 / 065086 can be cited. In addition, as a method for introducing PEG into an amino acid residue having a chemical reactivity for site-specific PEGylation of a protein in a peptide fragment, the methods described in [Biomaterials 22 (2001) 405-417], [Int. J. Mol. Sci. 2015, 16, 25831-25864], and [J. Pharm. Sci., 105 (2016) 460-475] can be cited.

[0451] As an amino acid residue having a chemical reactivity for site-specific PEGylation of a protein, for example, an amino acid residue having a thiol group and an amino acid residue having an azide group can be cited. As an amino acid residue having a thiol group, for example, cysteine, acetylcysteine, homocysteine, etc. can be cited, but are not limited to these.

[0452] As an amino acid residue having an azide group, for example, o-Az-Z-Lys residue, m-Az-Z-Lys residue, N 6-Azido lysine, p-azidophenylalanine, but not limited to these. In addition, it can also be the unnatural amino acid residues described in International Publication No. 2017 / 030156, [Nature. 2017 Nov 29; 551(7682):644 - 647.], International Publication No. 2013 / 068874, U.S. Patent Application Publication No. 2014 / 0046030, [Bioconj.Chem., 2014, 25(2), pp351 - 361], International Publication No. 2014 / 044872, [Bioconj.Chem. 2015 Nov 18; 26(11):2249 - 60], International Publication No. 2014 / 124258, [Proc Natl Acad Sci U S A. 2011 Jun 28; 108(26):10437 - 42]. PEG can be bound to the above - mentioned unnatural amino acid residues via a linker.

[0453] The above - mentioned linker refers to a hydrocarbon group having 1 to 20 carbon atoms, and the carbon can be modified by oxygen, nitrogen, sulfur, etc. In addition, the carbon can also be substituted with oxygen, nitrogen, sulfur. This linker can be appropriately changed according to the types of PEG or unnatural amino acid residues.

[0454] As a chemical synthesis method, specifically, for example, the following methods can be listed: After chemically synthesizing a peptide in which the amino acid residue at the PEG - introducing position in the amino acid sequence of wild - type IL - 2 is replaced with a thiol - group - having amino acid residue such as cysteine and / or an azide - group - having amino acid residue such as o - Az - Z - Lys residue, folding is performed to obtain an IL - 2 variant, and PEG is introduced into the IL - 2 variant to produce a PEG - conjugated IL - 2 variant. As a method for binding PEG to the IL - 2 variant into which a cysteine residue has been introduced, the method described in U.S. Patent No. 5206344 can be listed, etc.

[0455] In addition, specifically, for example, the following methods can be listed: After chemically synthesizing a peptide in which the amino acid residue at the PEG - introducing position in the amino acid sequence of wild - type IL - 2 is replaced with cysteine or an unnatural amino acid residue, folding is performed to obtain an IL - 2 variant, and PEG is introduced into the IL - 2 variant to produce a PEG - conjugated IL - 2 variant.

[0456] In the synthesis of the PEG - conjugated IL - 2 variant, the PEG reagent shown in the following (Formula XX2) can be used.

[0457] X-(Linker) n -PEG-Me (Formula XX2)

[0458] In the formula, X represents a functional group reactive with a thiol group, a functional group reactive with an azide group, and a functional group selectively reactive with an N-terminal amino group.

[0459] As the functional group reactive with a thiol, specifically, for example, a thiol group, a maleimide group, an acryloyl group, an iodoacetyl group, a bromoacetyl group, a chloroacetyl group, etc. can be mentioned, and preferably an iodoacetyl group or a maleimide group is sufficient.

[0460] As the functional group reactive with an azide, specifically, for example, an ethynyl group, a DBCO group, a DBN group, a cycloalkyne containing a heteroatom in the medium ring structure (Angew. Chem. Int. Ed. 2015, 54, 1190 - 1194), a thioester group, etc. can be mentioned, and preferably DBCO is listed.

[0461] As the functional group selectively reactive with an N-terminal amino group, specifically, for example, an aldehyde, etc. can be mentioned.

[0462] In the formula, as the linker, it is a hydrocarbon group having 1 to 20 carbon atoms, and the carbon can be modified with oxygen, nitrogen, sulfur, etc., and the carbon can also be substituted with oxygen, nitrogen, sulfur.

[0463] In the formula, n represents 0 or 1.

[0464] In the formula, PEG refers to a water-soluble polymer containing a structure formed by polymerization of ethylene glycol represented by "-(CH2CH2O) m -"(m is 2 or more), that is, a poly(ethylene glycol) molecule. The molecular weight of PEG can be, for example, PEG4, an average molecular weight of 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, or 200 kDa, etc., but there is no particular limitation. In addition, as the shape of PEG, it can be linear or branched, but is not limited to these.

[0465] Depending on the PEG reagent used, sometimes stereoisomers, optical isomers, geometric isomers, etc. are formed, and these isomers can be separated and used by known methods or used as a mixture. Since the obtained IL-2 variant is a macromolecule, it is considered that the difference in the structure of these partial structure isomers hardly has an impact.

[0466] In addition to the commercially available PEG reagents, the above PEG reagents can also be PEG reagents prepared from commercially available PEG reagents. For example, for a PEG reagent having a carboxylic acid equivalent such as a carboxyl group or N-hydroxysuccinimide ester at the terminal, condensation with an amine reactive with a thiol group or azide group can be used to prepare a PEG reagent. In addition, it can also be synthesized by condensing a carboxylic acid equivalent such as a carboxyl group or N-hydroxysuccinimide ester reactive with a thiol group or azide group with a PEG reagent having an amino group at the terminal, but is not limited to these methods.

[0467] (Production of PEG-conjugated IL-2 variant by expression method)

[0468] As a method for producing a PEG-conjugated IL-2 variant by the expression method, for example, the following method can be cited: After expressing an IL-2 variant in which the amino acid residue at the PEG-introducing position in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue having a chemical reactivity capable of site-specific PEGylation of a protein by a host cell such as Escherichia coli, PEG is bound to the amino acid residue in the IL-2 variant by chemical modification, thereby producing a PEG-conjugated IL-2 variant.

[0469] Specifically, for example, the following method can be cited: Similar to the method described in the section of (production of glycosylated IL-2 variant by expression method), an expression cassette containing a base sequence encoding an amino acid sequence in which the PEG-introducing position in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue having a chemical reactivity capable of site-specific PEGylation of a protein is recombined into an appropriate expression vector, the expression vector is introduced into a host cell such as Escherichia coli to obtain a transformant, the transformant is made to express the IL-2 variant, and PEG is bound to the cysteine residue or unnatural amino acid residue of the IL-2 variant by chemical modification, thereby obtaining a PEG-conjugated IL-2 variant.

[0470] In the case of using Escherichia coli as a host cell, in order to improve the expression efficiency or purification of the produced protein, a linker can be introduced at the N-terminus of wild-type IL-2 to construct an expression cassette. As such a linker, for example, a methionine residue, eight polyhistidines, and eight polyhistidines containing a methionine residue can be cited.

[0471] As a method for preparing an IL-2 variant in which the amino acid residue of IL-2 is replaced with a cysteine residue, for example, the methods described in US Patent No. 5206344, International Publication No. 2016 / 025385, etc. can be cited.

[0472] As a method for preparing an IL-2 variant in which an amino acid residue of IL-2 is substituted with a non-natural amino acid residue having a chemical reactivity capable of site-specific PEGylation of a protein, the methods described in International Publication No. 2017 / 030156, [Nature. 2017 Nov 29; 551(7682):644-647.], International Publication No. 2013 / 068874, U.S. Patent Application Publication No. 2014 / 0046030, [Bioconj.Chem., 2014, 25(2), pp 351-361], International Publication No. 2014 / 044872, [Bioconj.Chem. 2015 Nov 18; 26(11):2249-60], International Publication No. 2014 / 124258, [Proc Natl Acad Sci U S A. 2011 Jun 28; 108(26):10437-42] can be mentioned.

[0473] In addition, as a method for producing an IL-2 variant in which an amino acid residue of IL-2 is substituted with an o-Az-Z-Lys residue or an m-Az-Z-Lys residue o-Az-Z-Lys residue, and a method for binding PEG to the IL-2 variant, for example, the method described in International Publication No. 2017 / 030156 can be mentioned.

[0474] As a method for introducing PEG into an IL-2 variant in which an amino acid residue at the PEG-introducing position in the amino acid sequence of IL-2 is substituted with an amino acid residue having a chemical reactivity capable of site-specific PEGylation of a protein, PEG can be introduced according to the methods described in Japanese Patent No. 5206344, International Publication No. 2012 / 065086, International Publication No. 2017 / 030156, [Nature. 2017 Nov 29; 551(7682):644-647.], International Publication No. 2013 / 068874, U.S. Application Publication No. 2014 / 0046030, [Bioconj.Chem., 2014, 25(2), pp 351-361], International Publication No. 2014 / 044872, [Bioconj.Chem. 2015 Nov 18; 26(11):2249-60], International Publication No. 2014 / 124258, [Proc Natl Acad Sci U S A. 2011 Jun 28; 108(26):10437-42] and the like.

[0475] [IL-2 variants in which PEG or a sugar chain is further bound to a sugar chain-binding IL-2 variant or a PEG-binding IL-2 variant, and a method for producing the same]

[0476] The above-mentioned sugar chain-binding IL-2 variant can further bind to PEG. Additionally, the above-mentioned PEG-binding IL-2 variant can also further bind to a sugar chain. These IL-2 variants can be produced by combining the above-mentioned [method for producing a sugar chain-binding IL-2 variant] and [method for producing a PEG-binding IL-2 variant]. Additionally, PEG can be selectively introduced onto the amino group at the N-terminus according to International Publication No. 2012 / 065086, etc.

[0477] When further introducing a sugar chain into the PEG-binding IL-2 variant, it is preferably an IL-2 variant having an amino acid sequence in which at least one amino acid residue selected from the amino acid residues at positions 11, 12, 13, 15, 16, 18, 19, 20, 84, 87, 88, 91, 92, 108, 115, 119, 122, 123, and 130 in the amino acid sequence of wild-type IL-2 is bound to a sugar chain, and more preferably an IL-2 variant in which at least one amino acid residue selected from the amino acid residues at positions 12, 115, and 119 is bound to a sugar chain.

[0478] When further introducing PEG into the sugar chain-binding IL-2 variant, it is preferably an IL-2 variant having an amino acid sequence in which at least one amino acid residue selected from the amino acid residues at positions 1, 3, 4, 5, 6, 7, 8, 51, 60, 78, 79, 99, 100, 101, and 129 in the amino acid sequence of wild-type IL-2 is substituted with an amino acid residue bound to PEG, and more preferably an IL-2 variant having an amino acid sequence in which at least one amino acid residue selected from the amino acid residues at positions 1, 3, 51, and 78 is substituted with an amino acid residue bound to PEG.

[0479] Examples of the amino acid residue bound to PEG include a group derived from a cysteine residue bound to PEG, a group derived from an N-terminal amino acid residue, and a non-natural amino acid residue.

[0480] The group derived from a cysteine residue or the group derived from an N-terminal amino acid residue refers to a group in which PEG is bound to the side-chain thiol group of a cysteine residue or the main-chain amino group of an N-terminal amino acid residue through chemical modification, etc. PEG and the group derived from a cysteine residue or the group derived from an N-terminal amino acid residue can be bound via a linker. The linker can be appropriately changed according to the type of PEG or non-natural amino acid residue.

[0481] As a non-natural amino acid residue conjugated with PEG, for example, a group in which PEG is conjugated to a group derived from an amino acid residue having a thiol group or a group derived from an amino acid residue having an azide group by chemical modification or the like can be mentioned. As a group derived from an amino acid residue having a thiol group, for example, a group derived from an acetylcysteine residue conjugated with PEG, a group derived from a homocysteine residue conjugated with PEG, etc. can be mentioned, but not limited to these.

