Il-2 mutein for treating autoimmune and inflammatory diseases

Abstract

To provide an innovative IL-2 mutein capable of more selectively activating IL-2R signals in different cell types for treating immune-mediated diseases, e.g., a biologically optimized IL-2 mutein that selectively binds to the IL-2Rα subunit.SOLUTION: Provided is an isolated IL-2 mutein comprising a first polypeptide comprising a specific amino acid sequence except for having N at position 19 instead of D, and S instead of E at position 67 by substitution, where the polypeptide optionally comprises one or more additional amino acid substitutions relative to the specific sequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] Field

[0002] The present invention relates to a method for the production of IL-2 receptor α subunits but not IL-2 receptor β subunits. The present invention relates to an IL-2 mutein that does not show measurable binding to IL-2. Also relates to compositions, kits, methods and uses comprising such IL-2 muteins.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application is a continuation of U.S. Provisional Patent Application No. 63 / 129,712, filed December 23, 2020. The benefit of this application is claimed, the contents of which are incorporated herein by reference in their entirety. do.

[0005] REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0006] The sequence listing of the present application is filed under the file name “25184WOPCT-SEQLIST-21NOV 2021.TXT", with a creation date of November 21, 2021 and a size of 132KB. Sequence listings in ASCII format will be submitted electronically via EFS-Web. - This sequence listing submitted via the web is incorporated herein by reference in its entirety. The disclosure is hereby incorporated by reference. [Background technology]

[0007] The biological activity of interleukin 2 (IL-2) is determined by the following two or three polypeptides: mediated by the multisubunit IL-2 receptor (IL-2R) complex, which contains That is, CD25 (IL-2R α subunit) is the IL-1 receptor for IL-2. 2 complex, while CD122 (IL-2R β subunit) and and CD132 (IL-2R γ subunit) are required for signal transduction. Dimer I The IL-2R β / γ complex and the trimeric IL-2R α / β / γ complex are involved in the differentiation of various immune cell subtypes. Differential expression of IL-2R types, e.g., high affinity trimeric IL-2R α / β / γ The complex is constitutively expressed at high levels by regulatory T (Treg) cells and CD4+ T It is expressed transiently at low levels by effector cells, whereas it has moderate affinity The dimeric IL-2R β / γ complex is expressed primarily on CD8+ T cells and natural killer cells. (Stauber et al., (2006) PNAS USA 103(8):27 88-93; Malek and Castro, (2010) Immunity 33(2 ):153-165; Ross and Cantrell,(2018)Annu Rev Immunol 36:418-433). Thus, dimeric or trimeric IL-2 IL-2 muteins with a propensity to bind to the R complex activate various cell types, It can result in immune activation or suppression. Different cells can be used to treat immune-mediated diseases. Innovative IL-2 muteins that can more selectively activate IL-2R signaling in the IL-2 type For example, the IL-2R α subunit for the treatment of autoimmune and inflammatory diseases. There remains an unmet need to identify selectively binding, biologically optimized IL-2 muteins. It hasn't been done yet. Summary of the Invention

[0008] Overview of the Invention

[0009] The present disclosure relates to a method for the detection of IL-2 receptor α subunits that bind to IL-2 receptor β subunits. The present invention provides IL-2 muteins that do not exhibit measurable binding to IL-2 receptors. Methods or uses involving IL-2 muteins, compositions comprising such IL-2 muteins or The present invention provides kits, isolated nucleic acids comprising polynucleotide sequences encoding such IL-2 muteins. isolated nucleic acids and vectors, cells containing such isolated nucleic acids or vectors (e.g. Also provided herein are host cells, as well as methods for producing such IL-2 muteins. Provide.

[0010] In one embodiment, the IL-2 receptor α subunit is bound to the IL-2 receptor. Provided herein are IL-2 muteins that do not bind directly to the β subunit.

[0011] In certain embodiments, the amino acid sequence comprises the amino acid sequence set forth in SEQ ID NO: 1 or 2. The present invention provides an IL-2 mutein comprising a first polypeptide, wherein the first polypeptide is selected from the group consisting of 19 of SEQ ID NO: 1 or 2. D at position 31 of SEQ ID NO: 1 or 2 is replaced with N, and P at position 33 of SEQ ID NO: 1 or 2 is replaced with R; wherein the polypeptide optionally comprises one or more additional sequences relative to SEQ ID NO: 1 or 2. In one embodiment, the IL-2 mutein comprises the sequence A first polypeptide comprising the amino acid sequence set forth in sequence number 1, wherein SEQ ID NO: D at position 19 of SEQ ID NO:1 is substituted with N, and P at position 33 of SEQ ID NO:1 is substituted with R. wherein the polypeptide optionally comprises one or more additional amino acids relative to SEQ ID NO:1. In another embodiment, the IL-2 mutein has the sequence 2, wherein the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: D at position 19 of SEQ ID NO:2 is substituted with N, and P at position 33 of SEQ ID NO:2 is substituted with R; wherein the polypeptide optionally contains one or more additional amino acid residues relative to SEQ ID NO:2. It may contain substitutions.

[0012] In particular embodiments of the IL-2 muteins, the IL-2 muteins are One or more additional amino acid substitutions to the 67th position of SEQ ID NO: 1 or 2 include an E to S substitution. substitution, V to A at position 68, N to R at position 70, and Q to P at position 73 Substituted is selected from the group consisting of:

[0013] In some embodiments, the antibody comprises the amino acid sequence set forth in SEQ ID NO: 1 or 2. The present invention provides an IL-2 mutein comprising a first polypeptide comprising one of SEQ ID NOs: 1 or 2, D at position 9 is replaced with N, and E at position 67 of SEQ ID NO: 1 or 2 is replaced with S. wherein the polypeptide optionally comprises one or more additional sequences relative to SEQ ID NO: 1 or 2. In one embodiment, the IL-2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, wherein the sequence D at position 19 of SEQ ID NO:1 is replaced with N, and E at position 67 of SEQ ID NO:1 is replaced with S. wherein the polypeptide optionally comprises one or more additional amino acids to SEQ ID NO:1. In another embodiment, the IL-2 mutein comprises the sequence A first polypeptide comprising the amino acid sequence set forth in sequence number 2, wherein SEQ ID NO: D at position 19 of SEQ ID NO:2 is substituted with N, and E at position 67 of SEQ ID NO:2 is substituted with S. wherein the polypeptide optionally comprises one or more additional amino acids relative to SEQ ID NO:2. It may contain acid substitutions.

[0014] In another embodiment, the nucleic acid sequence of ... present invention is a nucleic acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2. IL-2 muteins comprising a polypeptide comprising position 19 of SEQ ID NO: 1 or 2, D in SEQ ID NO: 1 or 2 is replaced with N, and P in position 33 of SEQ ID NO: 1 or 2 is replaced with R, and wherein E at position 67 of sequence number 1 or 2 is replaced by S, and the polypeptide optionally comprises It may contain one or more additional amino acid substitutions relative to SEQ ID NO: 1 or 2. In an embodiment, the IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:1. wherein D at position 19 of SEQ ID NO:1 is replaced with N; P at position 33 of SEQ ID NO:1 is replaced with R, and E at position 67 of SEQ ID NO:1 is replaced with S. and optionally, the polypeptide further comprises one or more additional amino acids relative to SEQ ID NO:1. In another embodiment, the IL-2 mutein has the sequence No. 2, wherein the first polypeptide comprises an amino acid sequence as set forth in SEQ ID NO:2. D at position 19 of SEQ ID NO:2 is replaced with N, and P at position 33 of SEQ ID NO:2 is replaced with R, and wherein E at position 67 of sequence number 2 is replaced with S, and the polypeptide optionally has the sequence of SEQ ID NO: It may contain one or more additional amino acid substitutions for the two.

[0015] In yet another embodiment of the IL-2 mutein, the first polypeptide has the sequence of SEQ ID NO:1 or is a substitution of V to A at position 68, N to R at position 70, or Q to P at position 73 of 2. It further includes the substitution:

[0016] In yet another embodiment of the IL-2 mutein, the first polypeptide has the following three positions: and any two of the following substitutions: V to A at position 68 of SEQ ID NO: 1 or 2; N to R substitution at position 0 or Q to P substitution at position 73.

[0017] In yet another embodiment of the IL-2 mutein, the first polypeptide is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2. or 2 substitutions of V to A at position 68, N to R at position 70, and Q to Further includes substitutions to P.

[0018] In certain embodiments, the IL-2 mutein is selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In one embodiment, the IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:3. In another embodiment, the IL-2 mutant comprises a first polypeptide comprising an amino acid sequence. The polypeptide comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:4. In another embodiment, the IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:5. In yet another embodiment, the polypeptide further comprises a first polypeptide comprising an IL-2 polypeptide sequence. The tein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:6. In another embodiment, the IL-2 mutein is an IL-2 mutein having the amino acid sequence set forth in SEQ ID NO:7. In one embodiment, the IL-2 mutant comprises a first polypeptide comprising an amino acid sequence. The polypeptide comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:8. In one embodiment, the IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:9. In yet another embodiment, the IL-2 mutant comprises a first polypeptide comprising a first sequence. The polypeptide comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 10. In another embodiment, the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:11. In yet another embodiment, the polypeptide further comprises a first polypeptide comprising an IL- The mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:12. nothing.

[0019] In some embodiments of the IL-2 mutein, the first polypeptide is SEQ ID NO: 13. 14 or 15. IL-2 muteins are selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. The amino acid sequence set forth in SEQ ID NO: 13, including the amino acid sequence set forth in SEQ ID NO: 13 In another embodiment, the first polypeptide further comprises an IL-2 mutein. is an amine as set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. The first polypeptide further comprises the amino acid sequence set forth in SEQ ID NO:14. In yet another embodiment, the IL-2 mutein comprises a peptide having the sequence set forth in SEQ ID NO:3. , 4, 5, 6, 7, 8, 9, 10, 11 or 12. and a first polypeptide further comprising the amino acid sequence set forth in SEQ ID NO:15. .

[0020] In another embodiment of the IL-2 mutein, the first polypeptide is set forth in SEQ ID NO:16. In one embodiment, the IL-2 mutein further comprises a linker having the sequence The amino acid sequence shown in column numbers 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 The sequence of the amino acid sequence set forth in SEQ ID NO:13 and the sequence set forth in SEQ ID NO:16. In another embodiment, the first polypeptide comprises an IL-2 The muteins are set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. The amino acid sequence depicted in SEQ ID NO: 14 and the amino acid sequence depicted in SEQ ID NO: 16 and a linker as described in claim 1. In the present invention, the IL-2 mutein is selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 and the amino acid sequence set forth in SEQ ID NO: 15. and a linker as set forth in SEQ ID NO:16.

[0021] In various embodiments, the IL-2 mutein further comprises a second polypeptide. In this embodiment, the first polypeptide and the second polypeptide are the same. In form, the first and second polypeptides are not the same.

[0022] In one embodiment, the IL-2 mutein is selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 and the amino acid sequence set forth in SEQ ID NO: 14 a first polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:17; and a second polypeptide comprising the sequence.

[0023] In one embodiment, the IL-2 mutein is selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 and the amino acid sequence set forth in SEQ ID NO: 14 a first polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:55; and a second polypeptide comprising the sequence.

[0024] In another embodiment, the IL-2 mutein is selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 and the amino acid sequence set forth in SEQ ID NO: 14 a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18; and a second polypeptide comprising the amino acid sequence.

[0025] In some embodiments, the IL-2 mutein comprises a first polypeptide and a second polypeptide. wherein the first polypeptide comprises a peptide selected from SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28, The tide comprises the amino acid sequence set forth in SEQ ID NO: 17. The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:19, and the second polypeptide comprises The peptide comprises the amino acid sequence set forth in SEQ ID NO: 17. In the present invention, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:20, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:21. The polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17. In an embodiment, the first polypeptide has the amino acid sequence set forth in SEQ ID NO:21. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17. In another embodiment, the first polypeptide is an amino acid sequence set forth in SEQ ID NO:22. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:17. In yet another embodiment, the first polypeptide is set forth in SEQ ID NO:23. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:17. In one embodiment, the first polypeptide comprises the sequence set forth in SEQ ID NO:24. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:17. In another embodiment, the first polypeptide comprises the amino acid sequence of SEQ ID NO:25. The second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:17. In yet another embodiment, the first polypeptide comprises an amino acid sequence 26, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17. In yet another embodiment, the first polypeptide comprises the amino acid sequence set forth in The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:27, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: In yet another embodiment, the first The polypeptide comprises the amino acid sequence set forth in SEQ ID NO:28, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:17.

[0026] In some embodiments, the IL-2 mutein comprises a first polypeptide and a second polypeptide. peptide, wherein the first polypeptide is selected from the group consisting of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28, The peptide comprises the amino acid sequence set forth in SEQ ID NO: 55. The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:19, and the second polypeptide comprises The peptide comprises the amino acid sequence set forth in SEQ ID NO: 55. In the present invention, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 20, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 21. The two polypeptides comprise the amino acid sequence set forth in SEQ ID NO:55. In an embodiment, the first polypeptide has the amino acid sequence set forth in SEQ ID NO:21. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:55. In another embodiment, the first polypeptide is an amino acid sequence as set forth in SEQ ID NO:22. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:55. In yet another embodiment, the first polypeptide comprises the polypeptide set forth in SEQ ID NO:23. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:55. In one embodiment, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:24. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:55. In another embodiment, the first polypeptide comprises the amino acid sequence of SEQ ID NO:25. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:55. In yet another embodiment, the first polypeptide comprises an amino acid sequence comprising the sequence 26, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:55. In yet another embodiment, the first polypeptide comprises the amino acid sequence set forth below. The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:27, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:28. In yet another embodiment, the amino acid sequence is The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:28, and the second polypeptide comprises This code comprises the amino acid sequence set forth in SEQ ID NO:55.

[0027] In certain embodiments, the IL-2 mutein comprises a first polypeptide and a second polypeptide. wherein the first polypeptide comprises a peptide selected from SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, 36, 37 or 38, The tide comprises the amino acid sequence set forth in SEQ ID NO: 18. The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:29, and the second polypeptide comprises The peptide comprises the amino acid sequence set forth in SEQ ID NO: 18. In the present invention, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 30, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 31. The polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 18. In an embodiment, the first polypeptide has the amino acid sequence set forth in SEQ ID NO:31. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 18. In another embodiment, the first polypeptide is an amino acid sequence set forth in SEQ ID NO:32. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:18. In yet another embodiment, the first polypeptide is set forth in SEQ ID NO:33. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:18. In one embodiment, the first polypeptide comprises the sequence set forth in SEQ ID NO:34. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:18. In another embodiment, the first polypeptide comprises the amino acid sequence of SEQ ID NO:35. The second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:18. In yet another embodiment, the first polypeptide comprises an amino acid sequence 36, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 18. In yet another embodiment, the first polypeptide comprises the amino acid sequence set forth in The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 37, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: In yet another embodiment, the first The polypeptide comprises the amino acid sequence set forth in SEQ ID NO:38, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:18.

[0028] In certain embodiments, the IL-2 mutein comprises a first polypeptide and a second polypeptide. wherein the first and second polypeptides comprise the same amino acid sequence. In certain embodiments, the amino acid sequence of the first and second polypeptides is Each of the columns corresponds to SEQ ID NO: 45, 46, 47, 48, 49, 50, 51, 52, 53 or In one embodiment, the first polypeptide comprises the amino acid sequence set forth in Each of the first polypeptide and the second polypeptide has the amino acid sequence set forth in SEQ ID NO:45. In one embodiment, each of the first and second polypeptides comprises It comprises the amino acid sequence set forth in SEQ ID NO: 46. In one embodiment, Each of the first and second polypeptides is set forth in SEQ ID NO:47. In one embodiment, the first and second polypeptides include Each of the peptides comprises the amino acid sequence set forth in SEQ ID NO: 48. wherein each of the first and second polypeptides is set forth in SEQ ID NO:49. In one embodiment, the first polypeptide and Each of the second polypeptides comprises the amino acid sequence set forth in SEQ ID NO:50. In one embodiment, each of the first and second polypeptides has the sequence In one embodiment, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:51. Each of the first and second polypeptides has the amino acid sequence set forth in SEQ ID NO:52. In one embodiment, the first and second polypeptides Each comprises the amino acid sequence set forth in SEQ ID NO: 53. In one embodiment Each of the first and second polypeptides is set forth in SEQ ID NO:54. It includes an amino acid sequence.

[0029] In another embodiment, any of the various IL-2 muteins described herein. A pharmaceutical composition comprising any one of these and a pharma- ceutically acceptable carrier is provided.

[0030] In yet another embodiment, a therapeutically effective amount of any of the ILs described herein is administered. any one of the -2 muteins or any of the various pharmaceutical compositions described herein. and administering to the subject one of the following: do.

[0031] In certain embodiments, the IL-2 mediated disease is an immune disease. In this case, the IL-2 mediated disease is an autoimmune disease.

[0032] In some embodiments, the immune disorder is rheumatoid arthritis, Crohn's disease, psoriasis, psoriatic arthritis, or psoriatic joint disease. Arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), cutaneous lupus erythematosus (CL E), lupus nephritis, ankylosing spondylitis, type I diabetes, Sjogren's syndrome, ulcerative colitis, Neuromyelitis optica, celiac disease, scleroderma, temporal arteritis, atopic dermatitis, alopecia areata, migratory Graft-versus-host disease (GVHD), autoimmune hepatitis, primary sclerosing cholangitis, or inflammatory myopathy In one embodiment, the immune disease is rheumatoid arthritis. In one embodiment, the immune disease is Crohn's disease. In yet another embodiment, the immune disorder is psoriasis. In yet another embodiment, the immune disease is multiple sclerosis. In one embodiment, the immune disorder is SLE. In another embodiment, the immune disorder is CLE. In yet another embodiment, the immune disease is lupus nephritis. In another embodiment, the immune disease is ankylosing spondylitis. In one embodiment, the immune disease is type I diabetes. In another embodiment, the immune disease is ulcerative colitis. In yet another embodiment, the immune disease is neuromyelitis optica. In one embodiment, the immune disorder is celiac disease. In one embodiment, the immune disease is scleroderma. In one embodiment, the immune disease is temporal arteritis. In another embodiment, the immune disorder is atopic dermatitis. In one embodiment, the immune disease is alopecia areata. In yet another embodiment, the immune disease is GVHD. In one embodiment, the immune disease is primary sclerosing cholangitis. In another embodiment, the immune disease is an inflammatory myopathy.

[0033] In yet another embodiment, the method comprises administering to the subject a therapeutically effective amount of CD8+ T cells without activating CD8+ T cells. A method for selectively activating regulatory T cells comprising administering to a subject a therapeutically effective amount of any of the methods described herein. Any one of the various IL-2 muteins available in the art or species described herein. The present invention also provides a method for the treatment of a subject in need thereof, comprising administering any one of the pharmaceutical compositions described herein. do.

[0034] In yet another embodiment, the subject is treated with a method for the treatment of autism spectrum disorder characterized by the presence of a IL-2 receptor alpha subunit. Selectively inhibits cells expressing the IL-2 receptor β subunit without activating cells expressing the IL-2 receptor β subunit. The method of activating IL-2 receptors in a mammalian subject comprises administering to the mammal a therapeutically effective amount of any of the various IL-2 receptors described herein. any one of the various pharmaceutical compositions described herein. to a subject in need thereof.

[0035] In one embodiment, the various polypeptides of the various IL-2 muteins described herein are The present invention provides an isolated nucleic acid comprising a nucleotide sequence encoding any one of the polypeptides. In one embodiment, the isolated nucleic acid is selected from the group consisting of the various I The L-2 mutein encodes any one of the various first polypeptides. In one embodiment, the isolated nucleic acid can be used to identify the various IL-2 muteins described herein. In yet another embodiment, the second polypeptide encodes any one of the various second polypeptides. In particular, the isolated nucleic acids may be used to identify the various IL-2 muteins described herein. The polypeptide may encode one polypeptide and any one of a variety of second polypeptides.

[0036] In another embodiment, any of the various isolated nucleic acids described herein. or one of the expression vectors.

[0037] In yet another embodiment, any of the various isolated nucleic acids described herein. or a host cell comprising any one of the various expression vectors described herein. Provide the host cells.

[0038] In yet another embodiment, the various IL-2 muteins described herein are In one embodiment, the method comprises the steps of: Any one of the various host cells described herein can be cultured under conditions in which the tein is expressed. In another embodiment, the method comprises culturing the cells in which the IL-2 mutein is expressed. Under the conditions described herein, any one of the various expression vectors described herein is expressed. In yet another embodiment, the method comprises: Under conditions as described herein, the nucleic acid expresses any one of the various isolated nucleic acids described herein. This includes making the person do so.

[0039] In yet another embodiment, a method for treating an IL-2 mediated disease in a subject, comprising: Any one of the various IL-2 muteins described herein or The present invention provides the use of any one of the various pharmaceutical compositions disclosed herein.

[0040] In one embodiment, a method for treating an IL-2 mediated disease in a subject, comprising administering to said subject a therapeutically effective amount of an IL-2 mediated disease .... Any one of the various IL-2 muteins described herein or The use of any one of a variety of pharmaceutical compositions is provided.

[0041] In another aspect, a pharmaceutical composition for treating an IL-2 mediated disease in a subject is provided. For the manufacture of any one of the various IL-2 muteins described herein or The use of any one of the various pharmaceutical compositions described herein is provided.

[0042] In some embodiments of the various uses described herein, IL-2 mediated The disease is an immune disease. In other embodiments of the various uses described herein , IL-2 mediated diseases are autoimmune diseases.

[0043] In certain embodiments of the various uses described herein, the immune disorder is lymphoma. Urine, Crohn's disease, psoriasis, psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus (S LE), cutaneous lupus erythematosus (CLE), lupus nephritis, ankylosing spondylitis, type I diabetes, Sjögren's syndrome, ulcerative colitis, neuromyelitis optica, celiac disease, scleroderma, temporal arteritis , atopic dermatitis, alopecia areata, graft-versus-host disease (GVHD), autoimmune hepatitis, primary In one embodiment, the immune disease is chronic sclerosing cholangitis or inflammatory myopathy. In another embodiment, the immune disease is Crohn's disease. In yet another embodiment, the immune disease is psoriasis. In yet another embodiment, the immune disorder is psoriatic arthritis. In one embodiment, the immune disease is SLE. In one embodiment, the immune disease is CLE. In yet another embodiment, the immune disorder is lupus nephritis. In yet another embodiment, the immune disease is type I diabetes. In one embodiment, the immune disease is Sjogren's syndrome. In yet another embodiment, the immune disease is ulcerative colitis. In yet another embodiment, the immune disorder is celiac disease. In yet another embodiment, the immune disease is scleroderma. In one embodiment, the immune disorder is temporal arteritis. In yet another embodiment, the immune disease is alopecia areata. In yet another embodiment, the immune disease is GVHD. In one embodiment, the immune disease is autoimmune hepatitis. In another embodiment, the immune disorder is inflammatory myelopathy. It is opacity. [Brief description of the drawings]

[0044] [Figure 1A]1A-1C show the ability of exemplary IL-2 muteins to activate pSTAT5 responses in primary human Treg cells (CD3+ CD4+ CD25+ FoxP3+) (FIG. 1A), CD8 T cells (CD3+ CD8+) (FIG. 1B), or CD4+ conventional T cells (Tconv) cells (CD3+ CD4+ CD25- FoxP3-) (FIG. 1C). [Figure 1B] the above [Figure 1C] the above

[0045] [Figure 2A] Figures 2A-2C show the ability of more exemplary IL-2 muteins to activate pSTAT5 responses in primary human Treg cells (CD3+ CD4+ CD25+ FoxP3+) (Figure 2A), CD8 T cells (CD3+ CD8+) (Figure 2B) or Tconv cells (CD3+ CD4+ CD25- FoxP3-) (Figure 2C). [Figure 2B] the above [Figure 2C] the above

[0046] [Figure 3A] Figures 3A-3C show the ability of more exemplary IL-2 muteins to activate pSTAT5 responses in primary human Treg cells (CD3+ CD4+ CD25+ FoxP3+) (Figure 2A), CD8 T cells (CD3+ CD8+) (Figure 2B) or Tconv cells (CD3+ CD4+ CD25- FoxP3-) (Figure 2C). [Figure 3B] the above [Figure 3C] the above

[0047] [Figure 4A] Figures 4A-4C show the ability of exemplary IL-2 muteins to activate pSTAT5 responses in primary rhesus Treg cells (CD3+ CD4+ CD25+ CD127- FoxP3+) (Figure 4A), CD8 T cells (CD3+ CD8+) (Figure 4B) or Tconv cells (CD3+ CD4+ CD25- FoxP3-) (Figure 4C). [Figure 4B] the above [Figure 4C] the above

[0048] [Figure 5A] 5A-5C show the ability of exemplary IL-2 muteins to activate pSTAT5 responses in primary rhesus Treg cells (CD3+ CD4+ CD25+ CD127- FoxP3+) (FIG. 5A), CD8 T cells (CD3+ CD8+) (FIG. 5B) or Tconv cells (CD3+ CD4+ CD25- FoxP3-) (FIG. 5C). [Figure 5B] the above [Figure 5C] the above

[0049] [Figure 6A] Figures 6A-6D show the expansion of human Tregs in xenogeneic GvHD mice treated with exemplary IL-2 muteins. Figure 6A shows the results of 21BMT, Figure 6B shows the results of DNB558, Figure 6C shows the results of 48BMP, and Figure 6D shows the results of DNB557. The percentage of Tregs in human CD4+ T cells in the spleen was quantified by FACS. The fold change of Tregs is calculated as (Treg% in human CD4 T cells from treated mice / mean of Treg% in human CD4 T cells from isotype control group). The concentration of the muteins in the blood was measured daily and plotted with the right Y-axis. *Statistically significant difference compared to isotype control by one-way ANOVA with Sidak's multiple comparison test. [Figure 6B] the above [Figure 6C] the above [Figure 6D] the above

[0050] [Figure 7A]Figures 7A-7D show increased proliferation of CD25 on human Tregs in xenogeneic GvHD models treated with exemplary IL-2 muteins. Figure 7A shows the results of 21BMT, Figure 7B shows the results of DNB558, Figure 7C shows the results of 48BMP, and Figure 7D shows the results of DNB557. Mean fluorescence intensity (MFI) of CD25 on human CD4+ Treg cells in the spleen was quantified by FACS. Fold change is calculated as (MFI of CD25 on human CD4+ Treg cells from treated mice / mean of MFI of CD25 on human CD4+ Treg cells from isotype control group). *Statistically significant difference compared to isotype control by one-way ANOVA with Sidak's multiple comparison test. [Figure 7B] the above [Figure 7C] the above [Figure 7D] the above

[0051] [Figure 8A] Figures 8A-8D show concentration (nM) over time and fold change from baseline in Tregs over time from the PK / PD study described in Example 8. Figure 8A shows the results for 21BMT, Figure 8B shows the results for DNB558, Figure 8C shows the results for 48BMP, and Figure 8D shows the results for DNB557. [Figure 8B] the above [Figure 8C] the above [Figure 8D] the above

[0052] [Figure 9]9A-9D are non-limiting schematic diagrams showing the orientation of monovalent and bivalent IL-2 muteins. FIG. 9A shows a bivalent molecule having an IL-2 mutein linked at its C-terminus to the N-terminus of an Fc. FIG. 9B shows a monovalent molecule having one chain having an IL-2 mutein linked at its C-terminus to the N-terminus of an Fc, the second chain being an Fc moiety, and the two Fc regions being linked via complementary knob and hole mutations. FIG. 9C shows a bivalent molecule having an Fc linked at its C-terminus to the N-terminus of an IL-2 mutein. FIG. 9D shows a monovalent molecule having an Fc linked at its C-terminus to the N-terminus of an IL-2 mutein, the second chain being an Fc moiety, and the two Fc regions being linked via complementary knob and hole mutations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] Detailed Description

[0054] definition

[0055] So that the present invention may be more readily understood, certain scientific and technical terms are specifically defined below. All other classes used herein are intended to include, but are not limited to, those listed above, unless specifically defined elsewhere in this specification. Terms of science and technology have the meanings commonly understood by those of ordinary skill in the art to which this invention pertains. do.

[0056] As used in this specification, including the appended claims, the singular forms "a," "an," and "the" are used unless the context clearly indicates otherwise. Likewise, unless the context clearly indicates otherwise, the plural forms are included. Unless otherwise indicated, plural words include the corresponding singular forms.

[0057] An "IL-2 mutein" is a partial or complete IL-2 mutein having one or more amino acid substitutions, deletions, or additions. "IL-2 mutein" refers to a molecule that contains the full-length human IL-2 amino acid sequence. The term refers to a partial or full-length human I polypeptide having one or more amino acid substitutions, deletions or additions. Any fusion protein, protein conjugate or polysaccharide comprising the L-2 amino acid sequence. In certain embodiments, the IL-2 A mutein is a partial or complete variant of a gene that has one or more amino acid substitutions, deletions or additions. These muteins contain the full length human IL-2 amino acid sequence and are therefore referred to as "monovalent" IL-2 muteins. In some embodiments, the IL-2 mutein comprises one or more amino acid substitutions, deletions or deletions. Two partial or full-length human IL-2 amino acid sequences, each having a different sequence or addition, are provided. and are therefore referred to as "bivalent" IL-2 muteins. The amino acid sequence is set forth in SEQ ID NO: 39. The partial human IL-2 amino acid sequence is At least 50% of the full-length wild-type human IL-2 amino acid sequence set forth in SEQ ID NO:39 %, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% include.

[0058] As used herein, "86BCH", "99BHY", "43BGO", "44BGO" ”, “54BGO”, “47BJO”, “48BJO”, “49BJO”, “65BJO ", "44BJP", "45BJP", "46BJP" or "47BJP". Each specific IL-2 mutein has its specific amino acid substitution, deletion, or deletion as shown in Table 1. or partial or full-length human IL-2 amino acid sequences with additions thereto, and their identification Partial or full-length human IL-2 amino acid sequence having amino acid substitutions, deletions or additions of Any fusion protein, protein conjugate or multisubunit protein comprising the sequence Proteins (e.g., dimers).

[0059] The "Fc" region or domain is the C region of an antibody. H 2 and C H Heavy chain flag containing 3 domains An antibody refers to any class of immunoglobulin molecule (e.g., IgG, IgE , IgM, IgD and IgA) or any subclass (e.g., IgG1, IgG2 The Fc region may be one of the following: IgG1, IgG2, IgG4, IgA1 and IgA2. or domains are bounded by two or more disulfide bonds and C H Three-domain hydrophobic phase An "Fc variant" is a variant of a wild-type Fc region or domain. It contains one or more amino acid substitutions, deletions or insertions compared to the original.

[0060] The term "bind" or "binding" refers to the binding between molecules, including, for example, the formation of a complex. The interaction means, for example, hydrogen bonds, ionic bonds, hydrophobic interactions, and The complex may be a complex of the nucleophile, a nucleophile, or a non-nucleophile, including covalent and / or van der Waals interactions. Also refers to the bond between two or more molecules that are joined by covalent or non-covalent bonds, interactions or forces. Unless otherwise indicated, "binding affinity" as used herein refers to binding 1 between members of a pair (e.g., an IL-2 mutein and an IL-2 receptor subunit) The specific binding affinity reflects the IL-2 receptor subunit interaction. The binding rate of IL-2 muteins to on ) to dissociation rate (k off ) ratio ( k off / k on ) is the "dissociation constant" (or "equilibrium dissociation constant" used interchangeably), KD which is inversely related to affinity. The lower the KD value, the higher the affinity. The KD value is increased by the complex between the IL-2 mutein and the IL-2 receptor subunit. k on and k off As provided herein, IL-2 is dependent on both The dissociation constant KD of the mutein can be determined by any of the methods provided herein or known to those of skill in the art. or any other method of using the same, such as a surface plasmon resonance (SPR) assay, e.g., Biacor The amount of chromatin can be determined using, but is not limited to, KinExA and KinExA.

[0061] "Administer" or "administer" as applied to an animal, human, subject, cell, tissue, organ, or biological fluid. Or "administration" means exogenous administration of a compound to an animal, human, subject, cell, tissue, organ, or biological fluid. Contacting refers to the contacting of a drug, therapeutic substance, diagnostic agent or composition.

[0062] The term "subject" refers to any living organism, preferably an animal, more preferably a mammal (e.g., Human, rat, mouse, dog, cat or rabbit). In a preferred embodiment The term "subject" refers to a human. A subject "in need thereof" is one who has an IL-2 mediated diagnosed with, suspected of having, or affected by a disease or disorder that is associated with IL-2 and individuals having a predisposition to an IL-2 mediated disease or disorder, or for whom prevention of an IL-2 mediated disorder is desired. It is a desired object.

[0063] "Effector functions" are attributed to the Fc region of antibodies, which vary depending on the antibody isotype. Examples of antibody effector functions include Clq binding and complement-dependent Cytotoxicity (CDC); Fc receptor binding; Antibody-dependent cellular cytotoxicity (ADCC); Phagocytosis downregulation of cell surface receptors (e.g., B cell receptors); and B cell Activation is included.

[0064] As used herein, the terms "cell," "host cell," "cell line," and "cell culture" are used interchangeably and all such terms include progeny.

[0065] "Treating" or "treatment" refers to administering to a subject a composition containing any of the IL-2 muteins of the present invention. The agent, such as a substance, is administered to a subject having or having one or more disease symptoms for which the agent exhibits therapeutic activity. This means administering the agent internally or externally to a subject or patient suspected of having a disease. In general, an agent is one that induces regression of such symptoms to any clinically measurable degree. Induction or inhibition, delay or slowing of the progression of such conditions may be of benefit to a treated subject or population. The therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in any particular disease. The amount of agent effective to alleviate symptoms will depend on the condition, age, and weight of the patient, as well as the individual's The duration of the disease may vary depending on factors such as the ability of the drug to elicit a desired response in the setting of The reduction in symptoms should be evaluated by a physician or other trained person to assess the severity or progression of the condition. Can be assessed by any clinical scale typically used by trained health care providers The term further refers to a delay in the onset of symptoms associated with a disorder and / or a reduction in the severity of symptoms of such a disorder. The term also includes reducing the severity of an existing uncontrolled or undesirable symptom, This includes preventing additional symptoms, as well as ameliorating or preventing the underlying causes of such symptoms. In this context, the term refers to a vertebrate subject having a disorder, disease or condition, or to a subject having such a disorder, disease or condition. A beneficial outcome is achieved in a vertebrate subject that has the potential to develop a disease or condition. This indicates that

[0066] The terms "prevent", "preventing" and "prevention" refer to a disease, disorder, medical condition or related symptom. (e.g., GVHD, SLE, CLE, multiple sclerosis, ulcerative colitis or Crohn's disease) This means reducing the likelihood of the occurrence (or recurrence) of

[0067] The terms "IL-2-mediated disease," "IL-2-mediated disorder," and "IL-2-mediated condition" are used interchangeably. are used interchangeably and are either completely or partially caused by IL-2 signaling or Any disease, disorder or condition that is a result of IL-2 signaling, and / or Inhibiting IL-2 signaling in the body or in selected cell types, tissues or organs It means any disease, disorder or condition which it is desirable to modulate.

[0068] As used herein, the term "therapeutically effective amount" refers to a therapeutically effective amount for a given disease, disorder or condition, and and / or related conditions (e.g., GVHD, SLE, CLE, multiple sclerosis, ulcers) Reduce and / or improve the severity and / or duration of chronic colitis or Crohn's disease An agent (e.g., an IL-2 mutein or means the amount of any other agent described herein). The "therapeutically effective amount" of an agent (e.g., an IL-2 mutein) will vary depending on the individual's condition, age, sex, and other factors. and body weight and the ability of the substance / molecule / agent to elicit a desired response in an individual. A therapeutically effective amount may vary depending on factors such as any toxicity of the substance / molecule / agent. In certain embodiments, the therapeutically beneficial effect outweighs any adverse or detrimental effects. In the present context, the term "therapeutically effective amount" refers to an amount effective to treat a disease, disorder, or condition in a subject or mammal. of an IL-2 mutein or other agent (e.g., a drug) that is effective to "treat" It means quantity.

[0069] As used herein, a "carrier" refers to a carrier that is capable of carrying cells exposed to the carrier at the doses and concentrations used. or a pharma- ceutically acceptable carrier, excipient, or stabilizer that is non-toxic to mammals. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of suitable carriers include: buffers, e.g., phosphate, citrate, and other organic acids; antioxidants, such as ascorbic acid; low molecular weight (e.g., about 10 amino Polypeptides (less than 100% acid residues); proteins, e.g., serum albumin, gelatin or immunoglobulins; immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine monosaccharides, disaccharides and other carbohydrates; chelating agents, such as glucose, mannose or dextrin; chelating agents, such as EDT A; a sugar alcohol, such as mannitol or sorbitol; a salt-forming counterion, such as and / or non-ionic surfactants, such as TWEEN®, poly Ethylene glycol (PEG) and PLURONICS™. The term "carrier" is rarely used. diluents, adjuvants [e.g., Freund's adjuvant (complete or incomplete)], excipients Such carriers, including pharmaceutical carriers, are sterile liquids, such as water. and oils, e.g., of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, da The oil may be canola oil, mineral oil, sesame oil, etc. The composition (e.g., pharmaceutical composition) is administered intravenously. In this case, water is a typical carrier. Saline as well as dextrose and glycerol Aqueous solutions of may also be used as liquid carriers, particularly for injectable solutions. For example, pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, Malt, rice, wheat flour, chalk, silica gel, sodium stearate, monostearin Glycerol acid, talc, sodium chloride, skimmed milk powder, glycerol, propylene, glycol Coal, water, ethanol, etc. The composition may optionally contain small amounts of wetting agents, emulsifiers, etc. The composition may also contain a solution, a suspension, an emulsion, a suspending agent, a pH buffering agent, or a pH adjusting agent. The formulation may take the form of a tablet, pill, capsule, powder, sustained release formulation, etc. The products may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, stearyl alcohol, etc. Contains magnesium phosphate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers can be found in Remington and Gennaro, Remington gton's Pharmaceutical Sciences(18th ed.1 Compositions containing pharmaceutical compounds can be formulated with a suitable number of carriers, e.g. It may contain the IL-2 mutein in isolated or purified form.

[0070] As used herein, the term "pharmaceutical acceptable" refers to any substance or substance that is approved by the U.S. Federal or State government. Approved by a regulatory agency or the U.S. for use in animals, especially humans It means that it is included in the Pharmacopoeia, the European Pharmacopoeia or another generally recognized pharmacopoeia. Taste.

[0071] An "isolated nucleic acid" is a nucleic acid that is not naturally associated with a native sequence, but is instead a nucleic acid that is naturally associated with a native sequence, such as a genomic DNA sequence or a polypeptide. Nucleic acid substantially isolated from proteins or complexes, such as ribosomes and polymerases. An "isolated" nucleic acid molecule is a nucleic acid molecule that is It is separated from other nucleic acid molecules that are present in the natural source. A molecule, such as a cDNA molecule, may be produced by recombinant techniques and may be synthesized from other cellular material or The medium may be substantially free of chemical precursors, or if chemically synthesized, may be It may be substantially free of precursors or other chemicals. In one embodiment, one or more nucleic acid molecules encoding the IL-2 muteins described herein are isolated. or purified. This term includes nucleic acid sequences that have been removed from their naturally occurring environment. , recombinant or cloned DNA isolates, and chemically synthesized analogs, or by heterologous systems. Substantially pure molecules include isolated forms of the molecule. I can see it.

[0072] "Polynucleotide" or "nucleic acid", as used interchangeably herein, refers to any It means a polymer of nucleotides of any length, including DNA and RNA. Deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and and / or their analogues, or by DNA or RNA polymerases It can be any substrate that can be incorporated into a polymer by a synthetic reaction. The nucleic acid sequence may include modified nucleotides, such as methylated nucleotides and their analogs. As used herein, an "oligonucleotide" is generally less than about 200 nucleotides in length. (not necessarily) means a short, usually single-stranded, synthetic polynucleotide The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above discussion of polynucleotides applies equally well to oligonucleotides. The cells producing the IL-2 muteins of the present disclosure contain a nucleic acid encoding the IL-2 mutein. Suitable host cells include host cells into which a gene has been introduced. Suitable host cells are disclosed below.

[0073] IL-2 muteins

[0074] The present invention relates to a method for the production of IL-2 receptor α subunits but not IL-2 receptor β subunits. The present invention provides an IL-2 mutein that does not show measurable binding to IL-2.

[0075] The various IL-2 muteins described herein are identical to the IL-2 reference amino acid sequence, e.g. For example, one or more amino acid substitutions relative to the wild-type amino acid sequence set forth in SEQ ID NO: 39; It may be a partial or full-length human IL-2 molecule containing deletions or additions. In embodiments, partial or full-length sequences containing one or more amino acid substitutions, deletions or additions are The human IL-2 molecule may be expressed as a polypeptide that is expressed by a human immunoglobulin (e.g., Ig The Fc region is fused to the wild-type IgG1, IgG2, IgG3 or IgG4. The antibody may be of the Fc type, or may be modified to have a desired characteristic or property, e.g., an altered circulating antibody. Serum half-life, complement fixation, Fc receptor binding and / or effector functions (e.g., antigen-dependent The Fc variant may have a specific cytotoxicity (dependent cytotoxicity).

[0076] In one embodiment, the Fc region comprises a wild-type Fc region that has reduced / eliminated effector function. Two amino acid substitutions to the Fc polypeptide sequence, namely L234A and L235 A (sometimes referred to as a "LALA" mutation). In this embodiment, the Fc region contains three amino acid substitutions that reduce / eliminate effector function, namely, L234A, L235A, and D265S (referred to as "LALADS" mutations) In yet another embodiment, the Fc region is modified with a serotype Three amino acid substitutions, namely M252Y, S254T and T, increase the half-life in It is modified with 256E (sometimes called the "YTE" mutation). In certain embodiments, the Fc variants comprise a combination of the various mutations described herein. For example, LALA and YTE mutations, or LALADS and YTE mutations. Partial or complete Fc variants containing one or more substitutions, deletions or additions fused to the Fc variant. The full-length human IL-2 molecule can form homodimers via the Fc variant. The dimer contains two IL-2 mutant molecules and is therefore a bivalent IL-2 mutein. Figures 9A and 9C show non-limiting schematic diagrams of bivalent IL-2 muteins.