[0482] As a group derived from an amino acid residue having an azide group conjugated with PEG, for example, a group derived from an o-Az-Z-Lys residue conjugated with PEG, a group derived from an m-Az-Z-Lys residue conjugated with PEG, a group derived from an N 6 -diazo lysine residue conjugated with PEG, a group derived from a p-azidophenylalanine residue conjugated with PEG, etc. can be mentioned, but not limited to these.

[0483] As non-natural amino acid residues, in addition, they can also be non-natural amino acid residues described in WO 2017 / 030156, [Nature. 2017 Nov 29; 551(7682): 644-647.], WO 2013 / 068874, US Patent Application Publication No. 2014 / 0046030, [Bioconj. Chem., 2014, 25(2), pp 351-361], WO 2014 / 044872, [Bioconj. Chem. 2015 Nov 18; 26(11): 2249-60], WO 2014 / 124258, [Proc Natl Acad Sci U S A. 2011 Jun 28; 108(26): 10437-42], etc. PEG can be conjugated to the above non-natural amino acid residues via a linker. The linker can be appropriately changed according to the types of PEG or non-natural amino acid residues.

[0484] When the amino acid residue at the 1st position in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with PEG, preferably a group derived from an N-terminal amino acid residue conjugated with PEG, a group derived from a cysteine residue conjugated with PEG, a group derived from an acetylcysteine residue conjugated with PEG, a group derived from an o-Az-Z-Lys residue conjugated with PEG, a group derived from an m-Az-Z-Lys residue conjugated with PEG, and more preferably a group derived from an N-terminal amino acid residue conjugated with PEG, a group derived from an acetylcysteine residue conjugated with PEG.

[0485] When at least one amino acid residue selected from the amino acid residues at positions 3, 4, 5, 6, 7, 8, 51, 60, 78, 79, 99, 100, 101, and 129 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with PEG, preferably a group derived from a cysteine residue conjugated with PEG, a group derived from an o-Az-Z-Lys residue conjugated with PEG, or a group derived from an m-Az-Z-Lys residue conjugated with PEG, and more preferably a group derived from a cysteine residue conjugated with PEG.

[0486] Specific examples of the group derived from the N-terminal amino acid residue conjugated with PEG include, for example, the following structure (Formula Z0) conjugated with PEG via a linker obtained by reacting an aldehyde with the backbone amino group of an alanine residue.

[0487]

[0488] In the formula, specific examples of PEG include, but are not limited to, the following structure (Formula X00) when the average molecular weight is 20 kDa.

[0489]

[0490] Specific examples of the group derived from a cysteine residue conjugated with PEG include, for example, the following structure (Formula X4) conjugated with PEG via a linker obtained by reacting a haloacetyl group with the side-chain thiol group of a cysteine residue, the following (Formula X5) and / or (Formula X6) and / or (Formula X7) conjugated with PEG via a linker obtained by reacting a maleimide.

[0491]

[0492] Specific examples of PEG in the formula include, but are not limited to, the following (Formula X11) when the average molecular weight is 20 kDa, the following (Formula X11) when the average molecular weight is 40 kDa, the following (Formula X13) when the average molecular weight is 40 kDa, the following (Formula X13) when the average molecular weight is 80 kDa, the following (Formula X14) when the average molecular weight is 40 kDa, the following (Formula X14) when the average molecular weight is 80 kDa, and the following (Formula X15) when the average molecular weight is 50 kDa.

[0493]

[0494] Groups derived from acetylcysteine residues conjugated with PEG. Specifically, for example, structures shown in the following (Formula XX3) conjugated with PEG via a linker obtained by reacting a haloacetyl group with the side-chain thiol group of an acetylcysteine residue, structures shown in the following (Formula X8) and / or (Formula X9) and / or (Formula X10) conjugated with PEG via a linker obtained by reacting a maleimide, etc. can be cited.

[0495]

[0496] In the formula, specific examples of PEG include the above (Formula X11) when the average molecular weight is 40 kDa, the above (Formula X13) when the average molecular weight is 40 kDa, the above (Formula X13) when the average molecular weight is 80 kDa, the above (Formula X14) when the average molecular weight is 80 kDa, and the structure shown in the above (Formula X15) when the average molecular weight is 50 kDa, but are not limited to these.

[0497] As an embodiment of the IL-2 variant of the present invention, the following-described IL-2 variants are preferred.

[0498] · An IL-2 variant in which the amino acid residue at position 11 in the amino acid sequence of wild-type IL-2 is substituted with a sugar chain-conjugated amino acid residue and the amino acid residue at position 1 is substituted with a PEG-conjugated amino acid residue.

[0499] · An IL-2 variant in which the amino acid residue at position 12 in the amino acid sequence of wild-type IL-2 is substituted with a sugar chain-conjugated amino acid residue and the amino acid residue at position 1 is substituted with a PEG-conjugated amino acid residue.

[0500] · An IL-2 variant in which the amino acid residue at position 19 in the amino acid sequence of wild-type IL-2 is substituted with a sugar chain-conjugated amino acid residue and the amino acid residue at position 1 is substituted with a PEG-conjugated amino acid residue.

[0501] · An IL-2 variant in which the amino acid residue at position 38 in the amino acid sequence of wild-type IL-2 is substituted with a sugar chain-conjugated amino acid residue and the amino acid residue at position 1 is substituted with a PEG-conjugated amino acid residue.

[0502] · An IL-2 variant in which the amino acid residue at position 91 in the amino acid sequence of wild-type IL-2 is substituted with a sugar chain-conjugated amino acid residue and the amino acid residue at position 1 is substituted with a PEG-conjugated amino acid residue.

[0503] · An IL-2 variant in which the amino acid residues at positions 12 and 91 in the amino acid sequence of wild-type IL-2 are replaced with amino acid residues conjugated with sugar chains, and the amino acid residue at position 1 is replaced with an amino acid residue conjugated with PEG.

[0504] · An IL-2 variant in which the amino acid residues at positions 91 and 119 in the amino acid sequence of wild-type IL-2 are replaced with amino acid residues conjugated with sugar chains, and the amino acid residue at position 1 is replaced with an amino acid residue conjugated with PEG.

[0505] · An IL-2 variant in which the amino acid residues at positions 12 and 91 in the amino acid sequence of wild-type IL-2 are replaced with amino acid residues conjugated with sugar chains, and the amino acid residue at position 3 is replaced with an amino acid residue conjugated with PEG.

[0506] · An IL-2 variant in which the amino acid residue at position 15 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with sugar chains, and the amino acid residue at position 3 is replaced with an amino acid residue conjugated with PEG.

[0507] · An IL-2 variant in which the amino acid residues at positions 12 and 119 in the amino acid sequence of wild-type IL-2 are replaced with amino acid residues conjugated with sugar chains, and the amino acid residue at position 3 is replaced with an amino acid residue conjugated with PEG.

[0508] · An IL-2 variant in which the amino acid residues at positions 12 and 91 in the amino acid sequence of wild-type IL-2 are replaced with amino acid residues conjugated with sugar chains, and the amino acid residue at position 51 is replaced with an amino acid residue conjugated with PEG.

[0509] · An IL-2 variant in which the amino acid residue at position 12 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with sugar chains, and the amino acid residue at position 78 is replaced with an amino acid residue conjugated with PEG.

[0510] · An IL-2 variant in which the amino acid residues at positions 12 and 119 in the amino acid sequence of wild-type IL-2 are replaced with amino acid residues conjugated with sugar chains, and the amino acid residue at position 78 is replaced with an amino acid residue conjugated with PEG.

[0511] · An IL-2 variant in which the amino acid residue at position 15 in the amino acid sequence of wild-type IL-2 is replaced with an amino acid residue conjugated with sugar chains, and the amino acid residue at position 78 is replaced with an amino acid residue conjugated with PEG.

[0512] The PEGylation of the present invention, the sugar chain bound to the sugar chain-binding IL-2 variant, and PEG can be used in combination in the above various ways.

[0513] · An IL-2 variant in which the amino acid residue at position 11 in the amino acid sequence of wild-type IL-2 is replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), the structure of Saccharide in (Formula 1) is the structure represented by (Formula 7), the amino acid residue at position 1 is replaced with an N-terminal amino acid residue-derived group bound to PEG represented by (Formula X0), and the structure of PEG in (Formula X0) is the structure represented by (Formula X00) when the average molecular weight is 20 kDa.

[0514] · An IL-2 variant in which the amino acid residue at position 12 in the amino acid sequence of wild-type IL-2 is replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), the structure of Saccharide in (Formula 1) is the structure represented by (Formula 7), the amino acid residue at position 1 is replaced with an N-terminal amino acid residue-derived group bound to PEG represented by (Formula X0), and the structure of PEG in (Formula X0) is the structure represented by (Formula X00) when the average molecular weight is 20 kDa.

[0515] · An IL-2 variant in which the amino acid residue at position 38 in the amino acid sequence of wild-type IL-2 is replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), the structure of Saccharide in (Formula 1) is the structure represented by (Formula 7), the amino acid residue at position 1 is replaced with an N-terminal amino acid residue-derived group bound to PEG represented by (Formula X0), and the structure of PEG in (Formula X0) is the structure represented by (Formula X00) when the average molecular weight is 20 kDa.

[0516] · An IL-2 variant in which the amino acid residue at position 91 in the amino acid sequence of wild-type IL-2 is replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), the structure of Saccharide in (Formula 1) is the structure represented by (Formula 7), the amino acid residue at position 1 is replaced with an N-terminal amino acid residue-derived group bound to PEG represented by (Formula X0), and the structure of PEG in (Formula X0) is the structure represented by (Formula X00) when the average molecular weight is 20 kDa.

[0517] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 12 and the amino acid residue at position 91 are replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of Saccharide in (Formula 1) is the structure represented by (Formula 8), and the amino acid residue at position 1 is replaced with a PEG-bound acetylcysteine residue-derived group represented by (Formula XX3), and the structure of PEG in (Formula XX3) is the structure represented by (Formula X13) when the average molecular weight is 40 kDa or the structure represented by (Formula X15) when the average molecular weight is 50 kDa, an IL-2 variant.

[0518] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 12 and the amino acid residue at position 91 are replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of Saccharide in (Formula 1) is the structure represented by (Formula 8), and the amino acid residue at position 1 is replaced with a PEG-bound acetylcysteine residue-derived group represented by (Formula X8) and / or (Formula X9) and / or (Formula X10), and the structure of PEG in (Formula X8) and / or (Formula X9) and / or (Formula X10) is the structure represented by (Formula X13) when the average molecular weight is 80 kDa, or the structure represented by (Formula X15) when the average molecular weight is 50 kDa, or the structure represented by (Formula X14) when the average molecular weight is 80 kDa, an IL-2 variant.

[0519] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 19 is replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of Saccharide in (Formula 1) is the structure represented by (Formula 8), and the amino acid residue at position 1 is replaced with a PEG-bound acetylcysteine residue-derived group represented by (Formula XX3), and the structure of PEG in (Formula XX3) is the structure represented by (Formula X15) when the average molecular weight is 50 kDa or the structure represented by (Formula X13) when the average molecular weight is 40 kDa, an IL-2 variant.

[0520] · In the amino acid sequence of wild-type IL-2, the amino acid residues at positions 91 and 119 are replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of Saccharide in (Formula 1) is the structure represented by (Formula 8), and the amino acid residue at position 1 is replaced with a PEG-bound acetylcysteine residue-derived group represented by (Formula XX3), and the structure of PEG in (Formula XX3) is the structure represented by (Formula X15) when the average molecular weight is 50 kDa, an IL-2 variant.

[0521] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 12 and the amino acid residue at position 91 are replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is the structure represented by (Formula 8), and the amino acid residue at position 3 is replaced with a PEG-bound cysteine residue-derived group represented by (Formula X4), and the structure of the PEG in (Formula X4) is (Formula X11) when the average molecular weight is 20 kDa, or (Formula X11) when the average molecular weight is 40 kDa, or (Formula X13) when the average molecular weight is 40 kDa, or (Formula X14) when the average molecular weight is 40 kDa, or the structure represented by (Formula X15) when the average molecular weight is 50 kDa, an IL-2 variant.