[0077] In other embodiments, the pair of Fc variants comprises one Fc region containing a knob mutation ( The Fc region includes a first Fc region that contains a hole mutation (the "Fc knob") and another Fc region that contains a hole mutation (the "Fc hole"). The c knob and Fc hole can form heterodimers (known as “knob-in-hole” mutations). In certain embodiments, the Fc region contains two amino acid substitutions, modified with S354C and T366W (sometimes referred to as "knob" mutations) In another embodiment, the Fc region contains four amino acid substitutions, namely Y349 C, T366S, L368A and Y407V (sometimes called "hole" mutations) In certain embodiments, the Fc region is modified with a "hole" mutation. It is modified with four amino acid substitutions, designated as AA, ... Further containing H435R and Y435F. A target or full-length human IL-2 molecule can be fused to an Fc knob or an Fc hole. In one embodiment, a partial polypeptide comprising one or more substitutions, deletions or additions fused to an Fc knob is provided. The intact or full-length human IL-2 molecule is paired with an Fc hole to form a monovalent IL-2 mutein. In a specific embodiment, one or more substitutions, deletions or deletions are formed in the Fc hole fused thereto. Partial or full-length human IL-2 molecules containing Fc knobs or additions are paired with the Fc knob to form monovalent 9B and 9D show non-limiting examples of monovalent IL-2 muteins. In another embodiment, a partial sequence containing one or more substitutions, deletions or additions is provided. The intact or full-length human IL-2 molecule is fused to an Fc that has no knob or hole, and c The fusion IL-2 molecule may form a homodimeric bivalent IL-2 mutein (e.g., a bivalent IL For non-limiting examples of -2 muteins, see Figures 9A and 9C).

[0078] Partial or full-length human IL-2 molecules containing one or more substitutions, deletions or additions are The N-terminus of the Fc variant is linked via various peptide linkers between the L-2 molecule and the Fc variant. or C-terminally. In one embodiment, one or more substitutions, deletions or additions a partial or full-length human IL-2 molecule comprising: The peptide linker is located between the C-terminus of the IL-2 molecule and the N-terminus of the Fc variant. In one embodiment, a partial or full-length sequence containing one or more substitutions, deletions or additions. The human IL-2 molecule is fused to the C-terminus of the Fc variant, and the peptide linker is It lies between the C-terminus of the host and the N-terminus of the IL-2 molecule.

[0079] In certain embodiments, the amino acid sequence set forth in SEQ ID NO: 1, 2 or 40. and a first polypeptide comprising: D at position 19 of SEQ ID NO: 1, 2 or 40 is substituted with N, and P at position 33 of SEQ ID NO: 1, 2 or 40 is substituted with R In one embodiment, the IL-2 mutein is set forth in SEQ ID NO:1. a first polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:1, wherein D is replaced by N, and P at position 33 of SEQ ID NO: 1 is replaced by R. In an embodiment, the IL-2 mutein comprises the amino acid sequence set forth in SEQ ID NO:2. wherein D at position 19 of SEQ ID NO:2 is replaced with N; In yet another embodiment, P at position 33 of sequence number 2 is replaced with R. The -2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:40. wherein D at position 19 of SEQ ID NO:40 is replaced with N; and In the formula, P is replaced by R.

[0080] In some embodiments, the amino acid sequence set forth in SEQ ID NO: 1, 2 or 40 is The present invention provides an IL-2 mutein comprising a first polypeptide comprising a sequence selected from the group consisting of SEQ ID NOs: 1, 2 and 3, D at position 19 of SEQ ID NO: 1, 2 or 40 and E at position 67 of SEQ ID NO: 1, 2 or 40 are substituted with S. In one embodiment, the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:1. a first polypeptide comprising the amino acid sequence of SEQ ID NO:1, wherein D at position 19 of SEQ ID NO:1 is replaced with N; In another embodiment, E at position 67 of SEQ ID NO:1 is replaced with S. The IL-2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2. tide, wherein D at position 19 of SEQ ID NO:2 is replaced with N, and In yet another embodiment, the IL-2 mutein is a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:40, wherein D at position 19 of SEQ ID NO:40 is substituted with N, and E at position 67 of SEQ ID NO:40 is substituted with S It has been done.

[0081] In another embodiment, the amino acid sequence set forth in SEQ ID NO: 1, 2 or 40 is The present invention provides an IL-2 mutein comprising a first polypeptide comprising SEQ ID NO: 1, 2 or D at position 19 of SEQ ID NO: 1, 2 or 40 is substituted with N, and P at position 33 of SEQ ID NO: 1, 2 or 40 is substituted with R. and E at position 67 of SEQ ID NO: 1, 2 or 40 is replaced with S. In an embodiment, the IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:1. wherein D at position 19 of SEQ ID NO:1 is replaced with N; P at position 33 of SEQ ID NO:1 is replaced with R, and E at position 67 of SEQ ID NO:1 is replaced with S. In another embodiment, the IL-2 mutein is set forth in SEQ ID NO:2. wherein D at position 19 of SEQ ID NO:2 is N, and P at position 33 of SEQ ID NO:2 is replaced by R, and P at position 67 of SEQ ID NO:2 is replaced by is replaced with S. In yet another embodiment, the IL-2 mutein is A first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:40, D at position 19 of sequence number 40 is replaced with N, and P at position 33 of sequence number 40 is replaced with R. In this case, E at position 67 of SEQ ID NO:40 is replaced with S.

[0082] In yet another embodiment of the IL-2 mutein, the first polypeptide is selected from the group consisting of SEQ ID NOs: 1, 2, or substitution of V to A at position 68, substitution of N to R at position 70, or Q at position 73 to P.

[0083] In yet another embodiment of the IL-2 mutein, the first polypeptide has the following three positions: any two of the following substitutions: V to A at position 68 of SEQ ID NO: 1, 2 or 40 substitution, N to R at position 70 or Q to P at position 73.

[0084] In yet another embodiment of the IL-2 mutein, the first polypeptide has the sequence set forth in SEQ ID NO:1, 2 or 40 V to A substitution at position 68, N to R substitution at position 70, and Q at position 73 to P.

[0085] In one embodiment, the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:1. a first polypeptide comprising the sequence of SEQ ID NO:1, wherein D at position 19 of SEQ ID NO:1 is replaced with N; P at position 33 of SEQ ID NO:1 is substituted with R, and V at position 68 of SEQ ID NO:1 is substituted with A. In another embodiment, the IL-2 mutein is a IL-2 mutein having the amino acid sequence set forth in SEQ ID NO:1. a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, wherein position 19 of SEQ ID NO:1 D in SEQ ID NO:1 is replaced with N, P in position 33 in SEQ ID NO:1 is replaced with R, In yet another embodiment, N at position 70 of the IL-2 mutein is substituted with R. The nucleic acid comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, In this case, D at position 19 of SEQ ID NO:1 is replaced with N, and P at position 33 of SEQ ID NO:1 is replaced with R. In yet another embodiment, Q at position 73 of SEQ ID NO:1 is replaced with P. In the present invention, the IL-2 mutein comprises a first polypeptide having the amino acid sequence set forth in SEQ ID NO:1. and a polypeptide comprising: SEQ ID NO:1, in which D at position 19 is replaced by N; and SEQ ID NO:3, P at position 3 is replaced with R, V at position 68 of SEQ ID NO: 1 is replaced with A, No. 1, N at position 70 is substituted with R. In one embodiment, the IL-2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, wherein D at position 19 of SEQ ID NO:1 is replaced with N, and P at position 33 of SEQ ID NO:1 is replaced with R. V at position 68 of SEQ ID NO:1 is substituted with A, and Q at position 73 of SEQ ID NO:1 is substituted with P. In another embodiment, the IL-2 mutein is a IL-2 mutein having the amino acid sequence set forth in SEQ ID NO:1. a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, wherein position 19 of SEQ ID NO:1 D in SEQ ID NO:1 is replaced with N, P in position 33 in SEQ ID NO:1 is replaced with R, N at position 70 of SEQ ID NO: 1 is substituted with R, and Q at position 73 of SEQ ID NO: 1 is substituted with P. In yet another embodiment, the IL-2 mutein is an IL-2 mutein having the amino acid sequence set forth in SEQ ID NO:1. a first polypeptide comprising an amino acid sequence, wherein D at position 19 of SEQ ID NO:1 is replaced with N; P at position 33 of SEQ ID NO:1 is substituted with R, and V at position 68 of SEQ ID NO:1 is N at position 70 of SEQ ID NO:1 is replaced with R, and N at position 73 of SEQ ID NO:1 is replaced with Position Q is replaced with P.

[0086] In one embodiment, the IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:2. A first polypeptide comprising the sequence of SEQ ID NO:2, wherein D at position 19 of SEQ ID NO:2 is replaced with N. P at position 33 of SEQ ID NO:2 is substituted with R, and V at position 68 of SEQ ID NO:2 is substituted with A. In another embodiment, the IL-2 mutein is a IL-2 mutein having the amino acid sequence set forth in SEQ ID NO:2. a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, wherein position 19 of SEQ ID NO:2 D in SEQ ID NO:2 is replaced with N, P in position 33 in SEQ ID NO:2 is replaced with R, In yet another embodiment, N at position 70 of the IL-2 mutein is substituted with R. The nucleic acid comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, In SEQ ID NO:2, D at position 19 is replaced with N, and P at position 33 is replaced with R. In yet another embodiment, the Q at position 73 of SEQ ID NO:2 is replaced with a P. In the present invention, the IL-2 mutein comprises a first polypeptide having the amino acid sequence set forth in SEQ ID NO:2. and a polypeptide comprising the peptide of SEQ ID NO:2, in which D at position 19 is replaced with N, P at position 33 of SEQ ID NO: 2 is substituted with R, and V at position 68 of SEQ ID NO: 2 is substituted with A, In one embodiment, the N at position 70 of sequence number 2 is substituted with R. The nucleic acid comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, In SEQ ID NO:2, D at position 19 is replaced with N, and P at position 33 is replaced with R. V at position 68 of SEQ ID NO:2 is substituted with A, and Q at position 73 of SEQ ID NO:2 is In another embodiment, the IL-2 mutein is SEQ ID NO:2 wherein the first polypeptide comprises an amino acid sequence as set forth in SEQ ID NO:2. D at position 9 is replaced with N, and P at position 33 of SEQ ID NO: 2 is replaced with R, N at position 70 of SEQ ID NO:2 is substituted with R, and Q at position 73 of SEQ ID NO:2 is substituted with P In yet another embodiment, the IL-2 mutein is set forth in SEQ ID NO:2. wherein D at position 19 of SEQ ID NO:2 is N. P at position 33 of SEQ ID NO:2 is replaced with R, and P at position 68 of SEQ ID NO:2 is replaced with R V is replaced by A, N at position 70 of SEQ ID NO:2 is replaced by R, Q at position 73 is replaced with P.

[0087] In one embodiment, the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:40. a first polypeptide comprising the amino acid sequence of SEQ ID NO:40, wherein D at position 19 of SEQ ID NO:40 is replaced with N; At position 33 of SEQ ID NO:40, P is replaced with R, and at position 68 of SEQ ID NO:40, V is replaced with A. In another embodiment, the IL-2 mutein has SEQ ID NO: No. 40, wherein the first polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: D at position 19 of SEQ ID NO:40 is substituted with N, and P at position 33 of SEQ ID NO:40 is substituted with R. and N at position 70 of SEQ ID NO: 40 is substituted with R. The IL-2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:40. wherein D at position 19 of SEQ ID NO:40 is replaced with N; P at position 33 of SEQ ID NO:40 is substituted with R, and Q at position 73 of SEQ ID NO:40 is substituted with P. In yet another embodiment, the IL-2 mutein is set forth in SEQ ID NO:40. wherein D at position 19 of SEQ ID NO:40 is N, and P at position 33 of SEQ ID NO:40 is replaced by R; and V at position 8 is substituted with A, and N at position 70 of SEQ ID NO:40 is substituted with R. In this embodiment, the IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:40. A first polypeptide comprising a sequence, wherein D at position 19 of SEQ ID NO:40 is replaced with N. P at position 33 of SEQ ID NO:40 is replaced with R, and V at position 68 of SEQ ID NO:40 is replaced with A. and Q at position 73 of SEQ ID NO: 40 is replaced with P. In one embodiment, the IL-2 mutein comprises the amino acid sequence set forth in SEQ ID NO:40. a first polypeptide, wherein D at position 19 of SEQ ID NO:40 is replaced with N; P at position 33 of sequence number 40 is replaced with R, and N at position 70 of sequence number 40 is replaced with R. In yet another embodiment, Q at position 73 of SEQ ID NO: 40 is replaced with P. In the present invention, the IL-2 mutein comprises a first amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO:40. and a polypeptide comprising: P at position 33 of SEQ ID NO:40 is substituted with R, and V at position 68 of SEQ ID NO:40 is substituted with A. N at position 70 of SEQ ID NO:40 is substituted with R, and Q at position 73 of SEQ ID NO:40 is substituted with P has been replaced with.

[0088] In one embodiment, the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:1. a first polypeptide comprising the sequence of SEQ ID NO:1, wherein D at position 19 of SEQ ID NO:1 is replaced with N; In this case, E at position 67 of SEQ ID NO:1 is replaced with S, and V at position 68 of SEQ ID NO:1 is replaced with A. In another embodiment, the IL-2 mutein is a IL-2 mutein having the amino acid sequence set forth in SEQ ID NO:1. a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, wherein position 19 of SEQ ID NO:1 D in SEQ ID NO:1 is replaced with N, and E in position 67 of SEQ ID NO:1 is replaced with S, In yet another embodiment, N at position 70 of the IL-2 mutein is substituted with R. The nucleic acid comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, In this example, D at position 19 of SEQ ID NO:1 is replaced with N, and E at position 67 of SEQ ID NO:1 is replaced with S. In yet another embodiment, Q at position 73 of SEQ ID NO:1 is replaced with P. In the present invention, the IL-2 mutein comprises a first polypeptide having the amino acid sequence set forth in SEQ ID NO:1. and a polypeptide comprising the polypeptide of SEQ ID NO:1, in which D at position 19 of SEQ ID NO:1 is replaced with N, The E at position 67 of SEQ ID NO:1 is replaced with S, and the V at position 68 of SEQ ID NO:1 is replaced with A. In one embodiment, the N at position 70 of sequence number 1 is substituted with R. The nucleic acid comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, In this example, D at position 19 of SEQ ID NO:1 is replaced with N, and E at position 67 of SEQ ID NO:1 is replaced with S. V at position 68 of SEQ ID NO:1 is substituted with A, and Q at position 73 of SEQ ID NO:1 is In another embodiment, the IL-2 mutein is SEQ ID NO: 1 wherein the first polypeptide comprises an amino acid sequence as set forth in SEQ ID NO:1. D at position 9 is replaced with N, and E at position 67 of SEQ ID NO: 1 is replaced with N, N at position 70 of SEQ ID NO:1 is substituted with R, and Q at position 73 of SEQ ID NO:1 is substituted with P In yet another embodiment, the IL-2 mutein is set forth in SEQ ID NO:1. wherein D at position 19 of SEQ ID NO:1 is N. The E at position 67 of SEQ ID NO:1 is replaced with S, and the E at position 68 of SEQ ID NO:1 is replaced with S. V is replaced by A, N at position 70 of SEQ ID NO:1 is replaced by R, Q at position 73 is replaced with P.

[0089] In one embodiment, the IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:2. A first polypeptide comprising the sequence of SEQ ID NO:2, wherein D at position 19 of SEQ ID NO:2 is replaced with N. In this case, E at position 67 of SEQ ID NO:2 is replaced with S, and V at position 68 of SEQ ID NO:2 is replaced with A. In another embodiment, the IL-2 mutein is a IL-2 mutein having the amino acid sequence set forth in SEQ ID NO:2. a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, wherein position 19 of SEQ ID NO:2 D in SEQ ID NO:2 is replaced with N, and E in position 67 of SEQ ID NO:2 is replaced with S, In yet another embodiment, N at position 70 of the IL-2 mutein is substituted with R. The nucleic acid comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, In this example, D at position 19 of SEQ ID NO:2 is replaced with N, and E at position 67 of SEQ ID NO:2 is replaced with S. In yet another embodiment, the Q at position 73 of SEQ ID NO:2 is replaced with a P. In the present invention, the IL-2 mutein comprises a first polypeptide having the amino acid sequence set forth in SEQ ID NO:2. and a polypeptide comprising the peptide of SEQ ID NO:2, in which D at position 19 is replaced with N, The E at position 67 of SEQ ID NO: 2 is replaced with S, and the V at position 68 of SEQ ID NO: 2 is replaced with A. In one embodiment, the N at position 70 of sequence number 2 is substituted with R. The nucleic acid comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, In this example, D at position 19 of SEQ ID NO:2 is replaced with N, and E at position 67 of SEQ ID NO:2 is replaced with S. V at position 68 of SEQ ID NO:2 is substituted with A, and Q at position 73 of SEQ ID NO:2 is In another embodiment, the IL-2 mutein is SEQ ID NO:2 wherein the first polypeptide comprises an amino acid sequence as set forth in SEQ ID NO:2. D at position 9 is replaced with A, and E at position 67 of SEQ ID NO:2 is replaced with S, N at position 70 of SEQ ID NO:2 is substituted with R, and Q at position 73 of SEQ ID NO:2 is substituted with P In yet another embodiment, the IL-2 mutein is set forth in SEQ ID NO:2. wherein D at position 19 of SEQ ID NO:2 is N. The E at position 67 of SEQ ID NO:2 is replaced with S, and the E at position 68 of SEQ ID NO:2 is replaced with S. V is replaced by A, N at position 70 of SEQ ID NO:2 is replaced by R, Q at position 73 is replaced with P.

[0090] In one embodiment, the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:40. a first polypeptide comprising the amino acid sequence of SEQ ID NO:40, wherein D at position 19 of SEQ ID NO:40 is replaced with N; The E at position 67 of SEQ ID NO:40 is replaced with S, and the V is replaced with A. In another embodiment, the IL-2 mutein has SEQ ID NO: No. 40, wherein the first polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: D at position 19 of SEQ ID NO:40 is substituted with N, and E at position 67 of SEQ ID NO:40 is substituted with S. and N at position 70 of SEQ ID NO: 40 is substituted with R. The IL-2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:40. wherein D at position 19 of SEQ ID NO:40 is replaced with N; E at position 67 of SEQ ID NO:40 is substituted with S, and Q at position 73 of SEQ ID NO:40 is substituted with P. In yet another embodiment, the IL-2 mutein is set forth in SEQ ID NO:40. wherein D at position 19 of SEQ ID NO:40 is N, and E at position 67 of SEQ ID NO:40 is replaced by S, and V at position 8 is substituted with A, and N at position 70 of SEQ ID NO:40 is substituted with R. In this embodiment, the IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:40. A first polypeptide comprising a sequence, wherein D at position 19 of SEQ ID NO:40 is replaced with N. and E at position 67 of SEQ ID NO:40 is replaced with S, and V at position 68 of SEQ ID NO:40 is replaced with A. and Q at position 73 of SEQ ID NO: 40 is replaced with P. In one embodiment, the IL-2 mutein comprises the amino acid sequence set forth in SEQ ID NO:40. a first polypeptide, wherein D at position 19 of SEQ ID NO:40 is replaced with N; E at position 67 of sequence number 40 is replaced with S, and N at position 70 of sequence number 40 is replaced with R. In yet another embodiment, Q at position 73 of SEQ ID NO: 40 is replaced with P. In the present invention, the IL-2 mutein comprises a first amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO:40. and a polypeptide comprising: E at position 67 of SEQ ID NO:40 is substituted with S, and V at position 68 of SEQ ID NO:40 is substituted with A. N at position 70 of SEQ ID NO:40 is substituted with R, and Q at position 73 of SEQ ID NO:40 is substituted with P has been replaced with.

[0091] In one embodiment, the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:1. a first polypeptide comprising the sequence of SEQ ID NO:1, wherein D at position 19 of SEQ ID NO:1 is replaced with N; P at position 33 of SEQ ID NO:1 is replaced with R, and E at position 67 of SEQ ID NO:1 is replaced with S. In another embodiment, the V at position 68 of SEQ ID NO:1 is replaced with A. In the present invention, the IL-2 mutein comprises a first polypeptide having the amino acid sequence set forth in SEQ ID NO:1. and a polypeptide comprising the polypeptide of SEQ ID NO:1, in which D at position 19 of SEQ ID NO:1 is replaced with N, P at position 33 of SEQ ID NO: 1 is replaced with R, and E at position 67 of SEQ ID NO: 1 is replaced with S, In yet another embodiment, N at position 70 of sequence number 1 is replaced with R. The -2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1. wherein D at position 19 of SEQ ID NO:1 is replaced with N, and P at position 33 of SEQ ID NO:1 is replaced with R, E at position 67 of SEQ ID NO:1 is replaced by S, and In yet another embodiment, the IL-2 mutein is a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, D at position 19 of sequence number 1 is replaced with N, and P at position 33 of sequence number 1 is replaced with R. and E at position 67 of SEQ ID NO:1 is replaced with S, and V at position 68 of SEQ ID NO:1 is replaced with A. In one embodiment, N at position 70 of SEQ ID NO:1 is replaced with R. The IL-2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1. tide, in which D at position 19 is replaced with N and P at position 33 of SEQ ID NO:1 is replaced with R. The E at position 67 of SEQ ID NO:1 is replaced with S, and the E at position 68 of SEQ ID NO:1 is replaced with S. V is replaced by A, and Q at position 73 of SEQ ID NO: 1 is replaced by P. In an embodiment, the IL-2 mutein comprises the amino acid sequence set forth in SEQ ID NO:1. wherein D at position 19 of SEQ ID NO:1 is replaced with N; P at position 33 of sequence number 1 is replaced with R, and E at position 67 of sequence number 1 is replaced with S. N at position 70 of SEQ ID NO:1 is replaced with R, and Q at position 73 of SEQ ID NO:1 is replaced with P. In yet another embodiment, the IL-2 mutein is substituted with SEQ ID NO:1. a first polypeptide comprising the amino acid sequence set forth, wherein sequence 19 of SEQ ID NO:1 D at position 31 of SEQ ID NO: 1 is replaced with N, P at position 33 of SEQ ID NO: 1 is replaced with R, 1 is substituted with S for E at position 67, and SEQ ID NO: 1 is substituted with A for V at position 68; N at position 70 of SEQ ID NO:1 is substituted with R, and Q at position 73 of SEQ ID NO:1 is substituted with P. is.

[0092] In one embodiment, the IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:2. A first polypeptide comprising the sequence of SEQ ID NO:2, wherein D at position 19 of SEQ ID NO:2 is replaced with N. P at position 33 of SEQ ID NO:2 is replaced with R, and E at position 67 of SEQ ID NO:2 is replaced with S. In another embodiment, the V at position 68 of SEQ ID NO:2 is replaced with A. In the present invention, the IL-2 mutein comprises a first polypeptide having the amino acid sequence set forth in SEQ ID NO:2. and a polypeptide comprising the peptide of SEQ ID NO:2, in which D at position 19 is replaced with N, P at position 33 of SEQ ID NO: 2 is substituted with R, and E at position 67 of SEQ ID NO: 2 is substituted with R, In yet another embodiment, N at position 70 of sequence number 2 is replaced with R. The -2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2. wherein D at position 19 of SEQ ID NO:2 is replaced with N, and P at position 33 of SEQ ID NO:2 is replaced with and E at position 67 of SEQ ID NO:2 is replaced with S. and E at position 73 of SEQ ID NO:2 is replaced with R. In yet another embodiment, the IL-2 mutein is a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, D at position 19 of sequence number 2 is replaced with N, and P at position 33 of sequence number 2 is replaced with R. and E at position 67 of SEQ ID NO:2 is replaced with S, and V at position 68 of SEQ ID NO:2 is replaced with A. In one embodiment, N at position 70 of SEQ ID NO:2 is replaced with S. The IL-2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2. wherein D at position 19 of SEQ ID NO:2 is replaced with N, and P at position 67 of SEQ ID NO:2 is replaced with R, E at position 67 of SEQ ID NO:2 is replaced with S, V at position 68 of SEQ ID NO:2 is substituted with A, and Q at position 73 of SEQ ID NO:2 is substituted with P. In another embodiment, the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:2. a first polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein D at position 19 of SEQ ID NO:2 is replaced with N; P at position 33 of SEQ ID NO:2 is replaced with R, and E at position 67 of SEQ ID NO:2 is replaced with S. N at position 70 of SEQ ID NO:2 is replaced with R, and N at position 73 of SEQ ID NO:2 is replaced with wherein Q is replaced with P. In yet another embodiment, the IL-2 mutein is a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, wherein the sequence D at position 19 of SEQ ID NO:2 is replaced with N, and P at position 33 of SEQ ID NO:2 is replaced with R. In this case, E at position 67 of SEQ ID NO:2 is replaced with S, and V at position 68 of SEQ ID NO:2 is replaced with A. N at position 70 of SEQ ID NO:2 is substituted with R, and Q at position 73 of SEQ ID NO:2 is It is substituted with P.

[0093] In one embodiment, the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:40. a first polypeptide comprising the amino acid sequence of SEQ ID NO:40, wherein D at position 19 of SEQ ID NO:40 is replaced with N; P at position 33 of SEQ ID NO:40 is replaced with R, and E is replaced by S, and V at position 68 of SEQ ID NO: 40 is replaced by A. In an embodiment, the IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:40. wherein D at position 19 of SEQ ID NO:40 is replaced with N; In this case, P at position 33 of SEQ ID NO:40 is replaced with R, and E at position 67 of SEQ ID NO:40 is replaced with S. In yet another embodiment, the IL The -2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:40. wherein D at position 19 of SEQ ID NO:40 is replaced with N; and P is replaced with R, E at position 67 of SEQ ID NO: 40 is replaced with S, In yet another embodiment, Q at position 73 of IL-2.40 is substituted with P. The mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:40. wherein D at position 19 of SEQ ID NO:40 is replaced with N, and P at position 33 of SEQ ID NO:40 is replaced with is replaced by R, E at position 67 of SEQ ID NO: 40 is replaced by S, and V at position 68 is and N at position 70 of SEQ ID NO:40 is substituted with R. In the present invention, the IL-2 mutein is a nucleotide sequence comprising the amino acid sequence set forth in SEQ ID NO:40. 1 polypeptide, wherein D at position 19 of SEQ ID NO:40 is replaced with N, P at position 33 of SEQ ID NO:40 is substituted with R, and E at position 67 of SEQ ID NO:40 is substituted with S. V at position 68 of SEQ ID NO:40 is substituted with A, and Q at position 73 of SEQ ID NO:40 is In another embodiment, the IL-2 mutein is SEQ ID NO:4 0, wherein the first polypeptide comprises an amino acid sequence set forth in SEQ ID NO:40 D at position 19 of SEQ ID NO: 40 is substituted with N, and P at position 33 of SEQ ID NO: 40 is substituted with R; E at position 67 of SEQ ID NO:40 is substituted with S, and N at position 70 of SEQ ID NO:40 is substituted with R and Q at position 73 of SEQ ID NO: 40 is replaced with P. In the present invention, the IL-2 mutein is a nucleotide sequence comprising the amino acid sequence set forth in SEQ ID NO:40. 1 polypeptide, wherein D at position 19 of SEQ ID NO:40 is replaced with N, P at position 33 of SEQ ID NO:40 is substituted with R, and E at position 67 of SEQ ID NO:40 is substituted with S. V at position 68 of SEQ ID NO:40 is substituted with A, and N at position 70 of SEQ ID NO:40 is and Q at position 73 of SEQ ID NO:40 is substituted with P.

[0094] In one embodiment, the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:1. a first polypeptide comprising the sequence of SEQ ID NO:1, wherein D at position 19 of SEQ ID NO:1 is replaced with N; V at position 68 of SEQ ID NO:1 is substituted with A, and Q at position 73 of SEQ ID NO:1 is substituted with P. In one embodiment, the IL-2 mutein is set forth in SEQ ID NO:2. a first polypeptide comprising an amino acid sequence as set forth in claim 1, wherein D at position 19 of SEQ ID NO:2 is is substituted with N, V ​​at position 68 of SEQ ID NO:2 is substituted with A, and Q at position 3 is replaced with P. In one embodiment, the IL-2 mutein has the sequence 40, wherein the first polypeptide comprises an amino acid sequence set forth in SEQ ID NO: D at position 19 of SEQ ID NO:40 is substituted with N, and V at position 68 of SEQ ID NO:40 is substituted with A. and Q at position 73 of SEQ ID NO: 40 is substituted with P. In certain embodiments, I The L-2 mutein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. In one embodiment, the first polypeptide comprises an amino acid sequence as defined above. The L-2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:3. In another embodiment, the IL-2 mutein is set forth in SEQ ID NO:4. In yet another embodiment, the first polypeptide comprises an amino acid sequence comprising: The L-2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:5. In yet another embodiment, the IL-2 mutein is set forth in SEQ ID NO:6. In yet another embodiment, the first polypeptide comprises an amino acid sequence The IL-2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:7. In one embodiment, the IL-2 mutein is set forth in SEQ ID NO:8. In another embodiment, the first polypeptide comprises an amino acid sequence The mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:9. In yet another embodiment, the IL-2 mutein is set forth in SEQ ID NO: 10. In yet another embodiment, the first polypeptide comprises an amino acid sequence comprising: The IL-2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:11. In yet another embodiment, the IL-2 mutein comprises the sequence set forth in SEQ ID NO: 12. The first polypeptide comprises an amino acid sequence as set forth in the above.

[0095] In some embodiments of the IL-2 mutein, the first polypeptide is SEQ ID NO: 13. 14 or 15. IL-2 muteins are selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. The amino acid sequence set forth in SEQ ID NO: 13, including the amino acid sequence set forth in SEQ ID NO: 13 In another embodiment, the first polypeptide further comprises an IL-2 mutein. is an amine as set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. The first polypeptide further comprises the amino acid sequence set forth in SEQ ID NO:14. In yet another embodiment, the IL-2 mutein comprises a peptide having the sequence set forth in SEQ ID NO:3. , 4, 5, 6, 7, 8, 9, 10, 11 or 12. and a first polypeptide further comprising the amino acid sequence set forth in SEQ ID NO:15. .

[0096] In one embodiment, the IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:3. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:13. In another embodiment, the IL-2 mutein is an IL-2 mutein having the amino acid sequence set forth in SEQ ID NO:4. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:13. In yet another embodiment, the IL-2 mutein is set forth in SEQ ID NO:5. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:13. In yet another embodiment, the IL-2 mutein comprises a peptide set forth in SEQ ID NO:6. and the amino acid sequence set forth in SEQ ID NO:13. In yet another embodiment, the IL-2 mutein comprises the sequence The amino acid sequence set forth in SEQ ID NO: 7 and the amino acid sequence set forth in SEQ ID NO: 13 In one embodiment, the IL-2 mutein comprises a first polypeptide comprising the sequence The amino acid sequence set forth in SEQ ID NO: 8 and the amino acid sequence set forth in SEQ ID NO: 13 In another embodiment, the IL-2 mutein comprises a first polypeptide comprising: The amino acid sequence shown in SEQ ID NO: 9 and the amino acid sequence shown in SEQ ID NO: 13 In yet another embodiment, the first polypeptide comprises an IL-2 mutein. The amino acid sequence of SEQ ID NO: 10 and the amino acid sequence of SEQ ID NO: 13 are In yet another embodiment, the first polypeptide comprises an amino acid sequence of I The L-2 mutein has the amino acid sequence set forth in SEQ ID NO:11 and the amino acid sequence set forth in SEQ ID NO:13. and a first polypeptide comprising an amino acid sequence as set forth in claim 1. In this case, the IL-2 mutein has the amino acid sequence set forth in SEQ ID NO: 12 and the amino acid sequence set forth in SEQ ID NO: 13.

[0097] In one embodiment, the IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:3. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:14. In another embodiment, the IL-2 mutein is an IL-2 mutein having the amino acid sequence set forth in SEQ ID NO:4. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:14. In yet another embodiment, the IL-2 mutein is set forth in SEQ ID NO:5. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:14. In yet another embodiment, the IL-2 mutein comprises a peptide set forth in SEQ ID NO:6. The amino acid sequence of SEQ ID NO: 14 is also included in the In yet another embodiment, the IL-2 mutein comprises the sequence The amino acid sequence set forth in SEQ ID NO: 7 and the amino acid sequence set forth in SEQ ID NO: 14 In one embodiment, the IL-2 mutein comprises a first polypeptide comprising the sequence The amino acid sequence set forth in SEQ ID NO: 8 and the amino acid sequence set forth in SEQ ID NO: 14 In another embodiment, the IL-2 mutein comprises a first polypeptide comprising: The amino acid sequence shown in SEQ ID NO: 9 and the amino acid sequence shown in SEQ ID NO: 14 In yet another embodiment, the first polypeptide comprises an IL-2 mutein. The amino acid sequence of SEQ ID NO: 10 and the amino acid sequence of SEQ ID NO: 14 are In yet another embodiment, the first polypeptide comprises an amino acid sequence of I The L-2 mutein has the amino acid sequence set forth in SEQ ID NO:11 and the amino acid sequence set forth in SEQ ID NO:14. and a first polypeptide comprising an amino acid sequence as set forth in claim 1. In this case, the IL-2 mutein has the amino acid sequence set forth in SEQ ID NO: 12 and the amino acid sequence set forth in SEQ ID NO: 14.

[0098] In one embodiment, the IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:3. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:15. In another embodiment, the IL-2 mutein is an IL-2 mutein having the amino acid sequence set forth in SEQ ID NO:4. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 15. In yet another embodiment, the IL-2 mutein is set forth in SEQ ID NO:5. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:15. In yet another embodiment, the IL-2 mutein comprises a peptide set forth in SEQ ID NO:6. The amino acid sequence of SEQ ID NO: 15 is also included in the In yet another embodiment, the IL-2 mutein comprises the sequence The amino acid sequence set forth in SEQ ID NO: 7 and the amino acid sequence set forth in SEQ ID NO: 15 In one embodiment, the IL-2 mutein comprises a first polypeptide comprising the sequence The amino acid sequence set forth in SEQ ID NO: 8 and the amino acid sequence set forth in SEQ ID NO: 15 In another embodiment, the IL-2 mutein comprises a first polypeptide comprising: The amino acid sequence shown in SEQ ID NO: 9 and the amino acid sequence shown in SEQ ID NO: 15 In yet another embodiment, the first polypeptide comprises an IL-2 mutein. The amino acid sequence of SEQ ID NO: 10 and the amino acid sequence of SEQ ID NO: 15 are In yet another embodiment, the first polypeptide comprises an amino acid sequence of I The L-2 mutein has the amino acid sequence set forth in SEQ ID NO:11 and the amino acid sequence set forth in SEQ ID NO:15. and a first polypeptide comprising an amino acid sequence as set forth in claim 1. In this case, the IL-2 mutein has the amino acid sequence set forth in SEQ ID NO: 12 and the amino acid sequence set forth in SEQ ID NO: 15. A first polypeptide comprising the amino acid sequence set forth in

[0099] In another embodiment of the IL-2 mutein, the first polypeptide is selected from the group consisting of SEQ ID NOs: 16, 4 1 or 42. In one embodiment, the first polypeptide further comprises a linker as described in The polypeptide further comprises a linker as set forth in SEQ ID NO: 16. In the above, the first polypeptide further comprises a linker as set forth in SEQ ID NO:41. In yet another embodiment, the first polypeptide is set forth in SEQ ID NO:42. In some embodiments, the nucleic acid sequence of SEQ ID NO: 3, 4, 5, 6, 7, 8, or 9 further comprises a linker. The first amino acid sequence as set forth in SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 15, and is fused to the N-terminus of a second amino acid sequence as set forth in SEQ ID NO: 16, The linker described in 41 or 42 is a C-terminus of the first amino acid sequence and a C-terminus of the second amino acid sequence. In another embodiment, the sequence is located between the N-terminus of SEQ ID NO: 3, 4, 5, 6 The first amino acid sequence set forth in any one of claims 1 to 4 is SEQ ID NO: 1 3, 14 or 15, and The linker is located between the C-terminus of the second amino acid sequence and the N-terminus of the first amino acid sequence.

[0100] In one embodiment, the IL-2 mutein is selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 and further the amino acid sequence set forth in SEQ ID NO: 13. and a linker as set forth in SEQ ID NO:16. In another embodiment, the IL-2 mutein comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, , 6, 7, 8, 9, 10, 11 or 12, and further comprising the amino acid sequence set forth in SEQ ID NO: No. 14 and a linker as set forth in SEQ ID NO: 16. In yet another embodiment, the IL-2 mutein comprises a first polypeptide comprising Amino acids set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 The sequence, further comprising the amino acid sequence set forth in SEQ ID NO:15 and the amino acid sequence set forth in SEQ ID NO:16. In one embodiment, the first polypeptide comprises an IL-2 polypeptide having a linker. Tein has the amino acid sequence set forth in SEQ ID NO:7 and further set forth in SEQ ID NO:13. and a linker as set forth in SEQ ID NO:16. In another embodiment, the IL-2 mutein comprises the sequence set forth in SEQ ID NO:8. The amino acid sequence depicted in SEQ ID NO: 13 and the amino acid sequence depicted in SEQ ID NO: 16 and a linker as described in The IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:7 and further has the amino acid sequence set forth in SEQ ID NO:14 and a linker as set forth in SEQ ID NO:16. In another embodiment, the IL-2 mutein comprises a polypeptide of SEQ ID NO:8. and the amino acid sequence set forth in SEQ ID NO: 14. and a linker as set forth in SEQ ID NO: 16. In one embodiment, the IL-2 mutein comprises the amino acid sequence set forth in SEQ ID NO:7, and further comprises The amino acid sequence set forth in SEQ ID NO: 15 and the linker set forth in SEQ ID NO: 16 In another embodiment, the IL-2 mutein comprises a first polypeptide comprising The amino acid sequence set forth in SEQ ID NO:8 and the amino acid sequence set forth in SEQ ID NO:15 are The present invention includes a first polypeptide comprising the amino acid sequence and a linker set forth in SEQ ID NO:16. In some embodiments, the IL-2 amino acid sequence is an Fc or Fc variant amino acid sequence. The peptide linker is fused to the N-terminus of IL-2 and the C-terminus of Fc or Fc variants. In some embodiments, the Fc or Fc variant amino acid sequence is located between the N-terminus of the amino acid sequence. The amino acid sequence is fused to the N-terminus of the IL-2 amino acid sequence, and the peptide linker is Fc or is located between the C-terminus of the Fc variant and the N-terminus of the IL-2 amino acid sequence.

[0101] In one embodiment, the IL-2 mutein is selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 and further the amino acid sequence set forth in SEQ ID NO: 13. and a linker as set forth in SEQ ID NO:41. In another embodiment, the IL-2 mutein comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, , 6, 7, 8, 9, 10, 11 or 12, and further comprising the amino acid sequence set forth in SEQ ID NO: No. 14 and a linker as set forth in SEQ ID NO: 41. In yet another embodiment, the IL-2 mutein comprises a first polypeptide comprising Amino acids set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 The sequence, further comprising the amino acid sequence set forth in SEQ ID NO: 15 and the sequence set forth in SEQ ID NO: 41. In one embodiment, the first polypeptide comprises an IL-2 polypeptide having a linker. Tein has the amino acid sequence set forth in SEQ ID NO:7 and further set forth in SEQ ID NO:13. and a linker as set forth in SEQ ID NO:41. In another embodiment, the IL-2 mutein comprises the sequence set forth in SEQ ID NO:8. The amino acid sequence depicted in SEQ ID NO: 13 and the amino acid sequence depicted in SEQ ID NO: 41 and a linker as described in The IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:7 and further has the amino acid sequence set forth in SEQ ID NO:14 and a linker as set forth in SEQ ID NO:41. In another embodiment, the IL-2 mutein comprises a polypeptide of SEQ ID NO:8. and the amino acid sequence set forth in SEQ ID NO: 14. and a linker as set forth in SEQ ID NO:41. In one embodiment, the IL-2 mutein comprises the amino acid sequence set forth in SEQ ID NO:7, and further comprises The amino acid sequence set forth in SEQ ID NO: 15 and the linker set forth in SEQ ID NO: 41 In another embodiment, the IL-2 mutein comprises a first polypeptide comprising The amino acid sequence set forth in SEQ ID NO:8 and the amino acid sequence set forth in SEQ ID NO:15 are The present invention includes a first polypeptide comprising the amino acid sequence and a linker set forth in SEQ ID NO:41. In some embodiments, the IL-2 amino acid sequence is an Fc or Fc variant amino acid sequence. The peptide linker is fused to the N-terminus of IL-2 and the C-terminus of Fc or Fc variants. In some embodiments, the Fc or Fc variant amino acid sequence is located between the N-terminus of the amino acid sequence. The amino acid sequence is fused to the N-terminus of the IL-2 amino acid sequence, and the peptide linker is Fc or is located between the C-terminus of the Fc variant and the N-terminus of the IL-2 amino acid sequence.

[0102] In one embodiment, the IL-2 mutein is selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 and further the amino acid sequence set forth in SEQ ID NO: 13. and a linker as set forth in SEQ ID NO:42. In another embodiment, the IL-2 mutein comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, , 6, 7, 8, 9, 10, 11 or 12, and further comprising the amino acid sequence set forth in SEQ ID NO: No. 14 and a linker as set forth in SEQ ID NO: 42. In yet another embodiment, the IL-2 mutein comprises a first polypeptide comprising Amino acids set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 The sequence, further comprising the amino acid sequence set forth in SEQ ID NO: 15 and the sequence set forth in SEQ ID NO: 42. In one embodiment, the first polypeptide comprises an IL-2 polypeptide having a linker. Tein has the amino acid sequence set forth in SEQ ID NO:7 and further set forth in SEQ ID NO:13. and a linker as set forth in SEQ ID NO:42. In another embodiment, the IL-2 mutein comprises the sequence set forth in SEQ ID NO:8. The amino acid sequence depicted in SEQ ID NO: 13 and the amino acid sequence depicted in SEQ ID NO: 42 and a linker as described in The IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:7 and further has the amino acid sequence set forth in SEQ ID NO:14 and a linker as set forth in SEQ ID NO:42. In another embodiment, the IL-2 mutein comprises a polypeptide of SEQ ID NO:8. and the amino acid sequence set forth in SEQ ID NO: 14. and a linker as set forth in SEQ ID NO: 42. In one embodiment, the IL-2 mutein comprises the amino acid sequence set forth in SEQ ID NO:7, and further comprises The amino acid sequence set forth in SEQ ID NO: 15 and the linker set forth in SEQ ID NO: 42 In another embodiment, the IL-2 mutein comprises a first polypeptide comprising The amino acid sequence set forth in SEQ ID NO:8 and the amino acid sequence set forth in SEQ ID NO:15 are The present invention includes a first polypeptide comprising the amino acid sequence and a linker set forth in SEQ ID NO:42. In some embodiments, the IL-2 amino acid sequence is an Fc or Fc variant amino acid sequence. The peptide linker is fused to the N-terminus of IL-2 and the C-terminus of Fc or Fc variants. In some embodiments, the Fc or Fc variant amino acid sequence is located between the N-terminus of the amino acid sequence. The amino acid sequence is fused to the N-terminus of the IL-2 amino acid sequence, and the peptide linker is Fc or is located between the C-terminus of the Fc variant and the N-terminus of the IL-2 amino acid sequence.