[0522] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 12 and the amino acid residue at position 91 are replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is the structure represented by (Formula 8), and the amino acid residue at position 3 is replaced with a PEG-bound cysteine residue-derived group represented by (Formula X5) and / or (Formula X6) and / or (Formula X7), and the structure of the PEG in (Formula X5) and / or (Formula X6) and / or (Formula X7) is the structure represented by (Formula X15) when the average molecular weight is 50 kDa, an IL-2 variant.

[0523] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 12 is replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is the structure represented by (Formula 8), the amino acid residue at position 91 is replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is the structure represented by (Formula 4), and the amino acid residue at position 3 is replaced with a PEG-bound cysteine residue-derived group represented by (Formula X5) and / or (Formula X6) and / or (Formula X7), and the structure of the PEG in (Formula X5) and / or (Formula X6) and / or (Formula X7) is the structure represented by (Formula X14) when the average molecular weight is 80 kDa, an IL-2 variant.

[0524] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 12 is replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is the structure represented by (Formula 8); the amino acid residue at position 91 is replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is the structure represented by (Formula 4); and the amino acid residue at position 3 is replaced with a PEG-bound cysteine residue-derived group represented by (Formula X4), and the structure of the PEG in (Formula X4) is the structure represented by (Formula X15) when the average molecular weight is 50 kDa. An IL-2 variant.

[0525] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 15 is replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is the structure represented by (Formula 8); and the amino acid residue at position 3 is replaced with a PEG-bound cysteine residue-derived group represented by (Formula X4), and the structure of the PEG in (Formula X4) is the structure represented by (Formula X13) when the average molecular weight is 40 kDa or the structure represented by (Formula X15) when the average molecular weight is 50 kDa. An IL-2 variant.

[0526] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 15 is replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is the structure represented by (Formula 8); and the amino acid residue at position 3 is replaced with a PEG-bound cysteine residue-derived group represented by (Formula X5) and / or (Formula X6) and / or (Formula X7), and the structure of the PEG in (Formula X5) and / or (Formula X6) and / or (Formula X7) is the structure represented by (Formula X13) when the average molecular weight is 80 kDa. An IL-2 variant.

[0527] · In the amino acid sequence of wild-type IL-2, the amino acid residues at positions 12 and 119 are replaced with a sugar chain-bound cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is the structure represented by (Formula 8); and the amino acid residue at position 3 is replaced with a PEG-bound cysteine residue-derived group represented by (Formula X5) and / or (Formula X6) and / or (Formula X7), and the structure of the PEG in (Formula X5) and / or (Formula X6) and / or (Formula X7) is the structure represented by (Formula X13) when the average molecular weight is 80 kDa or the structure represented by (Formula X14) when the average molecular weight is 80 kDa. An IL-2 variant.

[0528] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 12 and the amino acid residue at position 119 are replaced with a cysteine residue-derived group conjugated with a sugar chain as shown in (Formula 1), and the structure of Saccharide in (Formula 1) is the structure shown in (Formula 8), and the amino acid residue at position 3 is replaced with a cysteine residue-derived group conjugated with PEG as shown in (Formula X4), and the structure of PEG in (Formula X4) is the structure shown in (Formula X15) when the average molecular weight is 50 kDa, an IL-2 variant.

[0529] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 12 and the amino acid residue at position 91 are replaced with a cysteine residue-derived group conjugated with a sugar chain as shown in (Formula 1), and the structure of Saccharide in (Formula 1) is the structure shown in (Formula 8), and the amino acid residue at position 51 is replaced with a cysteine residue-derived group conjugated with PEG as shown in (Formula X4), and the structure of PEG in (Formula X4) is the structure shown in (Formula X11) when the average molecular weight is 40 kDa, or the structure shown in (Formula X15) when the average molecular weight is 50 kDa, an IL-2 variant.

[0530] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 12 is replaced with a cysteine residue-derived group conjugated with a sugar chain as shown in (Formula 1), and the structure of Saccharide in (Formula 1) is the structure shown in (Formula 8), and the amino acid residue at position 78 is replaced with a cysteine residue-derived group conjugated with PEG as shown in (Formula X4), and the structure of PEG in (Formula X4) is the structure shown in (Formula X11) when the average molecular weight is 40 kDa, or the structure shown in (Formula X13) when the average molecular weight is 40 kDa, an IL-2 variant.

[0531] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 12 is replaced with a cysteine residue-derived group conjugated with a sugar chain as shown in (Formula 1), and the structure of Saccharide in (Formula 1) is the structure shown in (Formula 8), and position 78 is replaced with a cysteine residue-derived group conjugated with PEG as shown in (Formula X5) and / or (Formula X6) and / or (Formula X7), and the structure of PEG in (Formula X5) and / or (Formula X6) and / or (Formula X7) is the structure shown in (Formula X13) when the average molecular weight is 40 kDa or 80 kDa, or the structure shown in (Formula X14) when the average molecular weight is 80 kDa, an IL-2 variant.

[0532] · In the amino acid sequence of wild-type IL-2, the amino acid residues at positions 12 and 119 are replaced with a sugar chain-linked cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is the structure represented by (Formula 8), and the amino acid residue at position 78 is replaced with a PEG-linked cysteine residue-derived group represented by (Formula X4), and the structure of the PEG in (Formula X4) is the structure represented by (Formula X13) when the average molecular weight is 40 kDa, an IL-2 variant.

[0533] · In the amino acid sequence of wild-type IL-2, the amino acid residues at positions 12 and 119 are replaced with a sugar chain-linked cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is the structure represented by (Formula 8), and position 78 is replaced with a PEG-linked cysteine residue-derived group represented by (Formula X5) and / or (Formula X6) and / or (Formula X7), and the structure of the PEG in (Formula X5) and / or (Formula X6) and / or (Formula X7) is the structure represented by (Formula X13) when the average molecular weight is 80 kDa, or the structure represented by (Formula X14) when the average molecular weight is 80 kDa, an IL-2 variant.

[0534] · In the amino acid sequence of wild-type IL-2, the amino acid residue at position 15 is replaced with a sugar chain-linked cysteine residue-derived group represented by (Formula 1), and the structure of the Saccharide in (Formula 1) is the structure represented by (Formula 8), and the amino acid residue at position 78 is replaced with a PEG-linked cysteine residue-derived group represented by (Formula X4), and the structure of the PEG in (Formula X4) is the structure represented by (Formula X12) when the average molecular weight is 40 kDa, an IL-2 variant.

[0535] In the present embodiment, the amino acid sequence of wild-type IL-2 is preferably the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence with a methionine residue bound to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence with the N-terminal alanine residue missing in the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence with the N-terminal alanine residue missing and a methionine residue bound in the amino acid sequence shown in SEQ ID NO: 1, or in the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence with a methionine residue bound to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence with the N-terminal alanine residue missing in the amino acid sequence shown in SEQ ID NO: 1, and the amino acid sequence with the N-terminal alanine residue missing and a methionine residue bound in the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence in which the amino acid residue at position 125 is replaced with a serine residue or an alanine residue.

[0536] By binding the IL-2 variant of the present invention to a pharmacokinetic improvement factor that is generally known to improve in vivo pharmacokinetics, the blood half-life can be controlled. As the pharmacokinetic improvement factor, sugar chains, peptides, proteins, lipids, etc. can be cited, and the methods described in [Therapeutic Proteins (edited by Roland Kontermann, Wiley Blackwell, 2012)] can be used in combination. Specifically, in a manner that avoids affecting the selectivity of the Treg cell proliferation activity of the IL-2 variant of the present invention, the blood half-life can be controlled by methods such as sialylation, hydroxyethyl starchylation (HESylation), O-glycosylation, fusion of peptides and proteins as PEG mimetics, fusion of the constant region or Fc region of an antibody, fusion with serum proteins such as albumin (also including methods of introducing lipids to fuse with albumin), or binding to phospholipids or nanoparticles, encapsulation in nanoparticles, etc.

[0537] [Evaluation of the biological activity of the IL-2 variant]

[0538] The biological activity of the IL-2 variant can be evaluated by any suitable method known in the art. The evaluation methods also include the methods described in the following examples. Specifically, as methods for evaluating the biological activity of the IL-2 variant, for example, the following methods (a) to (e) can be cited. These methods can also be used for the determination of the therapeutic effect, efficacy, and pharmacokinetic properties of the IL-2 variant.

[0539] (a) Method for measuring the proliferation activity of Treg cells stimulated by the IL-2 variant

[0540] Culture Treg cells in a medium supplemented with the IL-2 variant or wild-type IL-2, and measure the proliferation rate of Treg cells. In addition, as methods for measuring the proliferation activity of Treg cells, for example, the following can be cited: a method for measuring the increase in the number of Treg cells in a mixed cell population by flow cytometry, and a method for measuring the presence ratio of the CD4 + CD25 + FOXP3 + marker phenotype or CD4 + CD25 + CD127 low marker phenotype; a method for measuring by introducing deuterated thymidine into the isolated Treg cells; a method for measuring the increase in the expression of cell cycle proteins related to proliferation such as Ki-67 in Treg cells; a method for measuring the dilution related to cell division of a biological fluorescent pigment such as carboxyfluorescein succinimidyl ester (CFSE) in Treg cells by flow cytometry.

[0541] (b) Method for measuring the proliferative activity of NK cells stimulated by an IL-2 variant

[0542] NK cells are cultured in a medium supplemented with an IL-2 variant or wild-type IL-2, and the proliferation rate of the NK cells is measured. In addition, as a method for measuring the proliferative activity of NK cells, for example, the following can be mentioned: a method of measuring the increase in the number of NK cells in a mixed cell population by flow cytometry, and a method of measuring the presence ratio of the CD56 + marker phenotype; a method of measuring by introducing deuterated thymidine into the isolated NK cells; a method of measuring the increase in the expression of cell cycle proteins related to proliferation such as Ki-67 in NK cells; a method of measuring the dilution related to cell division of a biological fluorescent dye such as CFSE in NK cells by flow cytometry.

[0543] The IL-2 variant of the present invention preferably has a higher Treg proliferative activity and / or a lower NK cell proliferative activity than wild-type IL-2. An IL-2 variant having the same Treg proliferative activity and / or NK cell proliferative activity as wild-type IL-2 can also be used instead of wild-type IL-2.

[0544] (c) Method for measuring the inhibitory activity of effector T cells (Tresp) of Tregs stimulated by an IL-2 variant

[0545] Tregs are cultured in a medium supplemented with an IL-2 variant or wild-type IL-2, and the proliferation rate of Tresp is measured when they are co-cultured with Tresp (CD4 + Tresp, CD8 + Tresp) in the presence of appropriate TCR stimulation, and the inhibitory rate of Tresp proliferation using the IL-2 variant is evaluated by comparison with wild-type IL-2. The IL-2 variant of the present invention preferably proliferates Tregs having at least the same inhibitory activity of Tresp proliferation as wild-type IL-2. An IL-2 variant that proliferates Tregs having the same inhibitory activity of Tresp proliferation as wild-type IL-2 can also be used instead of wild-type IL-2.

[0546] (d) Ex vivo testing

[0547] Regarding inflammatory cytokines such as IL-4, IL-6, IFNγ, or TNFα, which are effector molecules of the functions of Teff or NK cells, PBMCs are cultured in a medium supplemented with an IL-2 variant or wild-type IL-2, and the amount of cytokine production in the culture supernatant is measured. Additionally, the amount of anti-inflammatory cytokine production can be measured in the same manner. The IL-2 variant of the present invention preferably reduces the amount of inflammatory cytokine production and / or increases the amount of anti-inflammatory cytokine production as compared with wild-type IL-2. An IL-2 variant that produces the same amount of inflammatory cytokine and / or anti-inflammatory cytokine as wild-type IL-2 can also be used in place of wild-type IL-2.