[0103] In various embodiments, the IL-2 mutein further comprises a second polypeptide. In this embodiment, the second polypeptide is the same as the first polypeptide. In another embodiment, the second polypeptide is different from the first polypeptide. In this case, the second polypeptide does not comprise a partial or full-length IL-2 amino acid sequence.

[0104] In one embodiment, the IL-2 mutein is selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 and the amino acid sequence set forth in SEQ ID NO: 13 a first polypeptide having an amino acid sequence identical to that of the first polypeptide; In another embodiment, the IL-2 mutein comprises the sequence of SEQ ID NO: 3, 4, 5 , 6, 7, 8, 9, 10, 11 or 12; the amino acid sequence set forth in SEQ ID NO: 13 The amino acid sequence set forth in SEQ ID NO: 16, 41 or 42 The nucleic acid comprises a first polypeptide having an anchor sequence and a second polypeptide having the same structure as the first polypeptide. In yet another embodiment, the IL-2 mutein is selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, , 7, 8, 9, 10, 11 or 12, the amino acid sequence set forth in SEQ ID NO: 13 The first peptide comprises the amino acid sequence set forth in SEQ ID NO:16 and the linker sequence set forth in SEQ ID NO:17. and a second polypeptide identical to the first polypeptide. In embodiments, the IL-2 mutein is selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, the amino acid sequence set forth in SEQ ID NO: 13 a first polypeptide comprising the sequence and a linker sequence set forth in SEQ ID NO:41; In yet another embodiment, the polypeptide comprises a second polypeptide identical to the I polypeptide. The L-2 mutein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. the amino acid sequence set forth in SEQ ID NO:13 and the amino acid sequence set forth in SEQ ID NO:4 2 and a first polypeptide having the same sequence as the first polypeptide. 2 polypeptide. In one embodiment, the IL-2 mutein comprises SEQ ID NO:3 the amino acid sequence set forth in SEQ ID NO: 13 and the amino acid sequence set forth in SEQ ID NO: A first polypeptide comprising a linker sequence as set forth in No. 16, and a first polypeptide and the same second polypeptide. In another embodiment, the IL-2 mutein comprises The amino acid sequence set forth in SEQ ID NO:4 and the amino acid sequence set forth in SEQ ID NO:13 and a linker sequence as set forth in SEQ ID NO: 16; In yet another embodiment, the peptide comprises a second polypeptide identical to the IL- The mutein has the amino acid sequence set forth in SEQ ID NO:5, the amino acid sequence set forth in SEQ ID NO:13, and a linker sequence as set forth in SEQ ID NO:16. In yet another embodiment, the method comprises the steps of: In this case, the IL-2 mutein is the amino acid sequence set forth in SEQ ID NO:6, SEQ ID NO:13 and a linker sequence as set forth in SEQ ID NO:16. The method includes the steps of: (a) determining whether a first polypeptide is identical to a second polypeptide; In one embodiment, the IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:7. No. 13 and the linker sequence described in SEQ ID NO: 16 and a second polypeptide that is the same as the first polypeptide. In one embodiment, the IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:8. The sequence of the amino acid sequence set forth in SEQ ID NO:13 and the linker sequence set forth in SEQ ID NO:16 The nucleic acid comprises a first polypeptide having an anchor sequence and a second polypeptide having the same structure as the first polypeptide. In yet another embodiment, the IL-2 mutein is set forth in SEQ ID NO:9. the amino acid sequence depicted in SEQ ID NO:13 and the amino acid sequence depicted in SEQ ID NO:16 and a second polypeptide identical to the first polypeptide, the first polypeptide including a linker sequence as described above. In yet another embodiment, the IL-2 mutein comprises a peptide having the sequence represented by SEQ ID NO: 10, the amino acid sequence set forth in SEQ ID NO: 13, and A first polypeptide comprising a linker sequence as set forth in SEQ ID NO: 16, and and a second polypeptide identical to the mutein. In one embodiment, the IL-2 mutein comprises The amino acid sequence shown in SEQ ID NO:11 and the amino acid sequence shown in SEQ ID NO:13 and a linker sequence as set forth in SEQ ID NO: 16; In another embodiment, the IL-2 polypeptide is a polypeptide of the present invention. The mutein has the amino acid sequence set forth in SEQ ID NO:12, SEQ ID NO:13, and a linker sequence as set forth in SEQ ID NO:16. and a second polypeptide that is the same as the first polypeptide. wherein the IL-2 amino acid sequence is fused to the N-terminus of the Fc or Fc variant amino acid sequence; The peptide linker is located between the C-terminus of IL-2 and the N-terminus of the Fc or Fc variant. In some embodiments, the Fc or Fc variant amino acid sequence comprises the IL-2 amino acid sequence. The peptide linker is fused to the N-terminus of the Fc or Fc variant and to the C-terminus of the IL-1 -2 amino acid sequence and the N-terminus of the

[0105] In one embodiment, the IL-2 mutein is selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 and the amino acid sequence set forth in SEQ ID NO: 14 a first polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:17; and a second polypeptide comprising the sequence. The sequences are set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. The amino acid sequence of SEQ ID NO: 14 and the amino acid sequence of SEQ ID NO: 16, 41 or 42 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:17. and a second polypeptide comprising an amino acid sequence that is For example, the IL-2 mutein is selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. 2, the amino acid sequence set forth in SEQ ID NO: 14, and the amino acid sequence set forth in SEQ ID NO: A first polypeptide comprising a linker sequence as set forth in sequence number 16 and a second polypeptide comprising a linker sequence as set forth in sequence number 17. and a second polypeptide comprising the amino acid sequence set forth in claim 1. In the present invention, the IL-2 mutein is selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, or The amino acid sequence described in SEQ ID NO:12, the amino acid sequence described in SEQ ID NO:14, and and a linker sequence as set forth in SEQ ID NO: 41; and a first polypeptide comprising the linker sequence as set forth in SEQ ID NO: 17. and a second polypeptide comprising the amino acid sequence set forth in claim 1. In certain embodiments, the IL-2 muteins are selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. or the amino acid sequence set forth in SEQ ID NO:12, the amino acid sequence set forth in SEQ ID NO:14 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO: 42, and a second polypeptide comprising a linker sequence as set forth in SEQ ID NO: and a second polypeptide comprising the amino acid sequence set forth in claim 17. In one embodiment, the IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:3. No. 14 and the linker sequence described in SEQ ID NO: 16 and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:17. In yet another embodiment, the IL-2 mutein comprises a peptide having the sequence represented by SEQ ID NO: 4, the amino acid sequence described in SEQ ID NO: 14, and the amino acid sequence described in SEQ ID NO: A first polypeptide comprising a linker sequence as set forth in sequence number 16 and a second polypeptide comprising a linker sequence as set forth in sequence number 17. and a second polypeptide comprising the amino acid sequence set forth in claim 1. The IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:14, The amino acid sequence set forth in SEQ ID NO:16 and the linker sequence set forth in SEQ ID NO:17 are included in the and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:17. In yet another embodiment, the IL-2 mutein comprises the sequence set forth in SEQ ID NO:6. the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:1 6 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:17. In another embodiment, the polypeptide comprises a first polypeptide comprising an amino acid sequence comprising: The L-2 mutein has the amino acid sequence set forth in SEQ ID NO:7, the amino acid sequence set forth in SEQ ID NO:14, and a linker sequence as set forth in SEQ ID NO:16. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:17. In yet another embodiment, the IL-2 mutein is set forth in SEQ ID NO:8. the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:16 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:17. and a second polypeptide comprising an amino acid sequence. Tein has the amino acid sequence shown in SEQ ID NO: 9 and the amino acid sequence shown in SEQ ID NO: 14. a first polypeptide comprising the amino acid sequence and a linker sequence set forth in SEQ ID NO:16; and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 17. In another embodiment, the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:10. the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:16 a first polypeptide comprising a linker sequence having an amino acid sequence as set forth in SEQ ID NO:17; and a second polypeptide comprising a sequence. The amino acid sequence shown in SEQ ID NO: 11 and the amino acid sequence shown in SEQ ID NO: 14 are a first polypeptide comprising the amino acid sequence and a linker sequence as set forth in SEQ ID NO: 16; and a second polypeptide comprising the amino acid sequence set forth in sequence number 17. In one embodiment, the IL-2 mutein comprises the amino acid sequence set forth in SEQ ID NO:12. Sequence, the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:16 A first polypeptide including a linker sequence and the amino acid sequence set forth in SEQ ID NO:17 In one embodiment of the first polypeptide, the first polypeptide comprises an IL- 2 amino acid sequence fused to the N-terminus of an Fc or Fc variant amino acid sequence, The linker is located between the C-terminus of IL-2 and the N-terminus of the Fc or Fc variant. In another embodiment of the polypeptide, the Fc or Fc variant amino acid sequence is The peptide linker is fused to the N-terminus of the -2 amino acid sequence of Fc or an Fc variant. It is located between the C-terminus and the N-terminus of the IL-2 amino acid sequence.

[0106] In one embodiment, the IL-2 mutein is selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 and the amino acid sequence set forth in SEQ ID NO: 14 a first polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:55; and a second polypeptide comprising the sequence. The sequences are set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. The amino acid sequence of SEQ ID NO: 14 and the amino acid sequence of SEQ ID NO: 16, 41 or 42 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:55. and a second polypeptide comprising an amino acid sequence that is For example, the IL-2 mutein is selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. 2, the amino acid sequence set forth in SEQ ID NO: 14, and the amino acid sequence set forth in SEQ ID NO: A first polypeptide comprising a linker sequence as set forth in sequence number 16 and a second polypeptide as set forth in SEQ ID NO:55. and a second polypeptide comprising the amino acid sequence set forth in claim 1. In the present invention, the IL-2 mutein is selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, or The amino acid sequence described in SEQ ID NO:12, the amino acid sequence described in SEQ ID NO:14, and and a linker sequence as set forth in SEQ ID NO: 41; and a first polypeptide comprising the linker sequence as set forth in SEQ ID NO: 55. and a second polypeptide comprising the amino acid sequence set forth in claim 1. In certain embodiments, the IL-2 muteins are selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. or the amino acid sequence set forth in SEQ ID NO:12, the amino acid sequence set forth in SEQ ID NO:14 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO: 42, and a second polypeptide comprising a linker sequence as set forth in SEQ ID NO: and a second polypeptide comprising the amino acid sequence set forth in claim 55. In one embodiment, the IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:3. No. 14 and the linker sequence described in SEQ ID NO: 16 and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. In yet another embodiment, the IL-2 mutein comprises a peptide having the sequence represented by SEQ ID NO: 4, the amino acid sequence described in SEQ ID NO: 14, and the amino acid sequence described in SEQ ID NO: A first polypeptide comprising a linker sequence as set forth in sequence number 16 and a second polypeptide as set forth in SEQ ID NO:55. and a second polypeptide comprising the amino acid sequence set forth in claim 1. The IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:14, The amino acid sequence set forth in SEQ ID NO:16 and the linker sequence set forth in SEQ ID NO:17 are included in the and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. In yet another embodiment, the IL-2 mutein comprises the sequence set forth in SEQ ID NO:6. the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:1 6 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:55. In another embodiment, the polypeptide comprises a first polypeptide comprising an amino acid sequence comprising: The L-2 mutein has the amino acid sequence set forth in SEQ ID NO:7, the amino acid sequence set forth in SEQ ID NO:14, and a linker sequence as set forth in SEQ ID NO:16. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. In yet another embodiment, the IL-2 mutein is set forth in SEQ ID NO:8. the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:16 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:55. and a second polypeptide comprising an amino acid sequence. Tein has the amino acid sequence shown in SEQ ID NO: 9 and the amino acid sequence shown in SEQ ID NO: 14. a first polypeptide comprising the amino acid sequence and a linker sequence set forth in SEQ ID NO:16; and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. In another embodiment, the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:10. the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:16 and a first polypeptide comprising a linker sequence comprising the amino acid sequence set forth in SEQ ID NO:55. and a second polypeptide comprising a sequence. The amino acid sequence shown in SEQ ID NO: 11 and the amino acid sequence shown in SEQ ID NO: 14 are a first polypeptide comprising the amino acid sequence and a linker sequence as set forth in SEQ ID NO: 16; and a second polypeptide comprising the amino acid sequence set forth in sequence number 55. In one embodiment, the IL-2 mutein comprises the amino acid sequence set forth in SEQ ID NO:12. Sequence, the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:16 A first polypeptide including a linker sequence and an amino acid sequence as set forth in SEQ ID NO:55. In one embodiment of the first polypeptide, the first polypeptide comprises an IL- 2 amino acid sequence fused to the N-terminus of an Fc or Fc variant amino acid sequence, The linker is located between the C-terminus of IL-2 and the N-terminus of the Fc or Fc variant. In another embodiment of the polypeptide, the Fc or Fc variant amino acid sequence is The peptide linker is fused to the N-terminus of the -2 amino acid sequence of Fc or an Fc variant. It is located between the C-terminus and the N-terminus of the IL-2 amino acid sequence.

[0107] In one embodiment, the IL-2 mutein is selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 and the amino acid sequence set forth in SEQ ID NO: 15 a first polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:18; and a second polypeptide comprising the sequence. The sequences are set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. The amino acid sequence of SEQ ID NO: 15 and the amino acid sequence of SEQ ID NO: 16, 41 or 42 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:18. and a second polypeptide comprising an amino acid sequence that is For example, the IL-2 mutein is selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. 2, the amino acid sequence described in SEQ ID NO: 15, and the amino acid sequence described in SEQ ID NO: A first polypeptide comprising a linker sequence as set forth in sequence number 16 and a second polypeptide comprising a linker sequence as set forth in sequence number 18. and a second polypeptide comprising the amino acid sequence set forth in claim 1. In the present invention, the IL-2 mutein is selected from SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, or The amino acid sequence described in SEQ ID NO:12, the amino acid sequence described in SEQ ID NO:15, and and a linker sequence as set forth in SEQ ID NO: 41; and a first polypeptide comprising the linker sequence as set forth in SEQ ID NO: 18. and a second polypeptide comprising the amino acid sequence set forth in claim 1. In certain embodiments, the IL-2 muteins are selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. or the amino acid sequence described in SEQ ID NO:12, the amino acid sequence described in SEQ ID NO:15 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO: 42, and a second polypeptide comprising a linker sequence as set forth in SEQ ID NO: and a second polypeptide comprising the amino acid sequence set forth in claim 18. In one embodiment, the IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:3. No. 15 and the linker sequence described in SEQ ID NO: 16 and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18. In yet another embodiment, the IL-2 mutein comprises a peptide having the sequence represented by SEQ ID NO: 4, the amino acid sequence described in SEQ ID NO: 15, and the amino acid sequence described in SEQ ID NO: A first polypeptide comprising a linker sequence as set forth in sequence number 16 and a second polypeptide as set forth in SEQ ID NO: 18. and a second polypeptide comprising the amino acid sequence set forth in claim 1. The IL-2 mutein has the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:15, The amino acid sequence set forth in SEQ ID NO:16 and the linker sequence set forth in SEQ ID NO:17 are included in the and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18. In yet another embodiment, the IL-2 mutein comprises the sequence set forth in SEQ ID NO:6. the amino acid sequence set forth in SEQ ID NO:15 and the amino acid sequence set forth in SEQ ID NO:1 6 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:18. In another embodiment, the polypeptide comprises a first polypeptide comprising an amino acid sequence comprising: The L-2 mutein has the amino acid sequence set forth in SEQ ID NO:7, the amino acid sequence set forth in SEQ ID NO:15, and a linker sequence as set forth in SEQ ID NO:16. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18. In yet another embodiment, the IL-2 mutein is set forth in SEQ ID NO:8. the amino acid sequence set forth in SEQ ID NO: 15 and the amino acid sequence set forth in SEQ ID NO: 16 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:18. and a second polypeptide comprising an amino acid sequence. Tein has the amino acid sequence shown in SEQ ID NO: 9 and the amino acid sequence shown in SEQ ID NO: 15. a first polypeptide comprising the amino acid sequence and a linker sequence set forth in SEQ ID NO:16; and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 18. In another embodiment, the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:10. the amino acid sequence set forth in SEQ ID NO:15 and the amino acid sequence set forth in SEQ ID NO:16 a first polypeptide comprising a linker sequence having an amino acid sequence as set forth in SEQ ID NO:18; and a second polypeptide comprising a sequence. The amino acid sequence shown in SEQ ID NO: 11 and the amino acid sequence shown in SEQ ID NO: 15 are a first polypeptide comprising the amino acid sequence and a linker sequence as set forth in SEQ ID NO: 16; and a second polypeptide comprising the amino acid sequence set forth in sequence number 18. In one embodiment, the IL-2 mutein comprises the amino acid sequence set forth in SEQ ID NO:12. Sequence, the amino acid sequence set forth in SEQ ID NO:15 and the amino acid sequence set forth in SEQ ID NO:16 A first polypeptide including a linker sequence and the amino acid sequence set forth in SEQ ID NO:18 In one embodiment of the first polypeptide, the first polypeptide comprises an IL- 2 amino acid sequence fused to the N-terminus of an Fc or Fc variant amino acid sequence, The linker is located between the C-terminus of IL-2 and the N-terminus of the Fc or Fc variant. In another embodiment of the polypeptide, the Fc or Fc variant amino acid sequence is The peptide linker is fused to the N-terminus of the -2 amino acid sequence of Fc or an Fc variant. It is located between the C-terminus and the N-terminus of the IL-2 amino acid sequence. The IL-2 mutein comprises a first polypeptide and a second polypeptide, wherein the second polypeptide The polypeptide is the same as the first polypeptide, wherein the first polypeptide and the second polypeptide Each of the peptides is represented by SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 52, 5 3 or 54. In one embodiment, the first polypeptide comprises an amino acid sequence as set forth in Each of the first and second polypeptides has the amino acid sequence set forth in SEQ ID NO:45. In one embodiment, the first and second polypeptides include Each comprises the amino acid sequence set forth in SEQ ID NO: 46. In the present invention, each of the first and second polypeptides is set forth in SEQ ID NO:47. In one embodiment, the first and second polypeptides comprise an amino acid sequence Each of the polypeptides comprises the amino acid sequence set forth in SEQ ID NO: 48. In an embodiment, each of the first and second polypeptides has the sequence of SEQ ID NO:4. 9. In one embodiment, the first polypeptide comprises the amino acid sequence set forth in and the second polypeptide each comprises the amino acid sequence set forth in SEQ ID NO:50. In one embodiment, each of the first and second polypeptides is In one embodiment, the first Each of the polypeptide and the second polypeptide is an amino acid sequence set forth in SEQ ID NO:52. In one embodiment, the first polypeptide and the second polypeptide include Each of the above comprises the amino acid sequence set forth in SEQ ID NO: 53. In this case, each of the first and second polypeptides is set forth in SEQ ID NO:54. The amino acid sequence of the present invention is

[0108] In some embodiments, the IL-2 mutein comprises a first polypeptide and a second polypeptide. wherein the first polypeptide comprises a peptide selected from SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28, The tide comprises the amino acid sequence set forth in SEQ ID NO: 17. The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:19, and the second polypeptide comprises The peptide comprises the amino acid sequence set forth in SEQ ID NO: 17. In the present invention, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:20, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:21. The polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17. In an embodiment, the first polypeptide has the amino acid sequence set forth in SEQ ID NO:21. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17. In another embodiment, the first polypeptide is an amino acid sequence set forth in SEQ ID NO:22. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:17. In yet another embodiment, the first polypeptide is set forth in SEQ ID NO:23. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:17. In one embodiment, the first polypeptide comprises the sequence set forth in SEQ ID NO:24. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:17. In another embodiment, the first polypeptide comprises the amino acid sequence of SEQ ID NO:25. The second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:17. In yet another embodiment, the first polypeptide comprises an amino acid sequence 26, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17. In yet another embodiment, the first polypeptide comprises the amino acid sequence set forth in The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:27, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: In yet another embodiment, the first The polypeptide comprises the amino acid sequence set forth in SEQ ID NO:28, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:17.

[0109] In some embodiments, the IL-2 mutein comprises a first polypeptide and a second polypeptide. wherein the first polypeptide comprises a peptide selected from SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28, The tide comprises the amino acid sequence set forth in SEQ ID NO: 55. The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:19, and the second polypeptide comprises The peptide comprises the amino acid sequence set forth in SEQ ID NO: 55. In the present invention, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:20, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:21. The polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 55. In an embodiment, the first polypeptide has the amino acid sequence set forth in SEQ ID NO:21. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:55. In another embodiment, the first polypeptide is an amino acid sequence set forth in SEQ ID NO:22. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:55. In yet another embodiment, the first polypeptide is set forth in SEQ ID NO:23. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:55. In one embodiment, the first polypeptide comprises the sequence set forth in SEQ ID NO:24. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:55. In another embodiment, the first polypeptide comprises the amino acid sequence of SEQ ID NO:25. The second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:55. In yet another embodiment, the first polypeptide comprises an amino acid sequence 26, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:55. In yet another embodiment, the first polypeptide comprises the amino acid sequence set forth in The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:27, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: In yet another embodiment, the first The polypeptide comprises the amino acid sequence set forth in SEQ ID NO:28, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:55.