[0548] (e) Measurement of the Treg / Teff ratio

[0549] PMBCs cultured in a medium supplemented with an IL-2 variant or wild-type IL-2 are reacted with anti-human CD4 antibody, anti-human CD25 antibody, and anti-human Foxp3 antibody. In the CD4-positive fraction obtained using a flow cytometer, the CD25 + FOXP3 high fraction is used as Treg, and the CD25 + FOXP3 low fraction is used as effector T cells (Teff), and their abundance ratio [Treg(%) / Teff(%)] (Treg / Teff ratio) is calculated. Data analysis is performed using commercially available data analysis software (e.g., Flowjo, version 7.6.5, manufactured by TreeStar Inc.). The IL-2 variant of the present invention preferably has an increased Treg / Teff ratio as compared with wild-type IL-2. An IL-2 variant having the same Treg / Teff ratio as wild-type IL-2 can also be used in place of wild-type IL-2.

[0550] [Composition]

[0551] One embodiment of the present invention is a composition comprising an effective amount of the IL-2 variant of the present invention. Examples of the form of the composition include pharmaceutical compositions and reagents.

[0552] As shown in the examples described later, the IL-2 variant of the present invention selectively activates Treg, and thus the composition comprising the IL-2 variant of the present invention can be suitably used as a composition having an immunosuppressive effect. Additionally, as one embodiment of the present invention, a therapeutic agent for immune diseases comprising the IL-2 variant of the present invention is provided.

[0553] Examples of the conditions or diseases for which the composition of the present invention can be used include: systemic lupus erythematosus, psoriasis, chronic graft-versus-host disease, acute graft-versus-host disease, Crohn's disease, ulcerative colitis, inflammatory bowel disease, multiple sclerosis, celiac disease, idiopathic thrombocytopenic purpura, myasthenia gravis, Sjogren's syndrome, scleroderma, asthma, uveitis, epidermal hyperplasia, alopecia areata, Behcet's disease, Takayasu arteritis, cartilage inflammation, bone degeneration, arthritis, juvenile arthritis, juvenile rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Rett syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, pauciarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, dermatomyositis, psoriatic arthritis, vasculitis, myositis, polymyositis, dermatomyositis, osteoarthritis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary cirrhosis, sclerosing cholangitis, dermatitis, atopic dermatitis, atherosclerosis, Still's disease, chronic obstructive pulmonary disease, Guillain-Barré syndrome, type 1 diabetes, Graves' disease, Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, Wiskott-Aldrich syndrome, and other inflammatory diseases, autoimmune diseases, allergic diseases, etc.

[0554] The composition of the present invention can be formulated by known pharmaceutical methods. For example, it can be used orally or parenterally as capsules, tablets, pills, liquids, powders, granules, fine granules, film-coated tablets, precipitants, lozenges, sublingual tablets, chewable tablets, buccal tablets, pastes, syrups, suspensions, elixirs, emulsions, coatings, ointments, plasters, poultices, transdermal absorption preparations, lotions, inhalants, aerosols, injections, suppositories, etc.

[0555] In these formulations, it can be appropriately combined with a pharmaceutically acceptable carrier, specifically, for example, sterilized water or physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, fragrance, excipient, medium, preservative, binder, diluent, isotonic agent, analgesic, extender, disintegrant, buffer, coating agent, lubricant, coloring agent, sweetening agent, thickening agent, flavor and odor correcting agent, solubilizing aid, or other additives.

[0556] In addition, the administration of the composition of the present invention can be carried out using a syringe or other devices. As the devices, for example, an injection pen, an auto-injector, a needle-free device, a subcutaneous patch device, etc. can be mentioned.

[0557] The composition of the present invention can be used for animals including humans. However, for animals other than humans, there is no particular limitation, and various livestock, poultry, pets, laboratory animals, etc. can be used as the subjects. Specifically, for example, pigs, cows, horses, sheep, goats, chickens, wild ducks, ostriches, domestic ducks, dogs, cats, rabbits, hamsters, mice, rats, monkeys, etc. can be mentioned, but not limited thereto. In addition, it can be in a healthy state or a diseased state. Among them, when the composition of the present invention is a pharmaceutical composition, it can be used for animals in a diseased state.

[0558] The effective amount of the IL-2 variant in the composition depends, for example, on the treatment situation and purpose. The appropriate dosage can be adjusted according to the indication for using the IL-2 variant, the administration route, and the size (body weight, body surface area, or organ size) and / or condition (age and health status) of the administration subject.

[0559] For example, the dosage or intake amount per administration is usually 1 ng / kg body weight to 100 mg / kg body weight, preferably 0.01 μg / kg body weight to 1 mg / kg body weight.

[0560] The product (pharmaceutical, reagent) of the composition of the present invention or its instructions can indicate the meaning of being used for suppressing immunity, etc. Herein, "indicating in the product or instructions" means indicating on the main body, container, packaging, etc. of the product or indicating in the instructions, accompanying documents, promotional materials, other printed materials, etc. that disclose the product information.

[0561] [Selectivity for IL-2R]

[0562] One embodiment of the present invention is a method for improving the selectivity of IL-2 for IL-2R αβγ In this embodiment, by the above method, the sugar chain or PEG is bound to IL-2 for modification, whereby the selectivity of IL-2 for IL-2R αβγ can be improved.

[0563] When the selectivity of IL-2 for IL-2R αβγ is improved, there will be a case where the affinity of the IL-2 variant for the IL-2Rα subunit is increased compared to the affinity of the wild-type IL-2; and a case where the affinity of the IL-2 variant for at least one of the IL-2Rβ and γ subunits is decreased compared to the affinity of the wild-type IL-2.

[0564] One embodiment of the present invention is a method for increasing the affinity of IL-2 for the IL-2Rα subunit. "Increasing the affinity of IL-2 for the IL-2Rα subunit" means that the affinity of the IL-2 variant for the IL-2Rα subunit is increased compared to wild-type IL-2. In this embodiment, by modifying IL-2 by binding a sugar chain or PEG thereto by the above method, the affinity of the produced IL-2 variant for the IL-2Rα subunit can be increased compared to wild-type IL-2, and the selectivity of IL-2 for IL-2R αβγ is increased.

[0565] The affinity of IL-2 for the IL-2Rα subunit can be evaluated by measuring the binding of IL-2 to IL-2R α (CD25) by Biacore and determining the dissociation constant K D using a steady state model. The binding of IL-2 to IL-2Rα by Biacore can be measured by the method described in the following examples. In this embodiment, the K D of the IL-2 variant for L-2Rα is preferably lower than that of the wild-type IL-2 variant. Instead of wild-type IL-2, an IL-2 variant having an affinity for IL-2R α equivalent to that of wild-type IL-2 can also be used.

[0566] One embodiment of the present invention is a method for decreasing the affinity of IL-2 for at least one of the IL-2Rβ and γ subunits. "Decreasing the affinity of IL-2 for at least one of the IL-2Rβ and γ subunits" means that the affinity of the IL-2 variant for at least one of the IL-2Rβ and γ subunits is decreased compared to wild-type IL-2. In this embodiment, by modifying IL-2 by binding a sugar chain or PEG thereto by the above method, the affinity of the produced IL-2 variant for at least one of the IL-2Rβ and γ subunits can be decreased compared to wild-type IL-2, and the selectivity of IL-2 for IL-2R αβγ is increased.

[0567] For example, the affinity of IL-2 for the IL-2Rβγ subunit can be evaluated by measuring the binding of IL-2 to IL-2R βγ by Biacore and determining the dissociation constant K D using a 1:1 binding model. The binding of IL-2 to IL-2R βγ by Biacore can be measured by the method described in the following examples. In this embodiment, preferably, the K βγ of IL-2 for IL-2R DHigher than wild-type IL-2. It is also possible to use an IL-2 variant having the same affinity for the IL-2Rβγ subunit as wild-type IL-2 in place of wild-type IL-2.

[0568] [Method for selectively activating regulatory T cells]

[0569] One embodiment of the present invention is a method for selectively activating regulatory T cells using the IL-2 variant of the present invention. In this embodiment, by administering the IL-2 variant of the present invention to a subject to be measured, regulatory T cells can be selectively activated. Examples

[0570] Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to the following examples.

[0571] [Example 1] Synthesis of glycosylated IL-2 variant

[0572] Various IL-2 variants shown in Tables 1 to 5 were prepared by the method described below.

[0573] Table 1

[0574] The prepared glycosylated IL-2 variant

[0575]

[0576] Table 2

[0577] The prepared glycosylated IL-2 variant

[0578]

[0579] Table 3

[0580]

[0581] Table 4

[0582]

[0583] Table 5

[0584]

[0585] [Explanation of Tables 1 to 5]

[0586] · Glycosylation site: Position from the N-terminus of the amino acid sequence of wild-type mature human IL-2 (SEQ ID NO: 1) (hereinafter also simply referred to as wild-type IL-2)

[0587] · Cys mutation site: Position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1

[0588] ·Mutation at position 125: Indicates the presence or absence of a mutation in the amino acid residue at position 125 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1. It is denoted as "-" when no mutation is applied, and as "S" when a mutation substituting the amino acid residue from cysteine to serine is applied.

[0589] In the table, the structures described in the column of the amino acid residue after substitution at the sugar chain binding position are shown as follows.

[0590] C-glycan (GlcNAc, glucose, lactose, trisaccharide, pentasaccharide, asialo, disialo, tetrasialo) represents the structure shown in the following (Formula 1) in which a sugar chain is introduced via a CH2CONH linker to the side-chain thiol of cysteine.

[0591]

[0592] In the above (Formula 1), Saccharide represents a sugar chain.

[0593] N-glycan (GlcNAc, disialo) represents the structure shown in the following (Formula 2) in which a sugar chain is introduced to the side-chain amide of asparagine.

[0594]

[0595] In the above (Formula 2), Saccharide represents a sugar chain.

[0596] GlcNAc represents the structure shown in the following (Formula Y1).

[0597]

[0598] Glucose represents the structure shown in the following (Formula Y2).

[0599]

[0600] Lactose represents the structure shown in the following (Formula 4).

[0601]

[0602] Trisaccharide represents the structure shown in the following (Formula 5).

[0603]

[0604] The pentasaccharide represents the structure shown in the following (Formula 6).

[0605]

[0606] Asialo represents the structure shown in the following (Formula 7).

[0607]

[0608] Disialo represents the structure shown in the following (Formula 8).

[0609]

[0610] Tetrasialo represents the structure shown in the following (Formula Y3).

[0611]

[0612] · In the table, AcC recorded in the column of the amino acid residue after substitution at the Cys mutation position represents the structure shown in the following (Formula XXX).

[0613]

[0614] Table 6

[0615] The prepared IL-2 or IL-2 variant

[0616]

[0617] <Explanation of Table 6>

[0618] · Mutation position: The position from the N-terminus of the amino acid sequence shown in Sequence No. 1

[0619] · Mutation at the 125th position: Indicates the presence or absence of a mutation of the amino acid residue at the 125th position from the N-terminus of the amino acid sequence shown in Sequence No. 1. When a mutation is applied, it is denoted as -, and when a mutation of substituting the amino acid residue from cysteine to serine is applied, it is denoted as S.

[0620] In the table, C-acetamide recorded in the column of the amino acid residue after substitution represents the structure shown in the following (Formula 9).

[0621]

[0622] (Step 1) Synthesis of Peptide Segment 1

[0623] The peptide thioester or glycan-conjugated peptide thioester of the IL-2 amino acid sequence 1-57 prepared by the following method.

[0624] (Step 1-1a-1) Synthesis of Peptide Hydrazide

[0625] For the tritylhydrazide resin obtained by the method described in [Angew.Chem.Int.Ed.2014,53,6978-6981], the amino acid residue of the first residue was supported on the resin in DMF using Fmoc-Gln(Trt)-OH (5 equivalents), 1-hydroxybenzotriazole (5 equivalents), and N,N'-diisopropylcarbodiimide (5 equivalents). According to the conventional method, elongation of the amino acid using Fmoc amino acid (5.3 equivalents), HCTU (5 equivalents), N-methylmorpholine (5 equivalents) or 2,4,6-trimethylpyridine (5 equivalents) in DMF and deprotection by 20% piperidine-DMF solution were repeated, whereby the amino acids after the second residue were elongated. The elongated peptide was detached from the resin and the side chain protecting groups were removed using trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and water, and then dropped into ice-cold ether, and the resulting precipitate was recovered by centrifugation. Purification was performed using a reversed-phase HPLC column [Proteonavi (trade name), manufactured by Shiseido Company, Ltd.] to synthesize peptide hydrazide.

[0626] At this time, when introducing a sugar chain into cysteine, a peptide hydrazide in which the position for introducing the sugar chain is mutated to cysteine was prepared. In addition, when introducing two sugar chains, a peptide hydrazide in which one was mutated to cysteine and the other was mutated to S-acetamidomethylcysteine was prepared.

[0627] In addition, when preparing an IL-2 variant in which the 3rd or 51st position of the amino acid sequence is mutated to cysteine, a peptide hydrazide in which the position for introducing the sugar chain is mutated to cysteine and the 3rd or 51st position of the amino acid sequence is mutated to S-acetamidomethylcysteine was prepared.

[0628] In addition, when preparing an analogue in which the 1st position of the amino acid sequence is mutated to acetylcysteine, after elongating the peptide in which the position for introducing the sugar chain is mutated to cysteine and the 1st position of the amino acid sequence is mutated to S-acetamidomethylcysteine on the resin, the N-terminal amino group was acetylated using acetic anhydride and pyridine, and detachment from the resin, removal of the side chain protecting groups, and purification were performed according to the above method to prepare a peptide hydrazide in which the 1st position was mutated to acetylcysteine.

[0629] (Step 1-1a-2) Synthesis of Cys-Sugar Chain Conjugated Peptide Hydrazide or Cys-Acetamide Conjugated Peptide Hydrazide

[0630] The introduction of the sugar chain into the peptidyl hydrazide obtained in (Step 1-1a-1) using bromoacetyl sugar chain (prepared by the method described in International Publication No. 2005 / 010053) was carried out by the method described in [Tetrahedron Lett., 2004, 45, 3287-3290, Carbohydr.Res. 2009, 344, 762-770] to synthesize the target sugar chain-bound peptidyl hydrazide.

[0631] Using bromoacetamide instead of bromoacetyl sugar chain, Cys-acetamide-bound peptidyl hydrazide was synthesized by the same method as above.

[0632] (Step 1-1b) Synthesis of Asn-sugar chain-bound peptidyl hydrazide

[0633] For the trityl hydrazide resin obtained by the method described in [Angew.Chem.Int.Ed. 2014, 53, 6978-6981], the amino acid residue of the first residue was loaded onto the resin in DMF using Fmoc-Gln(Trt)-OH (5 equivalents), 1-hydroxybenzotriazole (5 equivalents), and N,N'-diisopropylcarbodiimide (5 equivalents). According to the conventional method, the elongation of the amino acid using Fmoc amino acid (5.3 equivalents), HCTU (5 equivalents), and N-methylmorpholine (5 equivalents) in DMF and the deprotection using 20% piperidine-DMF solution were repeated to elongate the amino acids other than Asn for the sugar chain binding of the second residue and later.

[0634] The sugar chain-bound Asn (prepared by the method described in International Publication No. 2004 / 005330) was elongated by the method described in International Publication No. 2004 / 005330. After the elongation, the resulting peptide was detached from the resin and the side chain protecting groups were removed using trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and water, and then dropped into ice-cold ether. The resulting precipitate was recovered by centrifugation. Purification was carried out using a reverse-phase HPLC column [Proteonavi (trade name), manufactured by Shiseido Company] to synthesize Asn-sugar chain-bound peptidyl hydrazide.

[0635] (Step 1-2a) Synthesis of peptide thioester or sugar chain-bound peptide thioester

[0636] Dissolve the peptide hydrazide obtained in (Process 1-1a-1), or the Cys-glycan-conjugated peptide hydrazide obtained in (Process 1-1a-2), or the Cys-acetamide-conjugated peptide hydrazide obtained in this step, or the Asn-glycan-conjugated peptide hydrazide obtained in (Process 1-1b) in 6 mol / L guanidine hydrochloride and 200 mmol / L phosphate buffer (pH 3). After cooling to -20°C, add 200 mmol / L sodium nitrite, 6 mol / L guanidine hydrochloride, and 200 mmol / L phosphate buffer (pH 7), and stir for 5 minutes. Add 400 mmol / L 2-mercaptoethanesulfonate, 6 mol / L guanidine hydrochloride, and 200 mmol / L phosphate buffer (pH 6), stir at -15°C for one and a half hours, and then purify using a reversed-phase HPLC column [Proteonavi (trade name), manufactured by Shiseido Company] to obtain a peptide thioester or a glycan-conjugated peptide thioester.

[0637] (Process 1-2b) Synthesis of two glycan-conjugated peptide thioesters

[0638] In the case of introducing two types of glycans, add silver acetate suspended in acetic acid to the reaction solution of the glycan-conjugated peptide hydrazide obtained in (Process 1-1a-2) and stir for 6 hours to remove S-acetamidomethyl. After adding dithiothreitol, change the solvent of the supernatant obtained by centrifugation to 4 mol / L guanidine hydrochloride and 5 mmol / L phosphate buffer (pH 5) by gel filtration (Superdex G-75). Add 6 mol / L guanidine hydrochloride and 200 mmol / L phosphate buffer (pH 3) to the eluate, adjust to pH 3 using 2 mol / L hydrochloric acid, and then cool to -15°C.

[0639] Add 6 mol / L guanidine hydrochloride, 200 mmol / L sodium nitrite, and 50 mmol / L phosphate buffer (pH 7), stir at -15°C for 5 minutes, then add 6 mol / L guanidine hydrochloride, 400 mmol / L 2-mercaptoethanesulfonate, and 200 mmol / L phosphate buffer (pH 6), stir at -15°C for one and a half hours, and purify using a reversed-phase HPLC column [Proteonavi (trade name), manufactured by Shiseido Company] to obtain a glycan-conjugated peptide thioester.

[0640] For the obtained glycan-conjugated peptide thioester, introduce the second type of glycan according to the method described in (Process 1-1a-2), and purify using a reversed-phase HPLC column [Proteonavi (trade name), manufactured by Shiseido Company] to obtain two glycan-conjugated peptide thioesters.

[0641] (Process 2) Synthesis of peptide segment 2

[0642] Prepare a peptide hydrazide or a glycan-conjugated peptide hydrazide of the amino acid sequence 58-104 of IL-2 by the following method.

[0643] (Step 2-1a-1) Synthesis of Peptide Hydrazide

[0644] For the tritylhydrazide resin obtained by the method described in [Angew. Chem. Int. Ed. 2014, 53, 6978 - 6981], the amino acid residue of the first residue was supported on the resin in DMF using Fmoc-Met-OH (5 equivalents), 1-hydroxybenzotriazole (5 equivalents), and N,N'-diisopropylcarbodiimide (5 equivalents).

[0645] According to the conventional method, the elongation of amino acids using Fmoc amino acids (5.3 equivalents), HCTU (5 equivalents), N-methylmorpholine (5 equivalents), or 2,4,6-trimethylpyridine (5 equivalents) and the deprotection by 20% piperidine-DMF solution were repeated in DMF, thereby elongating the amino acids after the second residue.

[0646] The elongated peptide was detached from the resin and the side chain protecting groups were removed using trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and water, and then dropped into ice-cold ether, and the resulting precipitate was recovered by centrifugation. It was purified using a reversed-phase HPLC column [Proteonavi (trade name), manufactured by Shiseido Company] to synthesize peptide hydrazide.

[0647] At this time, in the case of introducing a sugar chain, a peptide hydrazide in which the position for introducing the sugar chain was mutated to cysteine and further the amino acid sequence 58 was mutated to thioproline was prepared. When preparing an analogue in which the 78th position of the amino acid sequence was mutated to cysteine, a peptide hydrazide in which the 78th position of the amino acid sequence was mutated to cysteine was prepared. When preparing an analogue in which a sugar chain was introduced at the 91st position of the amino acid sequence and the 78th position of the amino acid sequence was mutated to cysteine, a peptide hydrazide in which the 91st position of the amino acid sequence was mutated to cysteine and the 78th position of the amino acid sequence was mutated to S-acetamidomethylcysteine was prepared.

[0648] (Step 2-1a-2) Synthesis of Cys-Sugar Chain-Bound Peptide Hydrazide or Cys-Acetamide-Bound Peptide Hydrazide

[0649] The sugar chain introduction of the peptidyl hydrazide obtained in (Step 2-1a-1) was carried out by the method described in [Tetrahedron Lett., 2004, 45, 3287-3290, Carbohydr.Res. 2009, 344, 762-770] using bromoacetyl sugar chain (prepared by the method described in International Publication No. 2005 / 010053). To the resulting reaction solution, after adding sodium 2-mercaptoethanesulfonate in an amount of 10 equivalents relative to the bromoacetyl sugar chain, an 8 mol / L guanidine hydrochloride aqueous solution, 2 mol / L hydrochloric acid, and methoxyamine hydrochloride were added to adjust the pH to 4, and the reaction was carried out at room temperature for 20 minutes. Purification was performed using a reversed-phase HPLC column [Proteonavi (trade name), manufactured by Shiseido Company] to synthesize Cys-sugar chain conjugated peptidyl hydrazide.

[0650] Using bromoacetamide instead of the bromoacetyl sugar chain, Cys-bromoacetamide conjugated peptidyl hydrazide was synthesized by the same method as described above.

[0651] (Step 2-1b) Synthesis of Asn-sugar chain conjugated peptidyl hydrazide

[0652] The Asn-sugar chain conjugated peptidyl hydrazide was prepared by the same method as (Step 1-1b).

[0653] (Step 3) Synthesis of Peptide Segment 3

[0654] The peptide or sugar chain conjugated peptide of the IL-2 amino acid sequence 105-133 was prepared by the following method.

[0655] (Step 3-1) Adjustment of Solubilizing Tag (H-C(Npys)RRRRR-NH2)

[0656] The elongation of amino acids using Fmoc amino acids (5.3 equivalents), HCTU (5 equivalents), and N-methylmorpholine (5 equivalents) in DMF and deprotection by a 20% piperidine-DMF solution were repeatedly performed on Rink-amide resin to elongate the amino acids. The resulting elongated peptide was detached from the resin and the side chain protecting groups were removed using trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and water, and then dropped into ice-cold ether. The resulting precipitate was recovered by centrifugation to prepare the solubilizing tag (H-C(Npys)RRRRR-NH2).

[0657] (Step 3-2) Synthesis of Solubilizing Tag-Introduced Peptide

[0658] The peptide of the IL-2 amino acid sequence 105-133 was prepared by the following method.

[0659] For the HMPB-ChemMatrix resin, Fmoc-Thr(tBu)-OH (5 equivalents), 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole (5 equivalents), and 1-methylimidazole (3.5 equivalents) were used to carry the amino acid residue of the first residue on the resin. According to the conventional method, the elongation of the amino acid using Fmoc amino acid (5.3 equivalents), HCTU (5 equivalents), N-methylmorpholine (5 equivalents), or 2,4,6-trimethylpyridine (5 equivalents) in DMF and the deprotection by 20% piperidine-DMF solution were repeated, whereby the amino acids after the second residue were elongated.

[0660] After the elongation-obtained peptide was detached from the resin and the side-chain protecting groups were removed using trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and water, it was dropped into ice-cold ether, and the resulting precipitate was recovered by centrifugation to obtain a crude purified product of the peptide. At this time, in the case of introducing a sugar chain into cysteine, a peptide in which the position where the sugar chain is introduced is mutated to cysteine and further the amino acid sequence 105 is mutated to thioproline was prepared.