[0110] In certain embodiments, the IL-2 mutein comprises a first polypeptide and a second polypeptide. wherein the first polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 4, 35, 36, 37 or 38, The domain comprises the amino acid sequence set forth in SEQ ID NO: 18. The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:29, and the second polypeptide comprises The peptide comprises the amino acid sequence set forth in SEQ ID NO: 18. In the present invention, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:30, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:30. The polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 18. In one embodiment, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:31. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 18. In one embodiment, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:32. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:18. In yet another embodiment, the first polypeptide is set forth in SEQ ID NO:33. and the second polypeptide comprises an amino acid sequence set forth in SEQ ID NO:18. In one embodiment, the first polypeptide comprises the sequence set forth in SEQ ID NO:34. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:18. In another embodiment, the first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:35. and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:18. In yet another embodiment, the first polypeptide comprises the amino acid sequence No. 36, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 18. In yet another embodiment, the first polypeptide comprises an amino acid sequence as set forth above. comprises the amino acid sequence set forth in SEQ ID NO:37, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 18. In yet another embodiment, the first polypeptide comprises the amino acid sequence set forth in The polypeptide comprises the amino acid sequence set forth in SEQ ID NO:38, and the second polypeptide comprises , comprising the amino acid sequence set forth in SEQ ID NO:18.

[0111] Methods for using IL-2 muteins

[0112] The present invention also relates to a method for preventing or treating an IL-2 mediated disease (e.g., an immune disease). , selective activation of regulatory T cells without activating CD8+ T cells, or IL IL-2 receptor α subunit expressing cells were not activated, and IL-2 receptor β subunit expressing cells were not activated. The present invention relates to a method for the selective activation of a cell, comprising the steps of: The present invention includes methods of using the various IL-2 muteins described herein.

[0113] Provided herein are methods for treating an IL-2 mediated disease in a subject. In one embodiment, the method comprises administering an IL-2 mutein provided herein to an IL-2 and administering to the subject a therapeutically effective amount to treat the vector-borne disease. Provided herein are methods for preventing IL-2 mediated disease in humans. In certain embodiments, In one embodiment, the method comprises administering an IL-2 mutein provided herein to a patient suffering from an IL-2 mediated disease. In some embodiments, the method comprises administering to the subject an effective amount of a compound effective to prevent the onset of the disease. In another embodiment, the subject has an IL-2 mediated disease. There is a risk of contracting sexually transmitted diseases.

[0114] In some embodiments of the various methods provided herein, IL-2-mediated Sexual disease is an immune disorder.

[0115] In certain embodiments, the immune disorder is an inflammatory disease. The inflammatory disease is uveitis.

[0116] In certain embodiments of the various methods provided herein, the immune disorder is an autoimmune disorder. Immune disorders, such as rheumatoid arthritis, Crohn's disease, psoriasis, psoriatic arthritis, multiple sclerosis, psoriasis, ankylosing spondylitis, type I diabetes, Sjogren's syndrome, ulcerative colitis, neuromyelitis optica, Celiac disease, scleroderma and temporal arteritis. is SLE, CLE or lupus nephritis. In one embodiment, the autoimmune disease In another embodiment, the autoimmune disease is Crohn's disease. In yet another embodiment, the autoimmune disease is psoriasis. In one embodiment, the autoimmune disease is multiple sclerosis. In yet another embodiment, the autoimmune disease is ankylosing spondylitis. In yet another embodiment, the autoimmune disease is Sjogren's disease. In another embodiment, the autoimmune disease is ulcerative colitis. In another embodiment, the autoimmune disease is neuromyelitis optica. In one embodiment, the autoimmune disease is celiac disease. In another embodiment, the autoimmune disease is scleroderma. In one embodiment, the autoimmune disease is lupus. In another embodiment, the autoimmune disease is SLE. In yet another embodiment, the autoimmune disease is lupus nephritis. do.

[0117] In other embodiments of the various methods provided herein, the immune disorder is hypersensitivity. Inflammatory diseases, such as atopic dermatitis, hypersensitivity vasculitis and allergies, such as allergic Asthma, allergic rhinitis (hay fever), urticaria (hives) and anaphylaxis In one embodiment, the immune disorder is atopic dermatitis. In yet another embodiment, the immune disorder is hypersensitivity vasculitis. The disorder is an allergy. In one embodiment, the immune disorder is allergic asthma. In another embodiment, the immune disorder is allergic rhinitis (hay fever). In yet another embodiment, the immune disorder is urticaria (hives). In some instances, the immune disorder is anaphylaxis.

[0118] In certain embodiments, the hypersensitivity disorder is a T cell hypersensitivity disorder. The term "T cell hypersensitivity" as used herein refers to transient or chronic abnormally high levels of T In certain embodiments, T cell effector function. Exemplary TH2 cytokines include IL-2, IL-3, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-21, IL-22, IL-23, IL-2 These include, but are not limited to, IL-4, IL-9, IL-13, IL-31, and TSLP. In one embodiment, the TH2 cytokine is IL-2. In some embodiments, the subject has transient or chronic abnormally high levels of 4, 5, 6, 7, 8, 9, or 10 different TH2 cytokines, One is IL-2.

[0119] In some embodiments, the immune disorder is alopecia areata, graft-versus-host disease (GVHD), The disease is autoimmune hepatitis, primary sclerosing cholangitis or inflammatory myopathy. In another embodiment, the immune disorder is alopecia areata. VHD. In yet another embodiment, the immune disease is autoimmune hepatitis. In yet another embodiment, the immune disorder is primary sclerosing cholangitis. In one embodiment, the immune disease is an inflammatory myopathy.

[0120] Thus, in some embodiments, a therapeutically effective amount of any of the compounds described herein may be used. Any one of the various IL-2 muteins or various pharmaceutical compositions described herein. and administering to the subject any one of the compounds of the present invention to treat an IL-2 mediated disease. A method is provided.

[0121] In certain embodiments, a therapeutically effective amount of the various IL-2 Any one of the muteins or any one of the various pharmaceutical compositions described herein. The present invention provides a method for treating an immune disorder in a subject, comprising administering to the subject one of the following:

[0122] In other embodiments, a therapeutically effective amount of the various IL-2 molecules described herein is administered. any one of the various pharmaceutical compositions described herein. The present invention provides a method for treating an autoimmune disease in a subject, comprising administering to the subject a

[0123] In yet other embodiments, a therapeutically effective amount of any of the various IL- 2 muteins or any of the various pharmaceutical compositions described herein. The present invention provides a method for treating an inflammatory disease in a subject comprising administering to the subject one of the following:

[0124] In certain embodiments, a therapeutically effective amount of the various IL-2 Any one of the muteins or any one of the various pharmaceutical compositions described herein. The present invention provides a method for treating GVHD in a subject, comprising administering to the subject one of the following:

[0125] In another specific embodiment, a therapeutically effective amount of any of the various compounds described herein is administered. or any one of the various pharmaceutical compositions described herein. A method of treating SLE in a subject comprising administering to the subject any one of the following is provided.

[0126] In yet another specific embodiment, a therapeutically effective amount of any of the compounds described herein. Any one of the various IL-2 muteins or various pharmaceutical compositions described herein. The present invention provides a method for treating CLE in a subject, comprising administering to the subject any one of the following: do.

[0127] In yet another specific embodiment, a therapeutically effective amount of any of the compounds described herein. Any one of the various IL-2 muteins or various pharmaceutical compositions described herein. and (c) administering to the subject any one of the compounds of the present invention to the subject. provide.

[0128] In yet another specific embodiment, a therapeutically effective amount of any of the compounds described herein. Any one of the various IL-2 muteins or various pharmaceutical compositions described herein. and administering to the subject any one of the compounds of the present invention. provide.

[0129] In yet another specific embodiment, a therapeutically effective amount of any of the compounds described herein. Any one of the various IL-2 muteins or various pharmaceutical compositions described herein. The present invention provides a method for treating Crohn's disease in a subject, comprising administering to the subject any one of the following: Provide.

[0130] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating GVHD in a subject, comprising administering to the subject an IL- 2 muteins are set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:14. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:17. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating GVHD in a subject, comprising administering to the subject an IL- 2 muteins are set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. the amino acid sequence shown in SEQ ID NO:14 and the amino acid sequence shown in SEQ ID NO:16 A first polypeptide comprising a linker sequence as set forth, and a second polypeptide as set forth in SEQ ID NO:17. and a second polypeptide comprising the amino acid sequence. A method for treating an IL-2 mutein infection in a subject, comprising administering to the subject an amount of an IL-2 mutein or a pharmaceutical composition thereof. A method of treating GVHD is provided, wherein the IL-2 mutein is set forth in SEQ ID NO:7. the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:16 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:17. and a second polypeptide comprising a nucleotide sequence of A method for treating GV in a subject, comprising administering to the subject an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating HD, wherein the IL-2 mutein is an agonist as set forth in SEQ ID NO:8. The amino acid sequence of SEQ ID NO:14 and the amino acid sequence of SEQ ID NO:16 a first polypeptide comprising a linker sequence having the amino acid sequence set forth in SEQ ID NO:17; and a second polypeptide comprising the sequence. A method for treating GVHD in a subject, comprising administering to the subject a -2 mutein or a pharmaceutical composition thereof. wherein the IL-2 mutein is SEQ ID NO: 19, 20, 21, 22, A first polypeptide comprising the amino acid sequence set forth in 23, 24, 25, 26, 27 or 28. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:17. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. and administering to the subject an IL-1 inhibitor, The -2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:23; and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 17. In an embodiment, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered to a subject. The present invention provides a method for treating GVHD in a subject, comprising administering to a subject an IL-2 mutant The polypeptide comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:24 and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: and a second polypeptide comprising the amino acid sequence set forth in claim 17.

[0131] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating GVHD in a subject, comprising administering to the subject an IL- 2 muteins are set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:14. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating GVHD in a subject, comprising administering to the subject an IL- 2 muteins are set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. the amino acid sequence shown in SEQ ID NO:14 and the amino acid sequence shown in SEQ ID NO:16 A first polypeptide comprising a linker sequence as set forth, and a second polypeptide as set forth in SEQ ID NO:55. and a second polypeptide comprising the amino acid sequence. A method for treating an IL-2 mutein infection in a subject, comprising administering to the subject an amount of an IL-2 mutein or a pharmaceutical composition thereof. A method of treating GVHD is provided, wherein the IL-2 mutein is set forth in SEQ ID NO:7. the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:16 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:55. and a second polypeptide comprising a nucleotide sequence of A method for treating GV in a subject, comprising administering to the subject an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating HD, wherein the IL-2 mutein is an agonist as set forth in SEQ ID NO:8. The amino acid sequence of SEQ ID NO:14 and the amino acid sequence of SEQ ID NO:16 a first polypeptide comprising a linker sequence having the amino acid sequence set forth in SEQ ID NO:55; and a second polypeptide comprising the sequence. A method for treating GVHD in a subject, comprising administering to the subject a -2 mutein or a pharmaceutical composition thereof. wherein the IL-2 mutein is SEQ ID NO: 19, 20, 21, 22, A first polypeptide comprising the amino acid sequence set forth in 23, 24, 25, 26, 27 or 28. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. and administering to the subject an IL-1 inhibitor, The -2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:23; and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. In an embodiment, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered to a subject. The present invention provides a method for treating GVHD in a subject, comprising administering to a subject an IL-2 mutant The polypeptide comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:24 and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: and a second polypeptide comprising the amino acid sequence set forth in claim 55.

[0132] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating GVHD in a subject, comprising administering to the subject an IL- 2 muteins are set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:15. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating GVHD in a subject, comprising administering to the subject an IL- 2 muteins are set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. the amino acid sequence shown in SEQ ID NO:15 and the amino acid sequence shown in SEQ ID NO:16 A first polypeptide comprising a linker sequence as set forth, and a second polypeptide as set forth in SEQ ID NO:18. and a second polypeptide comprising the amino acid sequence. A method for treating an IL-2 mutein infection in a subject, comprising administering to the subject an amount of an IL-2 mutein or a pharmaceutical composition thereof. A method of treating GVHD is provided, wherein the IL-2 mutein is set forth in SEQ ID NO:7. the amino acid sequence set forth in SEQ ID NO: 15 and the amino acid sequence set forth in SEQ ID NO: 16 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:18. and a second polypeptide comprising a nucleotide sequence of A method for treating GV in a subject, comprising administering to the subject an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating HD, wherein the IL-2 mutein is an agonist as set forth in SEQ ID NO:8. The amino acid sequence of SEQ ID NO:15 and the amino acid sequence of SEQ ID NO:16 a first polypeptide comprising a linker sequence having the amino acid sequence set forth in SEQ ID NO:18; and a second polypeptide comprising the sequence. A method for treating GVHD in a subject, comprising administering to the subject a -2 mutein or a pharmaceutical composition thereof. wherein the IL-2 mutein is SEQ ID NO: 29, 30, 31, 32, A first polypeptide comprising an amino acid sequence as set forth in any one of claims 33, 34, 35, 36, 37, or 38. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. and administering to the subject an IL-1 inhibitor, The -2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:33; and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 18. In an embodiment, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered to a subject. The present invention provides a method for treating GVHD in a subject, comprising administering to a subject an IL-2 mutant The polypeptide comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:34 and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: and a second polypeptide comprising the amino acid sequence set forth in claim 18.

[0133] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating GVHD in a subject, comprising administering to the subject an IL- 2 muteins are set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:13. In certain embodiments, the polypeptide comprises a first polypeptide and a second polypeptide that is identical to the first polypeptide. The method comprises administering to the subject a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. wherein the IL-2 mutein has the sequence Amino acid sequence set forth in numbers 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 13 and the amino acid sequence set forth in SEQ ID NO:16. a first polypeptide having a CAR sequence and an amino acid sequence identical to that of the first polypeptide; and a second polypeptide comprising an I amino acid sequence. A method for treating GVH in a subject, comprising administering to the subject an L-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating D, wherein the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:7. the amino acid sequence set forth in SEQ ID NO:13 and the amino acid sequence set forth in SEQ ID NO:16 a first polypeptide including a linker sequence and a second polypeptide having the same amino acid sequence as the first polypeptide. and a second polypeptide comprising an amino acid sequence that is The method comprises administering to the subject an effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating GVHD comprising administering to the patient an IL-2 mutein as set forth in SEQ ID NO:8. the amino acid sequence depicted in SEQ ID NO:13 and the amino acid sequence depicted in SEQ ID NO:16 a first polypeptide having a linker sequence as described above and a second polypeptide having the same amino acid sequence as the first polypeptide; and a second polypeptide comprising an amino acid sequence that is the sequence: administering to a subject a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method of treating GVHD in an elephant, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NO: 45, Amino acid sequences described in 46, 47, 48, 49, 50, 51, 52, 53, and 54 and a first polypeptide having an amino acid sequence having the same amino acid sequence as the first polypeptide. In certain embodiments, a therapeutically effective amount of IL-2 is administered to the subject. and administering to the subject a pharmaceutical composition thereof. The present invention provides a method for the preparation of an IL-2 mutein comprising administering to a mammalian subject the IL-2 mutein an amino acid sequence as set forth in SEQ ID NO:45. a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:45; and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:46. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or polypeptide is administered. The present invention provides a method for treating GVHD in a subject, comprising administering the pharmaceutical composition to the subject. wherein the IL-2 mutein is a nucleotide sequence comprising the amino acid sequence set forth in SEQ ID NO:46. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:46. Includes:

[0134] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating SLE in a subject, comprising administering to the subject IL-2 The muteins are set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:14. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:17. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered to The present invention provides a method for treating SLE in a subject, comprising administering to an elephant an IL-2 antibody. The tein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:16 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:17. and a second polypeptide comprising a nucleotide sequence of A method for treating IL-2 in a subject, comprising administering to the subject an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating E. coli, wherein the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:7. the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:16 a first polypeptide comprising a linker sequence having an amino acid sequence as set forth in SEQ ID NO:17; In certain embodiments, a therapeutically effective amount of an IL-1 polypeptide is administered. 2. A method for treating SLE in a subject, comprising administering to the subject a method ... The present invention provides a method of treating an IL-2 mutein comprising administering to a subject an IL-2 mutein having an amino acid sequence as set forth in SEQ ID NO:8. The sequence of the amino acid sequence set forth in SEQ ID NO:14 and the linker sequence set forth in SEQ ID NO:16 A first polypeptide comprising an anchor sequence and an amino acid sequence as set forth in SEQ ID NO:17. In certain embodiments, a therapeutically effective amount of an IL-2 mutein is administered. A method for treating SLE in a subject, comprising administering to the subject a compound or a pharmaceutical composition thereof. wherein the IL-2 muteins are SEQ ID NOs: 19, 20, 21, 22, 23, 24 25, 26, 27 or 28, and a first polypeptide comprising the amino acid sequence set forth in and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 17. In an embodiment, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered to a subject. The present invention provides a method for treating SLE in a subject, comprising administering to a subject an IL-2 mutein. a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:23 and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 and a second polypeptide comprising the amino acid sequence set forth in claim 7. The method comprises administering to a subject a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating SLE in a subject, comprising administering to said subject an IL-2 mutein having the sequence represented by SEQ ID NO: 24 and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:17. and a second polypeptide comprising an amino acid sequence corresponding to said first polypeptide.

[0135] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating SLE in a subject, comprising administering to the subject IL-2 The muteins are set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:14. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered to The present invention provides a method for treating SLE in a subject, comprising administering to an elephant an IL-2 antibody. The tein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:16 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:55. and a second polypeptide comprising a nucleotide sequence of A method for treating IL-2 in a subject, comprising administering to the subject an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating E. coli, wherein the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:7. the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:16 and a first polypeptide comprising a linker sequence comprising the amino acid sequence set forth in SEQ ID NO:55. In certain embodiments, a therapeutically effective amount of an IL-1 polypeptide is administered. 2. A method for treating SLE in a subject, comprising administering to the subject a method ... The present invention provides a method of treating an IL-2 mutein comprising administering to a subject an IL-2 mutein having an amino acid sequence as set forth in SEQ ID NO:8. The sequence of the amino acid sequence set forth in SEQ ID NO:14 and the linker sequence set forth in SEQ ID NO:16 A first polypeptide comprising an anchor sequence and an amino acid sequence as set forth in SEQ ID NO:55. In certain embodiments, a therapeutically effective amount of an IL-2 mutein is administered. A method for treating SLE in a subject, comprising administering to the subject a compound or a pharmaceutical composition thereof. wherein the IL-2 muteins are SEQ ID NOs: 19, 20, 21, 22, 23, 24 25, 26, 27 or 28, and a first polypeptide comprising the amino acid sequence set forth in and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. In an embodiment, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered to a subject. The present invention provides a method for treating SLE in a subject, comprising administering to a subject an IL-2 mutein. a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:23 and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:5 and a second polypeptide comprising the amino acid sequence set forth in claim 5. The method comprises administering to a subject a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating SLE in a subject, comprising administering to said subject an IL-2 mutein having the sequence represented by SEQ ID NO: 24 and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. and a second polypeptide comprising an amino acid sequence corresponding to said first polypeptide.

[0136] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating SLE in a subject, comprising administering to the subject IL-2 The muteins are set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:15. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered to The present invention provides a method for treating SLE in a subject, comprising administering to an elephant an IL-2 antibody. The tein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. the amino acid sequence set forth in SEQ ID NO: 15 and the amino acid sequence set forth in SEQ ID NO: 16 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:18. and a second polypeptide comprising a nucleotide sequence of A method for treating IL-2 in a subject, comprising administering to the subject an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating E. coli, wherein the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:7. the amino acid sequence set forth in SEQ ID NO:15 and the amino acid sequence set forth in SEQ ID NO:16 a first polypeptide comprising a linker sequence having an amino acid sequence as set forth in SEQ ID NO:18; In certain embodiments, a therapeutically effective amount of an IL-1 polypeptide is administered. 2. A method for treating SLE in a subject, comprising administering to the subject a method ... The present invention provides a method of treating an IL-2 mutein comprising administering to a subject an IL-2 mutein having an amino acid sequence as set forth in SEQ ID NO:8. The sequence of the amino acid sequence set forth in SEQ ID NO:15 and the linker sequence set forth in SEQ ID NO:16 A first polypeptide comprising an anchor sequence and an amino acid sequence as set forth in SEQ ID NO:18. In certain embodiments, a therapeutically effective amount of an IL-2 mutein is administered. A method for treating SLE in a subject, comprising administering to the subject a compound or a pharmaceutical composition thereof. wherein the IL-2 muteins are SEQ ID NOs: 29, 30, 31, 32, 33, 34 35, 36, 37 or 38; and and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 18. In an embodiment, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered to a subject. The present invention provides a method for treating SLE in a subject, comprising administering to a subject an IL-2 mutein. a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:33 and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 and a second polypeptide comprising the amino acid sequence set forth in claim 8. The method comprises administering to a subject a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating SLE in a subject, comprising administering to said subject an IL-2 mutein having the sequence represented by SEQ ID NO: 34 and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18. and a second polypeptide comprising an amino acid sequence corresponding to said first polypeptide.