[0661] The solubilizing tag obtained in (Step 3-1) (3 equivalents relative to the peptide crude product) was dissolved in 6.8 mol / L guanidine hydrochloride, 310 mmol / L phosphate buffer (pH 7), acetic anhydride (5 equivalents) was added, and the mixture was stirred at room temperature for 1 hour. After adding 10 equivalents of arginine hydrochloride, the above peptide crude product dissolved in 8 mol / L guanidine hydrochloride, 250 mmol / L tris(hydroxymethyl)aminomethane hydrochloride aqueous solution (pH 8) was added, and the mixture was stirred at room temperature for 1 hour. Purification was carried out using a reversed-phase HPLC column [Proteonavi (trade name), manufactured by Shiseido Company, Ltd.] to synthesize a solubilizing tag-introduced peptide.

[0662] (Step 3-3) Sugar chain introduction into the solubilizing tag-introduced peptide

[0663] The solubilizing tag-introduced peptide obtained in (Step 3-2) was dissolved in 8 mol / L guanidine hydrochloride, 5 mmol / L tris(2-carboxyethyl)phosphine, 200 mmol / L phosphate buffer (pH 6), and a 6 mol / L guanidine hydrochloride, 200 mmol / L phosphate buffer (pH 7) solution of bromoacetyl sugar chain (5 equivalents, prepared by the method described in International Publication No. 2005 / 010053) was added, and the reaction was carried out for 5 hours.

[0664] To 4 equivalents of the bromoacetyl glycan, 4 equivalents of sodium 2-mercaptoethanesulfonate were added, and after stirring for 1 hour, methoxyamine hydrochloride (300 equivalents) dissolved in 6 mol / L guanidine hydrochloride and 200 mmol / L phosphate buffer (pH 7) was added, and the pH was adjusted to pH 4 with 2 mol / L hydrochloric acid, followed by reaction for 1 hour. Purification was carried out using a reverse-phase HPLC column [Proteonavi (trade name), manufactured by Shiseido Company] to synthesize the glycan-conjugated peptide.

[0665] (Step 4) Synthesis of IL-2 variant and glycan-conjugated IL-2 variant

[0666] (Step 4-1) Ligation reaction of peptide segments 1 and 2

[0667] Peptide segment 1 obtained in the above (Step 1) and peptide segment 2 (1.1 equivalents) obtained in the above (Step 2) were dissolved in 8 mol / L guanidine hydrochloride, 100 mM tris(2-carboxyethyl)phosphine, 100 mM ascorbic acid, 50 mmol / L 4-mercaptophenylacetic acid, and 200 mmol / L phosphate buffer (pH 7), and after reaction, purification was carried out using a reverse-phase HPLC column [Proteonavi (trade name), manufactured by Shiseido Company] to synthesize the conjugate of peptide segments 1 and 2.

[0668] (Step 4-2) Thioesterification of the conjugate of peptide segments 1 and 2

[0669] The conjugate of peptide segments 1 and 2 obtained in the above (Step 4-1) was thioesterified by the same method as in (Step 1-2a).

[0670] (Step 4-3) Ligation with peptide segment 3

[0671] The peptide thioester obtained in the above (Step 4-2) and peptide segment 3 (1 equivalent) obtained in the above (Step 3) were dissolved in 8 mol / L guanidine hydrochloride, 100 mM tris(2-carboxyethyl)phosphine, 100 mM ascorbic acid, 50 mmol / L 4-mercaptophenylacetic acid, and 200 mmol / L phosphate buffer (pH 7), and after reaction, purification was carried out using a reverse-phase HPLC column [Proteonavi (trade name), manufactured by Shiseido Company] to synthesize the conjugate of peptide segments 1, 2, and 3.

[0672] (Step 4-4) Deprotection of acetamidomethyl

[0673] When the cysteine of the conjugate of peptide segments 1, 2, and 3 obtained in the above (Step 4-3) was protected with acetamidomethyl, the acetamidomethyl was removed by the method shown below.

[0674] Dissolve the conjugate of peptide segments 1, 2, and 3 in 6 mol / L urea and 5 mmol / L phosphate buffer (pH 5), add silver acetate (420 equivalents) suspended in acetic acid, and stir for 5 hours. After adding an excess amount of dithiothreitol, the supernatant obtained by centrifugation is purified using a reversed-phase HPLC column [Proteonavi (trade name), manufactured by Shiseido Company, Limited] to obtain the deacetamidomethylated product.

[0675] (Step 4-5) Deprotection of sialic acid benzyl ester

[0676] When the carboxylic acid of the sialic acid side chain on the sugar chain of the conjugate of peptide segments 1, 2, and 3 obtained in the above (Step 4-3) is protected by benzyl, after removing the benzyl group according to the method described in International Publication No. 2004 / 005330, it is purified using a reversed-phase HPLC column [Proteonavi (trade name), manufactured by Shiseido Company, Limited] to obtain the debenzylated product.

[0677] (Step 4-6) Synthesis of IL-2 variant and sugar chain-bound IL-2 variant

[0678] Dissolve the conjugate of peptide segments 1, 2, and 3 synthesized in (Step 4-3) or (Step 4-4) or (Step 4-5) in 6 mol / L guanidine hydrochloride and 100 mmol / L tris(hydroxymethyl)aminomethane hydrochloride (pH 8), then add 100 mmol / L tris(hydroxymethyl)aminomethane hydrochloride, 10 mmol / L reduced glutathione, and 1 mmol / L oxidized glutathione (pH 8), and stir at room temperature for 18 hours. Purify using a reversed-phase HPLC column [Proteonavi (trade name), manufactured by Shiseido Company, Limited] to obtain the IL-2 variant and the sugar chain-bound IL-2 variant.

[0679] Regarding the obtained IL-2 variant and sugar chain-bound IL-2 variant, it was confirmed by mass spectrometry that the calculated value was consistent with the measured value, and / or the CD spectrum was consistent with that of wild-type IL-2, and / or the band detected by SDS-PAGE was at the position of the assumed molecular weight band, so it was confirmed that there were no problems with the quality and purity.

[0680] [Example 2] Synthesis of N-terminal PEGylated and sugar chain-bound IL-2 variant

[0681] Prepare the N-terminal PEGylated and sugar chain-bound IL-2 shown in Table 7 using the method described below.

[0682] Table 7

[0683] The prepared N-terminal PEGylated and sugar chain-bound IL-2 variant

[0684]

[0685] <Explanation of Table 7>

[0686] · Sugar chain binding position, PEG binding position: The position from the N-terminus of the amino acid sequence of wild-type mature human IL-2 (SEQ ID NO: 1) (hereinafter also simply referred to as wild-type IL-2).

[0687] · Mutation at position 125: Indicates the presence or absence of a mutation of the amino acid residue at the 125th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1. When a mutation is introduced, it is denoted as -, and when a mutation substituting the amino acid residue from cysteine to serine is introduced, it is denoted as S.

[0688] · The following represents the structure described in the column of the substituted amino acid residue at the sugar chain binding position in the table.

[0689] C-glycoside (asialo): Represents the structure shown in the following (Formula 1) in which a sugar chain is introduced via a CH2CONH linker to the side-chain thiol of cysteine.

[0690]

[0691] In the above (Formula X1), Saccharide represents a sugar chain.

[0692] Asialo represents the structure shown in the following (Formula 7).

[0693]

[0694] The following represents the structure described in the column of the substituted amino acid residue at the PEG binding position in the table.

[0695] A1-PEG(CHO)[Li20(CHO)] represents the structure shown in the following (Formula Z0) in which PEG is introduced via a (CH2)3 linker to the main-chain amino of alanine.

[0696]

[0697] Li20 represents the structure shown in the following (Formula X00) in which PEG has an average molecular weight of about 20 kDa in the above (Formula Z0).

[0698]

[0699] In 1 mM EDTA and 20 mmol / L phosphate buffer (pH 5.5) of the sugar chain-binding IL-2 variant, 20 mmol / L phosphate buffer (pH 5.5) of PEG-aldehyde (10 equivalents, PJK-241; Creative PEG Works) was added at room temperature. After stirring at room temperature for 30 minutes, NaBH3(CN) (1000 equivalents) was added and stirred for 3 hours.

[0700] After replacing the solvent with 0.05% trifluoroacetic acid and 2% aqueous acetonitrile solution by ultrafiltration using Amicon Ultra-0.5 (10 kDa), purification was performed by size exclusion chromatography (column; manufactured by Waters, connecting XBridge BEH450A, 3.5 μm, 7.8 × 150 mm and XBridge BEH200A, 3.5 μm, 7.8 × 150 mm), and N-terminal PEGylated and sugar chain-binding IL-2 variant was synthesized.

[0701] The purity of the purified N-terminal PEGylated and sugar chain-binding IL-2 variant was confirmed by SDS-PAGE. As a result, in all variants, a single band with an increased molecular weight of PEG was observed, and a highly pure N-terminal PEGylated and sugar chain-binding IL-2 variant was confirmed.

[0702] [Example 3] Synthesis of Cys-PEGylated and sugar chain-binding IL-2 variant

[0703] Cys-PEGylated and sugar chain-binding IL-2 shown in Table 8 was prepared by the method described below.

[0704] Table 8

[0705] The prepared Cys-PFGylated and sugar chain-binding IL-2 variant

[0706]

[0707] <Explanation of Table 8>

[0708] · PEG binding position, sugar chain binding positions 1 and 2: Positions from the N-terminus of the amino acid sequence of wild-type mature human IL-2 (SEQ ID NO: 1) (hereinafter also abbreviated as wild-type IL-2)

[0709] · Mutation at position 125: Indicates the presence or absence of mutation of the amino acid residue at position 125 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1. When no mutation is applied, it is denoted as -, and when a mutation that replaces the amino acid residue from cysteine to serine is applied, it is denoted as S.

[0710] ·The following represents the structure described in the column of the substituted amino acid residue regarding the sugar chain binding position in the table.

[0711] C-sugar (lactose, disialo) represents the structure shown in the following (Formula 1) in which a sugar chain is introduced via a CH2CONH linker to the side chain thiol of cysteine.

[0712]

[0713] In the above (Formula 1), Saccharide represents a sugar chain.

[0714] Lactose represents the structure shown in the following (Formula 4).

[0715]

[0716] Disialo represents the structure shown in the following (Formula 8).

[0717]

[0718] The following represents the structure described in the column of the substituted amino acid residue regarding the PEG binding position in the table.

[0719] C-PEG(IAc) [Li20(IAc), Li40(IAc), V40(IAc), W40(IAc), Y50(IAc)] represents the structure shown in the following (Formula X4) in which a PEG is introduced via a CH2CONH(CH2)3O linker to the cysteine side chain.

[0720]

[0721] C-PEG(Mal) [V40(Mal), V80(Mal), W80(Mal), Y50(Mal)] represents the structure shown in the following (Formula X5) in which a PEG is introduced via a 3-(3-thioxo-2,5-dioxopyrrolidin-1-yl)-propoxy linker to the cysteine side chain. At this time, C-PEG(Mal) can also be the structure shown in (Formula X6) or (Formula X7) in which the dioxopyrrolidine ring is opened.

[0722]

[0723]

[0724] AcC-PEG(IAc) [Li40(IAc), Y50(IAc)] represents the structure shown in the following (Formula XX3) in which a PEG is introduced via a CH2CONH(CH2)3O linker to the acetylcysteine side chain.

[0725]

[0726] AcC-PEG(Mal) [V80(Mal), W80(Mal), Y50(Mal)] represents the structure shown in the following (Formula X8) in which PEG is introduced via a 3-(3-thioxo-2,5-dioxopyrrolidin-1-yl)-propoxy linker into the side chain of N-acetylcysteine. At this time, AcC-PEG(Mal) may also be the structure shown in (Formula X9) or (Formula X10) formed by ring-opening of the dioxopyrrolidine ring.