[0137] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating SLE in a subject, comprising administering to the subject IL-2 The muteins are set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:13. In certain embodiments, the polypeptide comprises a first polypeptide and a second polypeptide that is the same as the first polypeptide. The method comprises administering to a subject a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating SLE in a subject, comprising administering to said subject an IL-2 mutein having the sequence represented by SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. The amino acid sequence set forth in column number 13 and the linker set forth in SEQ ID NO: 16 a first polypeptide having a sequence and a second polypeptide having an amino acid sequence that is the same amino acid sequence as the first polypeptide; In certain embodiments, a therapeutically effective amount of an IL-1 polypeptide is administered. 2. A method for treating SLE in a subject, comprising administering to the subject a method ... The present invention provides a method of treating an IL-2 mutein comprising administering to a subject an IL-2 mutein having an amino acid sequence as set forth in SEQ ID NO:7. The sequence of the amino acid sequence set forth in SEQ ID NO:13 and the linker sequence set forth in SEQ ID NO:16 A first polypeptide having an anchor sequence and an amino acid sequence identical to that of the first polypeptide. and a second polypeptide comprising a nucleotide sequence of A method for treating IL-2 in a subject, comprising administering to the subject an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating E. coli, wherein the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:8. the amino acid sequence set forth in SEQ ID NO:13 and the amino acid sequence set forth in SEQ ID NO:16 a first polypeptide including a linker sequence and a second polypeptide having the same amino acid sequence as the first polypeptide. and a second polypeptide comprising an amino acid sequence that is The method comprises administering to the subject an effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating SLE, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NOs: 45, 46, 47, , 48, 49, 50, 51, 52, 53, 54 a polypeptide and a second polypeptide that contains an amino acid sequence that is the same amino acid sequence as the first polypeptide. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or polypeptide is administered. and a method for treating SLE in a subject, comprising administering the pharmaceutical composition to the subject. wherein the IL-2 mutein comprises a first amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO:45. a polypeptide and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:45; In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating SLE in a subject, comprising administering to the subject a composition comprising: The L-2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:46. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:46.

[0138] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating CLE in a subject, comprising administering to the subject IL-2 The muteins are set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:14. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:17. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered to The present invention provides a method for treating CLE in a subject, comprising administering to an elephant a therapeutically effective amount of IL-2 The tein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:16 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:17. and a second polypeptide comprising a nucleotide sequence of A method for treating IL-2 in a subject, comprising administering to the subject an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating E. coli, wherein the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:7. the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:16 a first polypeptide comprising a linker sequence having an amino acid sequence as set forth in SEQ ID NO:17; In certain embodiments, a therapeutically effective amount of an IL-1 polypeptide is administered. 2. A method for treating CLE in a subject, comprising administering to the subject a mutein or a pharmaceutical composition thereof. The present invention provides a method of treating an IL-2 mutein comprising administering to a subject an IL-2 mutein having an amino acid sequence as set forth in SEQ ID NO:8. The sequence of the amino acid sequence set forth in SEQ ID NO:14 and the linker sequence set forth in SEQ ID NO:16 A first polypeptide comprising an anchor sequence and an amino acid sequence as set forth in SEQ ID NO:17. In certain embodiments, a therapeutically effective amount of an IL-2 mutein is administered. A method for treating CLE in a subject, comprising administering to the subject a compound or a pharmaceutical composition thereof. wherein the IL-2 muteins are SEQ ID NOs: 19, 20, 21, 22, 23, 24 25, 26, 27 or 28, and a first polypeptide comprising the amino acid sequence set forth in and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 17. In an embodiment, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered to a subject. The present invention provides a method for treating CLE in a subject, comprising administering to a subject an IL-2 mutein. a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:23 and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 and a second polypeptide comprising the amino acid sequence set forth in claim 7. The method comprises administering to a subject a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating CLE in a subject, comprising administering to said subject an IL-2 mutein having the sequence set forth in SEQ ID NO: 24 and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:17. and a second polypeptide comprising an amino acid sequence corresponding to said first polypeptide.

[0139] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating CLE in a subject, comprising administering to the subject IL-2 The muteins are set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:14. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered to The present invention provides a method for treating CLE in a subject, comprising administering to an elephant a therapeutically effective amount of IL-2 The tein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:16 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:55. and a second polypeptide comprising a nucleotide sequence of A method for treating IL-2 in a subject, comprising administering to the subject an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating E. coli, wherein the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:7. the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:16 and a first polypeptide comprising a linker sequence comprising the amino acid sequence set forth in SEQ ID NO:55. In certain embodiments, a therapeutically effective amount of an IL-1 polypeptide is administered. 2. A method for treating CLE in a subject, comprising administering to the subject a mutein or a pharmaceutical composition thereof. The present invention provides a method of treating an IL-2 mutein comprising administering to a subject an IL-2 mutein having an amino acid sequence as set forth in SEQ ID NO:8. The sequence of the amino acid sequence set forth in SEQ ID NO:14 and the linker sequence set forth in SEQ ID NO:16 A first polypeptide comprising an anchor sequence and an amino acid sequence as set forth in SEQ ID NO:55. In certain embodiments, a therapeutically effective amount of an IL-2 mutein is administered. A method for treating CLE in a subject, comprising administering to the subject a compound or a pharmaceutical composition thereof. wherein the IL-2 muteins are SEQ ID NOs: 19, 20, 21, 22, 23, 24 25, 26, 27 or 28, and a first polypeptide comprising the amino acid sequence set forth in and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. In an embodiment, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered to a subject. The present invention provides a method for treating CLE in a subject, comprising administering to a subject an IL-2 mutein. a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:23 and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:5 and a second polypeptide comprising the amino acid sequence set forth in claim 5. The method comprises administering to a subject a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating CLE in a subject, comprising administering to said subject an IL-2 mutein having the sequence set forth in SEQ ID NO: 24 and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. and a second polypeptide comprising an amino acid sequence corresponding to said first polypeptide.

[0140] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating CLE in a subject, comprising administering to the subject IL-2 The muteins are set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:15. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered to The present invention provides a method for treating CLE in a subject, comprising administering to an elephant a therapeutically effective amount of IL-2 The tein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. the amino acid sequence set forth in SEQ ID NO: 15 and the amino acid sequence set forth in SEQ ID NO: 16 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:18. and a second polypeptide comprising a nucleotide sequence of A method for treating IL-2 in a subject, comprising administering to the subject an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating E. coli, wherein the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:7. the amino acid sequence set forth in SEQ ID NO:15 and the amino acid sequence set forth in SEQ ID NO:16 a first polypeptide comprising a linker sequence having an amino acid sequence as set forth in SEQ ID NO:18; In certain embodiments, a therapeutically effective amount of an IL-1 polypeptide is administered. 2. A method for treating CLE in a subject, comprising administering to the subject a mutein or a pharmaceutical composition thereof. The present invention provides a method of treating an IL-2 mutein comprising administering to a subject an IL-2 mutein having an amino acid sequence as set forth in SEQ ID NO:8. The sequence of the amino acid sequence set forth in SEQ ID NO:15 and the linker sequence set forth in SEQ ID NO:16 A first polypeptide comprising an anchor sequence and an amino acid sequence as set forth in SEQ ID NO:18. In certain embodiments, a therapeutically effective amount of an IL-2 mutein is administered. A method for treating CLE in a subject, comprising administering to the subject a compound or a pharmaceutical composition thereof. wherein the IL-2 muteins are SEQ ID NOs: 29, 30, 31, 32, 33, 34 35, 36, 37 or 38; and and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 18. In an embodiment, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered to a subject. The present invention provides a method for treating CLE in a subject, comprising administering to a subject an IL-2 mutein. a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:33 and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1 and a second polypeptide comprising the amino acid sequence set forth in claim 8. The method comprises administering to a subject a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating CLE in a subject, comprising administering to said subject an IL-2 mutein having the sequence set forth in SEQ ID NO: 34 and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18. and a second polypeptide comprising an amino acid sequence corresponding to said first polypeptide.

[0141] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating CLE in a subject, comprising administering to the subject IL-2 The muteins are set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:13. In certain embodiments, the polypeptide comprises a first polypeptide and a second polypeptide that is the same as the first polypeptide. The method comprises administering to a subject a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating CLE in a subject, comprising administering to said subject an IL-2 mutein having the sequence set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. The amino acid sequence set forth in column number 13 and the linker set forth in SEQ ID NO: 16 a first polypeptide having a sequence and a second polypeptide having an amino acid sequence that is the same amino acid sequence as the first polypeptide; In certain embodiments, a therapeutically effective amount of an IL-1 polypeptide is administered. 2. A method for treating CLE in a subject, comprising administering to the subject a mutein or a pharmaceutical composition thereof. The present invention provides a method of treating an IL-2 mutein comprising administering to a subject an IL-2 mutein having an amino acid sequence as set forth in SEQ ID NO:7. The sequence of the amino acid sequence set forth in SEQ ID NO:13 and the linker sequence set forth in SEQ ID NO:16 A first polypeptide having an anchor sequence and an amino acid sequence identical to that of the first polypeptide. and a second polypeptide comprising a nucleotide sequence of A method for treating IL-2 in a subject, comprising administering to the subject an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating E. coli, wherein the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:8. the amino acid sequence set forth in SEQ ID NO:13 and the amino acid sequence set forth in SEQ ID NO:16 a first polypeptide including a linker sequence and a second polypeptide having the same amino acid sequence as the first polypeptide. and a second polypeptide comprising an amino acid sequence that is The method comprises administering to the subject an effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating CLE, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NOs: 45, 46, 47, , 48, 49, 50, 51, 52, 53, 54 a polypeptide and a second polypeptide that contains an amino acid sequence that is the same amino acid sequence as the first polypeptide. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or polypeptide is administered. and a method for treating CLE in a subject, comprising administering the pharmaceutical composition to the subject. wherein the IL-2 mutein comprises a first amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO:45. a polypeptide and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:45; In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating CLE in a subject, comprising administering to the subject a composition comprising: The L-2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:46. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:46.

[0142] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating multiple sclerosis in a subject, comprising administering to the subject a The L-2 mutein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:15. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:17. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof The present invention provides a method for treating multiple sclerosis in a subject, the method comprising administering to the subject a IL-2 muteins are selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. The amino acid sequence described in SEQ ID NO: 14 and the sequence number A first polypeptide comprising a linker sequence as set forth in SEQ ID NO:16 and a second polypeptide comprising a linker sequence as set forth in SEQ ID NO:17. and a second polypeptide comprising an amino acid sequence that is selected from the group consisting of ribozymes. A method for treating a patient comprising administering to the patient a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating multiple sclerosis in a patient in need thereof, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NO: 7 The amino acid sequence described in SEQ ID NO: 14 and the sequence number A first polypeptide comprising a linker sequence as set forth in SEQ ID NO:16 and a second polypeptide comprising a linker sequence as set forth in SEQ ID NO:17. and a second polypeptide comprising an amino acid sequence that is selected from the group consisting of ribozymes. A method for treating a patient comprising administering to the patient a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating multiple sclerosis in a patient in need thereof, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NO:8. The amino acid sequence described in SEQ ID NO: 14 and the sequence number A first polypeptide comprising a linker sequence as set forth in SEQ ID NO:16 and a second polypeptide comprising a linker sequence as set forth in SEQ ID NO:17. and a second polypeptide comprising an amino acid sequence that is selected from the group consisting of ribozymes. A method for treating a patient comprising administering to the patient a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating multiple sclerosis in a patient in need thereof, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NO: 19 , 20, 21, 22, 23, 24, 25, 26, 27 or 28 a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:17; In certain embodiments, a therapeutically effective amount of an IL-2 mutein is administered. or a pharmaceutical composition thereof. The present invention provides a method for the preparation of an IL-2 mutein comprising administering to a mammalian subject the present invention thereto an IL-2 mutein having the amino acid sequence set forth in SEQ ID NO:23. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:17. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or peptide is administered. The present invention provides a method for treating multiple sclerosis in a subject, comprising administering the pharmaceutical composition to the subject. wherein the IL-2 mutein comprises the amino acid sequence set forth in SEQ ID NO:24. A first polypeptide and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:17. This includes the.

[0143] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating multiple sclerosis in a subject, comprising administering to the subject a The L-2 mutein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:15. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof The present invention provides a method for treating multiple sclerosis in a subject, the method comprising administering to the subject a IL-2 muteins are selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. The amino acid sequence described in SEQ ID NO: 14 and the sequence number A first polypeptide comprising a linker sequence as set forth in SEQ ID NO: 55 and a second polypeptide comprising a linker sequence as set forth in SEQ ID NO: 56. and a second polypeptide comprising an amino acid sequence that is selected from the group consisting of ribozymes. A method for treating a patient comprising administering to the patient a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating multiple sclerosis in a patient in need thereof, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NO: 7 The amino acid sequence described in SEQ ID NO: 14 and the sequence number A first polypeptide comprising a linker sequence as set forth in SEQ ID NO: 55 and a second polypeptide comprising a linker sequence as set forth in SEQ ID NO: 56. and a second polypeptide comprising an amino acid sequence that is selected from the group consisting of ribozymes. A method for treating a patient comprising administering to the patient a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating multiple sclerosis in a patient in need thereof, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NO:8. The amino acid sequence described in SEQ ID NO: 14 and the sequence number A first polypeptide comprising a linker sequence as set forth in SEQ ID NO: 55 and a second polypeptide comprising a linker sequence as set forth in SEQ ID NO: 56. and a second polypeptide comprising an amino acid sequence that is selected from the group consisting of ribozymes. A method for treating a patient comprising administering to the patient a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating multiple sclerosis in a patient in need thereof, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NO: 19 , 20, 21, 22, 23, 24, 25, 26, 27 or 28 a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55; In certain embodiments, a therapeutically effective amount of an IL-2 mutein is administered. or a pharmaceutical composition thereof. The present invention provides a method for the preparation of an IL-2 mutein comprising administering to a mammalian subject the present invention thereto an IL-2 mutein having the amino acid sequence set forth in SEQ ID NO:23. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or peptide is administered. The present invention provides a method for treating multiple sclerosis in a subject, comprising administering the pharmaceutical composition to the subject. wherein the IL-2 mutein comprises the amino acid sequence set forth in SEQ ID NO:24. A first polypeptide and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. This includes the.

[0144] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating multiple sclerosis in a subject, comprising administering to the subject a The L-2 mutein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:15 and the amino acid sequence set forth in SEQ ID NO:16. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof The present invention provides a method for treating multiple sclerosis in a subject, the method comprising administering to the subject a IL-2 muteins are selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. The amino acid sequence described in SEQ ID NO: 15 and the sequence number A first polypeptide comprising a linker sequence as set forth in SEQ ID NO:16 and a second polypeptide comprising a linker sequence as set forth in SEQ ID NO:18. and a second polypeptide comprising an amino acid sequence that is selected from the group consisting of ribozymes. A method for treating a patient comprising administering to the patient a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating multiple sclerosis in a patient in need thereof, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NO: 7 The amino acid sequence described in SEQ ID NO: 15 and the sequence number A first polypeptide comprising a linker sequence as set forth in SEQ ID NO:16 and a second polypeptide comprising a linker sequence as set forth in SEQ ID NO:18. and a second polypeptide comprising an amino acid sequence that is selected from the group consisting of ribozymes. A method for treating a patient comprising administering to the patient a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating multiple sclerosis in a patient in need thereof, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NO:8. The amino acid sequence described in SEQ ID NO: 15 and the sequence number A first polypeptide comprising a linker sequence as set forth in SEQ ID NO:16 and a second polypeptide comprising a linker sequence as set forth in SEQ ID NO:18. and a second polypeptide comprising an amino acid sequence that is selected from the group consisting of ribozymes. A method for treating a patient comprising administering to the patient a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating multiple sclerosis in a patient in need thereof, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NO:29 , 30, 31, 32, 33, 34, 35, 36, 37 or 38 a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18; In certain embodiments, a therapeutically effective amount of an IL-2 mutein is administered. or a pharmaceutical composition thereof. The present invention provides a method for the preparation of an IL-2 mutein comprising administering to a mammalian subject the present invention thereto an IL-2 mutein having the amino acid sequence set forth in SEQ ID NO:33. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or peptide is administered. The present invention provides a method for treating multiple sclerosis in a subject, comprising administering the pharmaceutical composition to the subject. wherein the IL-2 mutein comprises the amino acid sequence set forth in SEQ ID NO:34. A first polypeptide and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18. This includes the.

[0145] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating multiple sclerosis in a subject, comprising administering to the subject a The L-2 mutein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:13 and the amino acid sequence set forth in SEQ ID NO:14. and a second polypeptide that is identical to the first polypeptide. In this embodiment, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered to the subject. The present invention provides a method for treating multiple sclerosis in a subject, comprising administering to a subject an IL-2 mutein. is an amine as set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. the amino acid sequence set forth in SEQ ID NO:13 and the amino acid sequence set forth in SEQ ID NO:16 a first polypeptide including a linker sequence and a second polypeptide having the same amino acid sequence as the first polypeptide. and a second polypeptide comprising an amino acid sequence that is The method comprises administering to the subject an effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating multiple sclerosis, wherein the IL-2 mutein is set forth in SEQ ID NO:7. the amino acid sequence depicted in SEQ ID NO:13 and SEQ ID NO:16 and a first polypeptide having the same amino acid sequence as the first polypeptide. and a second polypeptide comprising an amino acid sequence that is a nucleotide sequence of comprising administering to a subject a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method of treating multiple sclerosis in a subject, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NO: No. 8, the amino acid sequence set forth in SEQ ID NO: 13, and A first polypeptide comprising a linker sequence as set forth in SEQ ID NO: 16, and and a second polypeptide comprising an amino acid sequence that is the same amino acid sequence as the first polypeptide. In one embodiment, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered to a subject. The present invention provides a method for treating multiple sclerosis in a subject, comprising administering to a subject an IL-2 mutant The sequences are set forth in SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 52, 53, and 54. a first polypeptide having an amino acid sequence identical to that of the first polypeptide; and a second polypeptide comprising an amino acid sequence which is The method comprises administering to a subject a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating multiple sclerosis in a patient with multiple sclerosis, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NO: 45. a first polypeptide comprising the amino acid sequence set forth, and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:45; and a second polypeptide comprising the amino acid sequence. A method for treating an IL-2 mutein infection in a subject, comprising administering to the subject an amount of an IL-2 mutein or a pharmaceutical composition thereof. A method of treating multiple sclerosis is provided, wherein the IL-2 mutein is set forth in SEQ ID NO:46. a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:46; and a second polypeptide comprising an amino acid sequence.

[0146] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating ulcerative colitis in a subject, comprising administering to the subject a The L-2 mutein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:15. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:17. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof The present invention provides a method for treating ulcerative colitis in a subject, the method comprising administering to the subject a IL-2 muteins are selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. The amino acid sequence described in SEQ ID NO: 14 and the sequence number A first polypeptide comprising a linker sequence as set forth in SEQ ID NO:16 and a second polypeptide comprising a linker sequence as set forth in SEQ ID NO:17. and a second polypeptide comprising an amino acid sequence that is selected from the group consisting of ribozymes. A method for treating a patient comprising administering to the patient a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating ulcerative colitis in a patient in need thereof, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NO: 7. The amino acid sequence described in SEQ ID NO: 14 and the sequence number A first polypeptide comprising a linker sequence as set forth in SEQ ID NO:16 and a second polypeptide comprising a linker sequence as set forth in SEQ ID NO:17. and a second polypeptide comprising an amino acid sequence that is selected from the group consisting of ribozymes. A method for treating a patient comprising administering to the patient a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating ulcerative colitis in a patient in need thereof, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NO:8. The amino acid sequence described in SEQ ID NO: 14 and the sequence number A first polypeptide comprising a linker sequence as set forth in SEQ ID NO:16 and a second polypeptide comprising a linker sequence as set forth in SEQ ID NO:17. and a second polypeptide comprising an amino acid sequence that is selected from the group consisting of ribozymes. A method for treating a patient comprising administering to the patient a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating ulcerative colitis in a patient in need thereof, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NO: 19. , 20, 21, 22, 23, 24, 25, 26, 27 or 28 a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:17; In certain embodiments, a therapeutically effective amount of an IL-2 mutein is administered. or a pharmaceutical composition thereof. The present invention provides a method for the preparation of an IL-2 mutein comprising administering to a mammalian subject the present invention thereto an IL-2 mutein having the amino acid sequence set forth in SEQ ID NO:23. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:17. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or peptide is administered. The present invention provides a method for treating ulcerative colitis in a subject, comprising administering the pharmaceutical composition to the subject. wherein the IL-2 mutein comprises the amino acid sequence set forth in SEQ ID NO:24. A first polypeptide and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:17. This includes the.

[0147] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating ulcerative colitis in a subject, comprising administering to the subject a The L-2 mutein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:15. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof The present invention provides a method for treating ulcerative colitis in a subject, the method comprising administering to the subject a IL-2 muteins are selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. The amino acid sequence described in SEQ ID NO: 14 and the sequence number A first polypeptide comprising a linker sequence as set forth in SEQ ID NO: 55 and a second polypeptide comprising a linker sequence as set forth in SEQ ID NO: 56. and a second polypeptide comprising an amino acid sequence that is selected from the group consisting of ribozymes. A method for treating a patient comprising administering to the patient a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating ulcerative colitis in a patient in need thereof, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NO: 7. The amino acid sequence described in SEQ ID NO: 14 and the sequence number A first polypeptide comprising a linker sequence as set forth in SEQ ID NO: 55 and a second polypeptide comprising a linker sequence as set forth in SEQ ID NO: 56. and a second polypeptide comprising an amino acid sequence that is selected from the group consisting of ribozymes. A method for treating a patient comprising administering to the patient a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating ulcerative colitis in a patient in need thereof, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NO:8. The amino acid sequence described in SEQ ID NO: 14 and the sequence number A first polypeptide comprising a linker sequence as set forth in SEQ ID NO: 55 and a second polypeptide comprising a linker sequence as set forth in SEQ ID NO: 56. and a second polypeptide comprising an amino acid sequence that is selected from the group consisting of ribozymes. A method for treating a patient comprising administering to the patient a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating ulcerative colitis in a patient in need thereof, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NO: 19. , 20, 21, 22, 23, 24, 25, 26, 27 or 28 a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55; In certain embodiments, a therapeutically effective amount of an IL-2 mutein is administered. or a pharmaceutical composition thereof. The present invention provides a method for the preparation of an IL-2 mutein comprising administering to a mammalian subject the present invention thereto an IL-2 mutein having the amino acid sequence set forth in SEQ ID NO:23. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or peptide is administered. The present invention provides a method for treating ulcerative colitis in a subject, comprising administering the pharmaceutical composition to the subject. wherein the IL-2 mutein comprises the amino acid sequence set forth in SEQ ID NO:24. A first polypeptide and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. This includes the.