[0727]

[0728]

[0729] Li20 represents the structure shown in the following (Formula X11) in which PEG has an average molecular weight of about 20 kDa in the above (Formula X4) to (Formula X10).

[0730]

[0731] Li40 represents the structure shown in the above (Formula X11) in which PEG has an average molecular weight of about 40 kDa in the above (Formula X4) to (Formula X10).

[0732] V40 represents the structure shown in the following (Formula X13) in which PEG has an average molecular weight of about 40 kDa in the above (Formula X4) to (Formula X10).

[0733]

[0734] V80 represents the structure shown in the above (Formula X13) in which PEG has an average molecular weight of about 80 kDa in the above (Formula X4) to (Formula X10).

[0735] W40 represents that in the above (Formula X4) to (Formula X10), PEG is (CH2CH2O) m with an average molecular weight of 5 kDa and (CH2CH2O) n with an average molecular weight of 7.5 kDa, and has the structure shown in the following (Formula X14).

[0736]

[0737] W80 represents that in the above (Formula X4) to (Formula X10), PEG is (CH2CH2O) m with an average molecular weight of 5 kDa and (CH2CH2O) n with an average molecular weight of 17.5 kDa, and has the structure shown in the above (Formula X14).

[0738] In Y50, in the above (Formula X4) to (Formula X10), PEG is (CH2CH2O) m with an average molecular weight of 10 kDa, (CH2CH2O) n with an average molecular weight of 20 kDa, has the structure shown in the following (Formula X15).

[0739]

[0740] (Step 1) Preparation of PEG-haloacetyl

[0741] Dissolve PEG-amine (SUNBRIGHT GL2-400PA; NOF Corporation or SUNBRIGHT GL3-400PA100U; NOF Corporation or SUNBRIGHT GL4-400PA; NOF Corporation) in chloroform, add 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Hydrochloride (5 equivalents), 4-Dimethylaminopyridine (5 equivalents), and iodoacetic acid (5 equivalents), and stir at room temperature for 90 hours. Add ether / isopropanol = 1 / 1, and filter the precipitated solid. Dissolve the residue in water, remove iodoacetic acid by ultrafiltration using Amicon Ultra-0.5 (10 kDa), and freeze-dry to synthesize PEG-IAc.

[0742] (Step 2) Synthesis of Cys-PEGylated and sugar chain-conjugated IL-2 variant

[0743] In 1 mmol / L EDTA and 20 mmol / L phosphate buffer (pH 5.5) of the sugar chain-binding IL-2 variant shown in Table 5, PEG-haloacetyl (5 equivalents, the compound synthesized in Step 1 above or SUNBRIGHT ME-200IA; NOF Corporation or SUNBRIGHT ME-400IA; NOF Corporation) or PEG-maleimide (5.0 nmol, SUNBRIGHT GL2-800MA; NOF Corporation or SUNBRIGHT GL4-400MA100U; NOF Corporation or SUNBRIGHT GL4-800MA; NOF Corporation) was added at room temperature, and then using 0.1 mol / L aqueous sodium hydroxide solution, the pH was adjusted to 7.2 - 7.4, and the mixture was stirred for 2 hours. Purification was carried out by size exclusion chromatography (column; manufactured by Waters, connecting XBridge BEH450A, 3.5 μm, 7.8×150 mm and XBridge BEH200A, 3.5 μm, 7.8×150 mm) to synthesize Cys-PEGylated, sugar chain-binding IL-2 variant.

[0744] The purity of the purified Cys-PEGylated, sugar chain-binding IL-2 variant was confirmed by SDS-PAGE. As a result, in all variants, a single band with an increased molecular weight of PEG was observed, and it was confirmed that a highly pure Cys-PEGylated, sugar chain-binding IL-2 variant was obtained.

[0745] [Example 4] Preparation of 8His-IL-2 for Escherichia coli and human IL-2 expressing vectors imported with o-Az-Z-Lys and 8His-IL-2 expressing vectors imported with m-Az-Z-Lys

[0746] The 8His-IL-2 expressing vectors for Escherichia coli, the 8His-IL-2 expressing vectors imported with o-Az-Z-Lys, and the 8His-IL-2 expressing vectors imported with m-Az-Z-Lys shown in Table 9 were prepared by the following method.

[0747] Table 9

[0748] The prepared 8His-IL-2 expressing vectors for Escherichia coli, the 8His-IL-2 expressing vectors imported with o-Az-Z-Lys, and the 8His-IL-2 expressing vectors imported with m-Az-Z-Lys

[0749]

[0750] <Explanation of Table 9>

[0751] ·Az-Z-Lys Import Location: Position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1

[0752] ·Modification at the 1st Position: Represents the modification of the alanine residue at the 1st position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1. MHHHHHHHHA indicates that a methionine and a polyhistidine sequence (HHHHHHHH) tag are bound to the N-terminal alanine residue.

[0753] ·Mutation at the 125th Position: Represents the presence or absence of a mutation of the amino acid residue at the 125th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1. When no mutation is applied, it is denoted as -, and when a mutation that substitutes the amino acid residue from cysteine to serine is applied, it is denoted as S.

[0754] In the table, o-Az-Z-Lys described in the column of the substituted amino acid residue represents the structure shown in the following (Formula 10),

[0755]

[0756] m-Az-Z-Lys represents the structure shown in the following (Formula XX1).

[0757]

[0758] As IL-2, based on the amino acid sequence in which the amino acid residue at the 125th position of the wild-type mature human IL-2 amino acid sequence shown in SEQ ID NO: 1 is substituted from cysteine to serine, and a methionine and a polyhistidine sequence (HHHHHHHH) tag are bound to the N-terminus, 8His-IL-2 (amino acid sequence: SEQ ID NO: 2, base sequence encoding this amino acid sequence: SEQ ID NO: 3) is used to prepare the above expression vector.

[0759] Downstream of the lac repressor gene lacI of pFLAG-CTS (manufactured by SIGMA), between the NdeI restriction enzyme site and the SalI restriction enzyme site of pFLAG-CTS-Pyl TS (International Publication No. WO2017 / 030156) into which the base sequence encoding pyrrolysine tRNA and the base sequence of pyrrolysyl-tRNA synthetase (hereinafter also denoted as Pyl tRNA, tRNA Pyl ) are inserted, the base sequence encoding 8His-IL-2 (SEQ ID NO: 3) is inserted to prepare an 8His-IL-2 expression vector for Escherichia coli (hereinafter denoted as pFLAG-CTS-Pyl TS_8His-hIL-2).

[0760] Based on the base sequence of 8His-IL-2, a base sequence (SEQ ID NOs: 4-18, 27-37) in which the codons corresponding to the positions where o-Az-Z-Lys or m-Az-Z-Lys is introduced are replaced with amber (TAG) codons was prepared by PCR or artificial gene synthesis (GENEWIZ, Japan). The obtained base sequence was substituted for the 8His-IL-2 base sequence of pFLAG-CTS-Pyl TS-8His-hIL-2.

[0761] [Example 5] Preparation of 8His-IL-2, IL-2 into which o-Az-Z-Lys is introduced, and 8His-IL-2 into which m-Az-Z-Lys is introduced

[0762] The 8His-IL-2, 8His-IL-2 into which o-Az-Z-Lys is introduced, and 8His-IL-2 into which m-Az-Z-Lys is introduced shown in Table 10 were prepared by the following method.

[0763] Table 10

[0764] The prepared 8His-IL-2, IL-2 into which o-Az-Z-Lys is introduced, and 8His-IL-2 into which m-Az-Z-Lys is introduced

[0765]

[0766] <Explanation of Table 10>

[0767] · Az-Z-Lys introduction position: Position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1

[0768] · First position modification: Represents the modification of the alanine residue at the first position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1. MHHHHHHHHA indicates that a methionine and polyhistidine sequence (HHHHHHHH) tag is bound to the N-terminal alanine residue.

[0769] · 125th position mutation: Represents the presence or absence of a mutation in the amino acid residue at the 125th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1. When no mutation is applied, it is denoted as -, and when a mutation substituting the amino acid residue from cysteine to serine is applied, it is denoted as S.

[0770] In the table, o-Az-Z-Lys described in the column of the substituted amino acid residue represents the structure shown in the following (Formula 10),

[0771]

[0772] m-Az-Z-Lys represents the structure shown in the following (Formula XX1).

[0773]

[0774] The Escherichia coli prepared in Example 4 was transfected with the 8His-IL-2 expression vector and the 8His-IL-2 expression vector transfected with o-Az-Z-Lys or the 8His-IL-2 expression vector transfected with m-Az-Z-Lys into Escherichia coli B-95.delA [SciRep, 2015.5(9699)]. 100 ng of the 8His-IL-2 expression vector and various 8His-IL-2 expression vectors transfected with o-Az-Z-Lys or 8His-IL-2 expression vectors transfected with m-Az-Z-Lys were added to 100 μL of competent cells, gently mixed, and left standing on ice for 30 minutes.

[0775] Then, after heating in a water bath at 42 °C for 30 seconds, it was left standing on ice for 2 minutes again. 500 μL of LB medium was added, and after shaking culture in an incubator set at 37 °C for 60 minutes, the entire volume was plated on an LB plate (1.5 w / v% agarose) containing ampicillin (manufactured by Wako Pure Chemical Industries, Ltd.) at a final concentration of 100 μg / mL. After culturing overnight in an incubator set at 37 °C, the Escherichia coli growing on the plate was screened as the gene-transfected strain.

[0776] The entire amount of the obtained gene-transfected strain was recovered and inoculated into 800 mL of Super Broth [1 w / v% MOPS (manufactured by Nakarai Tesque), 3 w / v% Tryptone (manufactured by DIFCO), 2 w / v% Yeast Extract (manufactured by DIFCO)] supplemented with o-Az-Z-Lys or m-Az-Z-Lys at a final concentration of 1 mM (synthesized by GVK Biosciences according to the method described in International Publication No. 2017 / 030156) and ampicillin at a final concentration of 100 μg / mL, and cultured with shaking at 165 rpm in a bioshaker set at 37 °C.

[0777] When the absorbance value at 600 nm of the cell solution reached 1.5 - 2.0, isopropyl-β-thiogalactoside (IPTG) (manufactured by Nakarai Tesque) at a final concentration of 1.0 mmol / L was added, and the culture was continued with shaking at 165 rpm in a bioshaker set at 42 °C for 3 hours to express each human IL-2.

[0778] After centrifuging the cultured bacterial solution [CR21E (manufactured by Hitachi, Ltd.), 7000 rpm, 4 °C, 5 minutes], the Escherichia coli cells were recovered, and 40 mL of B-PER (Bacterial Protein Extraction Reagent) (manufactured by Thermo Scientific) was added for cell lysis. Then, centrifugation was performed (12000×g, 4 °C, 5 minutes) to obtain inclusion bodies.

[0779] The obtained inclusion bodies were dissolved in 32 mL of Inclusion Body Solubilization Reagent (manufactured by Thermo Scientific), and then centrifugation was performed again (12000×g, 4 °C, 30 minutes) to recover the supernatant.

[0780] The inclusion body solution was diluted to 3 times its volume with 100 mmol / L Tris-HCl buffer (manufactured by Wako Pure Chemical Industries, Ltd.) (pH 8.0) containing 6 mol / L guanidine hydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.), and then added to TALON Metal Affinity Resin (manufactured by Clontech). After washing with 100 mmol / L Tris-HCl buffer (pH 8.0) containing 6 mol / L guanidine hydrochloride, elution was performed with 100 mmol / L Tris-HCl buffer (pH 8.0) containing 250 mmol / L imidazole and 6 mol / L guanidine hydrochloride. The protein concentration of the eluate was measured by measuring the absorbance at 280 nm.

[0781] The above eluate was diluted 3 times with refolding buffer [100 mmol / L Tris-HCl buffer (pH 8.0) containing 1 mmol / L oxidized glutathione (manufactured by Sigma-Aldrich)] and allowed to stand overnight at 4 °C. Then, it was concentrated using Amicon Ultra-4 (3 kDa) (manufactured by Merck Millipore).

[0782] Superdex 75 10 / 300 GL (manufactured by GE Healthcare) was connected to AKTA FPLC (manufactured by GE Healthcare), and 100 mmol / L Tris-HCl buffer (pH 8.0) containing 2 mol / L guanidine hydrochloride was used as the mobile phase for delivery. The above concentrated solution was added to the SEC column to recover the monomer fraction.

[0783] The resulting component was diluted 2-fold with D-PBS (manufactured by Nakarai Tesque) and allowed to stand at room temperature for 6 hours. Then, the buffer was replaced with D-PBS by ultrafiltration using Amicon Ultra-4 (3 kDa).

[0784] All of the prepared 8His-IL-2 with o-Az-Z-Lys incorporated or 8His-IL-2 with m-Az-Z-Lys incorporated were confirmed by SDS-PAGE to have bands of the same molecular weight as 8His-IL-2.

[0785] [Example 6] Preparation of an IL-2 expression vector for Escherichia coli incorporating o-Az-Z-Lys

[0786] An IL-2 expression vector for Escherichia coli incorporating o-Az-Z-Lys as shown in Table 11 was prepared by the following method.

[0787] Table 11

[0788] The prepared IL-2 expression vector for Escherichia coli incorporating o-Az-Z-Lys

[0789]

[0790] <Explanation of Table 11>

[0791] · Az-Z-Lys incorporation position: Position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1

[0792] · Modification at the 1st position: Represents the modification of the alanine residue at the 1st position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1. MA indicates that methionine is bound to the N-terminal alanine residue. M indicates that a mutation of substituting alanine with methionine has been applied to the amino acid residue.

[0793] · Mutation at the 125th position: Represents the presence or absence of a mutation of the amino acid residue at the 125th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1. When no mutation is applied, it is denoted as -, and when a mutation of substituting the amino acid residue from cysteine to serine is applied, it is denoted as S.

[0794] In the table, o-Az-Z-Lys described in the column of the substituted amino acid residue refers to the structure shown in the following (Formula 10).

[0795]

[0796] As IL-2, an expression vector as described above was prepared using IL-2 composed of an amino acid sequence in which the amino acid residue at position 125 of the wild-type mature human IL-2 amino acid sequence shown in SEQ ID NO: 1 was substituted from cysteine to serine and methionine was bound to the N-terminus (amino acid sequence: SEQ ID NO: 38, base sequence encoding the amino acid sequence: SEQ ID NO: 39), or IL-2 composed of an amino acid sequence in which the amino acid residue at position 125 of the wild-type mature human IL-2 amino acid sequence shown in SEQ ID NO: 1 was substituted from cysteine to serine, the alanine residue at position 1 was deleted, and methionine was bound to the N-terminus (amino acid sequence: SEQ ID NO: 40, base sequence encoding the amino acid sequence: SEQ ID NO: 41).

[0797] Between the NdeI restriction enzyme site and the SalI restriction enzyme site of pFLAG-CTS-Pyl TS, a base sequence (SEQ ID NOs: 42 to 50) in which the codons corresponding to the sites for introducing o-Az-Z-Lys were substituted with amber (TAG) codons was inserted, thereby preparing various IL-2 expression vectors for Escherichia coli into which o-Az-Z-Lys was introduced (hereinafter referred to as pFLAG-CTS-PylTS_hIL-2).

[0798] [Example 7] Preparation of IL-2 into which o-Az-Z-Lys was introduced

[0799] IL-2 into which o-Az-Z-Lys was introduced in which any amino acid residue of IL-2 was substituted with an o-Az-Z-Lys residue as shown in Table 12 was prepared by the following method.

[0800] Table 12

[0801] The prepared IL-2 into which o-Az-Z-Lys was introduced

[0802]

[0803] <Explanation of Table 12>

[0804] · o-Az-Z-LysK introduction position: Position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1

[0805] · Modification at position 1: Represents the modification of the alanine residue at position 1 from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1. MA indicates that methionine is bound to the N-terminal alanine residue. M indicates that a mutation of amino acid residue substitution in which alanine was substituted with methionine was applied.

[0806] ·Mutation at position 125: Indicates the presence or absence of a mutation in the amino acid residue at the 125th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1. It is denoted as "-" when no mutation is applied, and as "S" when a mutation that substitutes the amino acid residue from cysteine to serine is applied.

[0807] In the table, o-Az-Z-Lys described in the column of the substituted amino acid residue refers to the structure shown in the following (Formula 10).

[0808]

[0809] The Escherichia coli prepared in Example 6 was transformed with an IL-2 expression vector into which o-Az-Z-Lys was introduced, and Escherichia coli B-95.delA [Sci Rep, 2015.5(9699)]. An inclusion body lysate was prepared by the method described in Example 5.

[0810] HiPrep 26 / 60 Sephacryl S-100HR (manufactured by GE Healthcare) was connected to AKTA FPLC, and a 100 mmol / L Tris-HCl buffer solution (pH 8.0) containing 2 mol / L guanidine hydrochloride was delivered as the mobile phase. The above inclusion body lysate was added to the SEC column, and the monomer component was recovered.

[0811] Oxidized glutathione was added to a concentration of 2 mmol / L, and the mixture was allowed to stand overnight at 4°C. Then, it was concentrated using Amicon Ultra-4 (3 kDa) (manufactured by Merck Millipore), and the buffer was exchanged to a 10 mmol / L acetic acid buffer solution (pH 4.5) containing 0.4 mol / L arginine hydrochloride and 5 w / v% trehalose using a NAP column (manufactured by GE Healthcare).

[0812] It was confirmed by SDS-PAGE that the prepared IL-2 into which o-Az-Z-Lys was introduced had the molecular weight predicted by the amino acid sequence.

[0813] [Example 8] PEGylation of 8His-IL-2 into which o-Az-Z-Lys was introduced, 8His-IL-2 into which m-Az-Z-Lys was introduced, or IL-2 into which o-Az-Z-Lys was introduced

[0814] The PEGylated products of 8His-IL-2 into which o-Az-Z-Lys was introduced, 8His-IL-2 into which m-Az-Z-Lys was introduced, or IL-2 into which o-Az-Z-Lys was introduced shown in Tables 13 to 15 (hereinafter referred to as PEG-bound IL-2 variants) were prepared by the following method.

[0815] Table 13

[0816] The prepared PEG-conjugated IL-2 variants

[0817]

[0818] Table 14

[0819] The prepared PEG-conjugated IL-2 variants

[0820]

[0821] Table 15

[0822]

[0823] <Explanation of Tables 13, 14 and 15>

[0824] · PEG-introducing position: Position from the N-terminus of SEQ ID NO: 1

[0825] · Modification at the 1st position: Represents the modification of the alanine residue at the 1st position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1. MA indicates that methionine is bound to the N-terminal alanine residue, and MHHHHHHHHA indicates that methionine and a polyhistidine sequence (HHHHHHHH) tag are bound to the N-terminal alanine residue.

[0826] · Mutation at the 125th position: Represents the presence or absence of a mutation of the amino acid residue at the 125th position from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1. When no amino acid residue mutation is applied, it is denoted as -, and when a mutation substituting the amino acid residue from cysteine to serine is applied, it is denoted as S.

[0827] The f...

Claims

1. An interleukin-2 (IL-2) variant conjugated with polyethylene glycol (PEG), which has an increased selectivity for the IL-2 receptor, and the IL-2 receptor is IL-2R αβγ , wherein the IL-2 variant consists of the following sequences: The amino acid sequence in which the amino acid residue at position 125 of the amino acid sequence of SEQ ID NO: 1 is substituted with a serine residue, and the amino acid residue at position 129 in the above amino acid sequence is substituted with a PEG-conjugated unnatural amino acid residue, wherein the PEG-conjugated unnatural amino acid residue is a group derived from an o-Az-Z-Lys residue conjugated with PEG and has a structure represented by the following (Formula 11) and / or (Formula 12): or wherein the PEG-conjugated unnatural amino acid residue is a group derived from an m-Az-Z-Lys residue conjugated with PEG and has a structure represented by the following (Formula Y4) and / or (Formula Y5): or wherein the PEG-conjugated unnatural amino acid residue is a group derived from a cysteine residue conjugated with PEG and has a structure represented by the following (Formula X11) and / or (Formula X12) and / or (Formula X13):

2. The IL-2 variant according to claim 1, wherein, PEG is linear.

3. The IL-2 variant according to claim 1, wherein, PEG is branched.

4. The IL-2 variant according to claim 1, wherein, PEG is a PEG having an average molecular weight of 10 kDa or more.

5. The IL-2 variant according to claim 1, wherein, PEG is a PEG having an average molecular weight of 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, or 80 kDa.

6. The IL-2 variant according to claim 1, wherein, PEG has a structure represented by at least one of the following (Formula 13), (Formula 14), (Formula 15), (Formula 16), (Formula X7), (Formula X8), (Formula X9), (Formula X10), (Formula X11), (Formula X13), (Formula X14), or (Formula X15):

7. The IL-2 variant according to any one of claims 1 to 6, wherein, A methionine residue is further conjugated to the N-terminus of the IL-2 variant.

8. An interleukin-2 (IL-2) variant conjugated with polyethylene glycol (PEG), which has an increased selectivity for the IL-2 receptor, and the IL-2 receptor is IL-2R αβγ , wherein the IL-2 variant consists of the following sequences: The amino acid sequence in which the amino acid residue at position 125 of the amino acid sequence of SEQ ID NO: 1 is substituted with a serine residue, and the amino acid residue at position 129 in the above amino acid sequence is substituted with a PEG-conjugated unnatural amino acid residue, and the alanine at the N-terminus of the IL-2 variant is deleted, wherein the PEG-conjugated unnatural amino acid residue is a group derived from an o-Az-Z-Lys residue conjugated with PEG and has a structure represented by the following (Formula 11) and / or (Formula 12): or wherein the PEG-conjugated unnatural amino acid residue is a group derived from an m-Az-Z-Lys residue conjugated with PEG and has a structure represented by the following (Formula Y4) and / or (Formula Y5): or wherein the PEG-conjugated unnatural amino acid residue is a group derived from a cysteine residue conjugated with PEG and has a structure represented by the following (Formula X11) and / or (Formula X12) and / or (Formula X13):

9. An interleukin-2 (IL-2) variant conjugated with polyethylene glycol (PEG), which has an increased selectivity for the IL-2 receptor, and the IL-2 receptor is IL-2R αβγ , wherein the IL-2 variant consists of the following sequence: The amino acid sequence in which the amino acid residue at position 125 of the amino acid sequence of SEQ ID NO: 1 is substituted with a serine residue, and the amino acid residue at position 129 in the above amino acid sequence is substituted with a PEG-conjugated unnatural amino acid residue, and the alanine at the N-terminus of the IL-2 variant is deleted, and a methionine is further conjugated, The PEG-conjugated unnatural amino acid residue is a group derived from an o-Az-Z-Lys residue conjugated with PEG and has the structure shown in the following (Formula 11) and / or (Formula 12): or The PEG-conjugated unnatural amino acid residue is a group derived from an m-Az-Z-Lys residue conjugated with PEG and has the structure shown in the following (Formula Y4) and / or (Formula Y5): or The PEG-conjugated unnatural amino acid residue is a group derived from a cysteine residue conjugated with PEG and has the structure shown in the following (Formula X11) and / or (Formula X12) and / or (Formula X13):

10. The IL-2 variant according to claim 8 or 9, wherein PEG has an average molecular weight of 80 kDa, and wherein PEG contains a structure represented by at least one of Formula 16 or Formula X9:

11. A method for manufacturing the IL-2 variant according to any one of claims 1 to 10.

12. A composition comprising the IL-2 variant according to any one of claims 1 to 10.

13. A therapeutic agent for immune diseases, comprising the IL-2 variant according to any one of claims 1 to 10.

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