[0148] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating ulcerative colitis in a subject, comprising administering to the subject a The L-2 mutein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:15 and the amino acid sequence set forth in SEQ ID NO:16. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof The present invention provides a method for treating ulcerative colitis in a subject, the method comprising administering to the subject a IL-2 muteins are selected from the group consisting of SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. The amino acid sequence described in SEQ ID NO: 15 and the sequence number A first polypeptide comprising a linker sequence as set forth in SEQ ID NO:16 and a second polypeptide comprising a linker sequence as set forth in SEQ ID NO:18. and a second polypeptide comprising an amino acid sequence that is selected from the group consisting of ribozymes. A method for treating a patient comprising administering to the patient a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating ulcerative colitis in a patient in need thereof, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NO: 7. The amino acid sequence described in SEQ ID NO: 15 and the sequence number A first polypeptide comprising a linker sequence as set forth in SEQ ID NO:16 and a second polypeptide comprising a linker sequence as set forth in SEQ ID NO:18. and a second polypeptide comprising an amino acid sequence that is selected from the group consisting of ribozymes. A method for treating a patient comprising administering to the patient a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating ulcerative colitis in a patient in need thereof, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NO:8. The amino acid sequence described in SEQ ID NO: 15 and the sequence number A first polypeptide comprising a linker sequence as set forth in SEQ ID NO:16 and a second polypeptide comprising a linker sequence as set forth in SEQ ID NO:18. and a second polypeptide comprising an amino acid sequence that is selected from the group consisting of ribozymes. A method for treating a patient comprising administering to the patient a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating ulcerative colitis in a patient in need thereof, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NO:29 , 30, 31, 32, 33, 34, 35, 36, 37 or 38 a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18; In certain embodiments, a therapeutically effective amount of an IL-2 mutein is administered. or a pharmaceutical composition thereof. The present invention provides a method for the preparation of an IL-2 mutein comprising administering to a mammalian subject the present invention thereto an IL-2 mutein having the amino acid sequence set forth in SEQ ID NO:33. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or peptide is administered. The present invention provides a method for treating ulcerative colitis in a subject, comprising administering the pharmaceutical composition to the subject. wherein the IL-2 mutein comprises the amino acid sequence set forth in SEQ ID NO:34. A first polypeptide and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18. This includes the.

[0149] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating ulcerative colitis in a subject, comprising administering to the subject a The L-2 mutein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:13 and the amino acid sequence set forth in SEQ ID NO:14. and a second polypeptide that is identical to the first polypeptide. In this embodiment, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered to the subject. The present invention provides a method for treating ulcerative colitis in a subject, comprising administering to the subject an IL-2 mutein. is an amine as set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. the amino acid sequence set forth in SEQ ID NO:13 and the amino acid sequence set forth in SEQ ID NO:16 a first polypeptide including a linker sequence and a second polypeptide having the same amino acid sequence as the first polypeptide. and a second polypeptide comprising an amino acid sequence that is The method comprises administering to the subject an effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating ulcerative colitis, wherein the IL-2 mutein is set forth in SEQ ID NO:7. the amino acid sequence depicted in SEQ ID NO:13 and SEQ ID NO:16 and a first polypeptide having the same amino acid sequence as the first polypeptide. and a second polypeptide comprising an amino acid sequence that is a nucleotide sequence of comprising administering to a subject a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating ulcerative colitis in a subject, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NO: No. 8, the amino acid sequence set forth in SEQ ID NO: 13, and A first polypeptide comprising a linker sequence as set forth in SEQ ID NO: 16, and and a second polypeptide comprising an amino acid sequence that is the same amino acid sequence as the first polypeptide. In one embodiment, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered to a subject. The present invention provides a method for treating ulcerative colitis in a subject, the method comprising administering to the subject an IL-2 mutant The sequences are set forth in SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 52, 53, and 54. a first polypeptide having an amino acid sequence identical to that of the first polypeptide; and a second polypeptide comprising an amino acid sequence which is The method comprises administering to a subject a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating ulcerative colitis in a patient with IL-2 mutein comprising administering to said patient a therapeutically effective amount of IL-2 mutein having the structure set forth in SEQ ID NO: 45. a first polypeptide comprising the amino acid sequence set forth, and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:45; and a second polypeptide comprising the amino acid sequence. A method for treating an IL-2 mutein infection in a subject, comprising administering to the subject an amount of an IL-2 mutein or a pharmaceutical composition thereof. A method of treating ulcerative colitis is provided, wherein the IL-2 mutein is set forth in SEQ ID NO:46. a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:46; and a second polypeptide comprising an amino acid sequence.

[0150] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating Crohn's disease in a subject, comprising administering to the subject an IL The -2 mutein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:14. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:17. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. and administering to the subject a compound comprising the steps of: The L-2 mutein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:1 6 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:17. and a second polypeptide comprising an amino acid sequence comprising administering to the subject an effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating Crohn's disease in a mammal comprising administering to said mammal an IL-2 mutein as set forth in SEQ ID NO:7. the amino acid sequence depicted in SEQ ID NO:14 and SEQ ID NO:16 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:17. and a second polypeptide comprising an amino acid sequence that is The method comprises administering to the subject an effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating Crohn's disease, wherein the IL-2 mutein is set forth in SEQ ID NO:8. the amino acid sequence shown in SEQ ID NO:14 and the amino acid sequence shown in SEQ ID NO:16 A first polypeptide comprising a linker sequence as set forth, and a second polypeptide as set forth in SEQ ID NO:17. and a second polypeptide comprising the amino acid sequence. A method for treating an IL-2 mutein infection in a subject, comprising administering to the subject an amount of an IL-2 mutein or a pharmaceutical composition thereof. A method for treating Crohn's disease is provided, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 1, 22, 23, 24, 25, 26, 27 or 28. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:17. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or an IL-2 mutein thereof is administered. A method of treating Crohn's disease in a subject is provided, comprising administering a pharmaceutical composition to the subject, The IL-2 mutein comprises a first polypeptide having the amino acid sequence set forth in SEQ ID NO:23. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:17. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof The present invention provides a method for treating Crohn's disease in a subject, comprising administering to the subject a The IL-2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:24. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:17.

[0151] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating Crohn's disease in a subject, comprising administering to the subject an IL The -2 mutein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:14. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. and administering to the subject a compound comprising the steps of: The L-2 mutein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. the amino acid sequence set forth in SEQ ID NO:14 and the amino acid sequence set forth in SEQ ID NO:1 6 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:55. and a second polypeptide comprising an amino acid sequence comprising administering to the subject an effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating Crohn's disease in a mammal comprising administering to said mammal an IL-2 mutein as set forth in SEQ ID NO:7. the amino acid sequence depicted in SEQ ID NO:14 and SEQ ID NO:16 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:55. and a second polypeptide comprising an amino acid sequence that is The method comprises administering to the subject an effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating Crohn's disease, wherein the IL-2 mutein is set forth in SEQ ID NO:8. the amino acid sequence shown in SEQ ID NO:14 and the amino acid sequence shown in SEQ ID NO:16 A first polypeptide comprising a linker sequence as set forth, and a second polypeptide as set forth in SEQ ID NO:55. and a second polypeptide comprising the amino acid sequence. A method for treating an IL-2 mutein infection in a subject, comprising administering to the subject an amount of an IL-2 mutein or a pharmaceutical composition thereof. A method of treating Crohn's disease is provided, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 1, 22, 23, 24, 25, 26, 27 or 28. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or an IL-2 mutein thereof is administered. A method of treating Crohn's disease in a subject is provided, comprising administering a pharmaceutical composition to the subject, The IL-2 mutein comprises a first polypeptide having the amino acid sequence set forth in SEQ ID NO:23. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof The present invention provides a method for treating Crohn's disease in a subject, comprising administering to the subject a The IL-2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:24. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:55.

[0152] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating Crohn's disease in a subject, comprising administering to the subject an IL The -2 mutein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:15. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. and administering to the subject a compound comprising the steps of: The L-2 mutein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. the amino acid sequence set forth in SEQ ID NO:15 and the amino acid sequence set forth in SEQ ID NO:1 6 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:18. and a second polypeptide comprising an amino acid sequence comprising administering to the subject an effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating Crohn's disease in a mammal comprising administering to said mammal an IL-2 mutein as set forth in SEQ ID NO:7. the amino acid sequence depicted in SEQ ID NO:15 and SEQ ID NO:16 and a first polypeptide comprising a linker sequence as set forth in SEQ ID NO:18. and a second polypeptide comprising an amino acid sequence that is The method comprises administering to the subject an effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating Crohn's disease, wherein the IL-2 mutein is set forth in SEQ ID NO:8. the amino acid sequence shown in SEQ ID NO:15 and the amino acid sequence shown in SEQ ID NO:16 A first polypeptide comprising a linker sequence as set forth, and a second polypeptide as set forth in SEQ ID NO:18. and a second polypeptide comprising the amino acid sequence. A method for treating an IL-2 mutein infection in a subject, comprising administering to the subject an amount of an IL-2 mutein or a pharmaceutical composition thereof. A method of treating Crohn's disease is provided, wherein the IL-2 mutein is selected from the group consisting of SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 1, 32, 33, 34, 35, 36, 37 or 38. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or an IL-2 mutein thereof is administered. A method of treating Crohn's disease in a subject is provided, comprising administering a pharmaceutical composition to the subject, The IL-2 mutein comprises a first polypeptide having the amino acid sequence set forth in SEQ ID NO:33. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18. In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof The present invention provides a method for treating Crohn's disease in a subject, comprising administering to the subject a The IL-2 mutein comprises a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:34. and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:18.

[0153] In certain embodiments, a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof is administered. The present invention provides a method for treating Crohn's disease in a subject, comprising administering to the subject an IL The -2 mutein is set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. and a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:13. A peptide and a second polypeptide that is identical to the first polypeptide. The method comprises administering to a subject a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. and (c) administering to a subject a dose of 100 mg of IL-2 mutein to said subject. Amino acids set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 Sequence, the amino acid sequence set forth in SEQ ID NO:13 and the amino acid sequence set forth in SEQ ID NO:16 A first polypeptide including a linker sequence and an amino acid sequence identical to that of the first polypeptide. and a second polypeptide comprising the amino acid sequence. or a pharmaceutical composition thereof, The present invention provides a method for treating Rohn's disease, wherein the IL-2 mutein is set forth in SEQ ID NO:7. the amino acid sequence set forth in SEQ ID NO:13 and the amino acid sequence set forth in SEQ ID NO:16 a first polypeptide having a linker sequence and a second polypeptide having the same amino acid sequence as the first polypeptide; and a second polypeptide comprising an amino acid sequence that is a A method for treating a patient comprising administering to the patient a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating Crohn's disease in a subject, wherein the IL-2 mutein is as set forth in SEQ ID NO:8. The amino acid sequence set forth in SEQ ID NO: 13 and SEQ ID NO: 16 and a first polypeptide having the same linker sequence as the first polypeptide. and a second polypeptide comprising an amino acid sequence that is an amino acid sequence. The method comprises administering to a subject a therapeutically effective amount of an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating Crohn's disease in a subject, comprising administering to the subject an IL-2 mutein having the sequence The items listed in numbers 45, 46, 47, 48, 49, 50, 51, 52, 53, and 54 a first polypeptide having an amino acid sequence and an amino acid sequence identical to that of the first polypeptide; and a second polypeptide comprising a nucleotide sequence of A method for treating IL-2 infection in a subject, comprising administering to the subject an IL-2 mutein or a pharmaceutical composition thereof. The present invention provides a method for treating Crohn's disease, wherein the IL-2 mutein is set forth in SEQ ID NO:45. a first polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:45; In certain embodiments, a therapeutically effective amount of IL-2 The method of treating Crohn's disease in a subject, comprising administering to the subject a mutein or a pharmaceutical composition thereof. The present invention provides a method of treatment, wherein the IL-2 mutein is an amino acid sequence set forth in SEQ ID NO:46. a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO:46; and a second polypeptide comprising the amino acid sequence set forth in SEQ ID NO:47. and 2 polypeptides.

[0154] In yet another embodiment, the method comprises administering to the subject a therapeutically effective amount of CD8+ T cells without activating CD8+ T cells. A method for selectively activating regulatory T cells comprising administering to a subject a therapeutically effective amount of any of the methods described herein. The various IL-2 muteins or pharmaceutical compositions described herein are In some embodiments, the method includes administering to a subject in need thereof a Selective activation of regulatory T cells without activating D8+ T cells was demonstrated in vitro. In certain embodiments, CD8+ T cells can be measured by in vivo or ex vivo methods. Selective activation of regulatory T cells without activating T cells is herein defined as pSTAT5 assays as described in the examples provided, including but not limited to The amount of the protein can be measured by any method known to those of skill in the art, including by measurement using a known method.

[0155] In yet another embodiment, the subject is treated with a method for the treatment of autism spectrum disorder characterized by the presence of a IL-2 receptor alpha subunit. Selectively inhibits cells expressing the IL-2 receptor β subunit without activating cells expressing the IL-2 receptor β subunit. The method of activating IL-2 receptors in a mammalian subject comprises administering to the mammal a therapeutically effective amount of any of the various IL-2 receptors described herein. The present invention relates to a method for administering a medicament or the various pharmaceutical compositions described herein to a subject in need thereof. In some embodiments, the method includes: of the various IL-2 muteins or various pharmaceutical compositions described herein. In an embodiment, the present invention is directed to a method for the production of IL-2 receptor α subunit-expressing cells without activating the cells. In addition, selective activation of cells expressing the IL-2 receptor β subunit is an in vivo In certain embodiments, the IL-2 receptor may be measured by in vitro or ex vivo methods. The IL-2 receptor β subunit was expressed without activating cells expressing the receptor α subunit. Selective activation of cells expressing the IL-1 gene is described in the Examples provided herein. Any method known to those skilled in the art can be used to assay the pSTAT5 signaling pathway, including, but not limited to, the pSTAT5 assay described in It may be measured by any known method.

[0156] In yet another embodiment, a method for treating an IL-2 mediated disease in a subject, comprising: Any one of the various IL-2 muteins described herein or The present invention provides the use of any one of the various pharmaceutical compositions disclosed herein.

[0157] In one embodiment, a method for treating an IL-2 mediated disease in a subject, comprising administering to said subject a therapeutically effective amount of an IL-2 mediated disease .... Any one of the various IL-2 muteins described herein or The use of any one of a variety of pharmaceutical compositions is provided.

[0158] In another aspect, a pharmaceutical composition for treating an IL-2 mediated disease in a subject is provided. For the manufacture of any one of the various IL-2 muteins described herein or The use of any one of the various pharmaceutical compositions described herein is provided.

[0159] In various uses of the IL-2 muteins described herein, The disease can be any immune disease described in this disclosure (e.g., an autoimmune disease or an inflammatory disease). ) may be possible.

[0160] Nucleic acids, expression vectors, cells, and methods for producing IL-2 muteins

[0161] Also disclosed herein are polynucleotides encoding such IL-2 muteins. Isolated nucleic acids and vectors containing nucleotide sequences, such isolated nucleic acids or vectors Cells (e.g., host cells) containing such targets, as well as methods for producing such IL-2 muteins. is provided herein.

[0162] In one embodiment, any one of the IL-2 muteins described herein Provided herein is an isolated nucleic acid encoding a polypeptide. In the present invention, the isolated nucleic acids are intended to represent the various IL-2 muteins described herein. In another embodiment, the first polypeptide encodes any one of the first polypeptides. The isolated nucleic acids may be used to identify the various second polypeptides of the various IL-2 muteins described herein. In yet another embodiment, the isolated nucleic acid encodes any one of the peptides. The acid may be any of the various first polypeptides of the various IL-2 muteins described herein. and any one of a variety of second polypeptides. In an embodiment, the isolated nucleic acid comprises one or more of the polypeptides disclosed in Table 9. Code.

[0163] In another embodiment, the first polypeptide of the IL-2 mutein described herein An isolated first nucleic acid encoding a polypeptide and an isolated second nucleic acid encoding a polypeptide. A second nucleic acid is provided.

[0164] In another embodiment, the isolated nucleic acid further encodes a signal sequence.

[0165] In another embodiment, the present invention comprises one or more of the various isolated nucleic acids disclosed herein. The present invention provides an expression vector comprising the nucleic acid, wherein the nucleic acid is capable of being expressed by a host cell transfected with the expression vector. The gene is operably linked to a control sequence that is recognized by the host cell when expressed.

[0166] In yet another embodiment, the various isolated nucleic acids or nucleic acids described herein are provides host cells comprising one or more of a variety of expression vectors. In some embodiments, In some embodiments, the host cell comprises one or more of the various isolated nucleic acids disclosed herein. In embodiments, the host cell is capable of expressing one or more of the various expression vectors disclosed herein. In yet other embodiments, the host cell is capable of expressing any of the various expression vectors disclosed herein. The expression vector may be any one of the various vectors disclosed herein. In yet another embodiment, the host cell comprises one or more of the isolated nucleic acids described herein. A first expression vector comprising a first nucleic acid encoding a first polypeptide of the IL-2 mutein described herein. A vector and a vector encoding a second polypeptide of the IL-2 mutein described herein. and a second expression vector comprising a second nucleic acid.

[0167] In yet another embodiment, the various IL-2 muteins described herein are In one embodiment, the method comprises the steps of: Any one of the various host cells described herein can be cultured under conditions in which the tein is expressed. In another embodiment, the method comprises culturing the cells in which the IL-2 mutein is expressed. Under the conditions described herein, any one of the various expression vectors described herein is expressed. In yet another embodiment, the method comprises: Under conditions as described herein, the nucleic acid expresses any one of the various isolated nucleic acids described herein. This includes making the person do so.

[0168] In certain embodiments of the various methods for producing an IL-2 mutein of the invention, the method comprises: Further isolating the IL-2 mutein from the host cell or medium or from the in vitro expression system. Included.

[0169] Mammals that can be used as hosts for expression of the IL-2 muteins disclosed herein Cell lines are well known in the art and are described in American Type Culture Collection (American Type Culture Collection). These include the numerous immortalized cell lines available from the American College of Cancer Cells (ATCC). Among others, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, He La cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma Cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and Other cell lines that may be used include insect cells, such as Sf 9 cells, amphibian cells, bacterial cells, plant cells and fungal cells. In order to achieve the desired properties of the expressed IL-2 mutein, Various modifications (e.g., glutamine synthetase knockout, nutritional Mutations such as auxotrophic mutations can be introduced.

[0170] When a recombinant expression vector encoding the or, more preferably, into the medium in which the host cells are cultured. Culturing the host cells for a period of time sufficient to allow secretion of the IL-2 mutein. Thus, an IL-2 mutein is produced.

[0171] The IL-2 mutein can be recovered from the culture medium using standard protein purification methods.

[0172] Generally, glycoproteins produced in individual cell lines or transgenic animals is a sequence characteristic of the glycoprotein produced in the cell line or transgenic animal. Thus, the individual glycosylation patterns of the IL-2 muteins The turn of events is based on the individual cell lines or transfectants used to produce the IL-2 muteins. The various IL-2 muteins or such IL-2 muteins are dependent on the genetic species of the animal. The disclosure of isolated nucleic acids encoding the IL-2 muteins provides information regarding the glycosylation patterns that the IL-2 muteins may have. This is irrelevant to the

[0173] Pharmaceutical Compositions and Administration

[0174] In another embodiment, the IL-2 muteins described herein are combined with a pharma- ceutical and an acceptable carrier.

[0175] In some embodiments, the composition further comprises an additional agent.

[0176] In certain embodiments, the additional agent is an IL-2 inhibitor as disclosed herein. These are agents that are effective in treating the same disorders that the muteins are used to treat. In this embodiment, the additional agent is an IL-2 mutant as disclosed herein. The present invention is an agent that is effective in ameliorating one or more of the side effects of

[0177] Producing Pharmaceutical or Sterile Compositions of the IL-2 Muteins Described Herein To this end, the IL-2 muteins of the present invention are mixed with a pharma- ceutically acceptable carrier or excipient. For example, Remington's Pharmaceutical Sciences and and US Pharmacopeia: National Formulary, Ma ck Publishing Company, Easton, PA (1984). I want to be.

[0178] Formulations of therapeutic and diagnostic agents may be, for example, lyophilized powders, slurries, aqueous solutions or or in the form of a suspension, by mixing with an acceptable carrier, excipient or stabilizer. (e.g., Hardman et al. (2001) Goodman and Gilman 's The Pharmacological Basis of Therapeu tics,McGraw-Hill,New York,NY;Gennaro(200 0)Remington: The Science and Practice of Pharmacy, Lippincott, Williams and Wilkins, N New York, NY; Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms:Parental Medications,Marc el Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical utical Dosage Forms:Tablets,Marcel Dekke r, NY; Lieberman et al. (eds.) (1990) Pharmaceutical D osage Forms:Disperse Systems,Marcel Dekk er, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety,Marcel Dekker,Inc.,N (See New York, NY).

[0179] Toxicity and therapeutic effects of IL-2 mutein compositions administered alone or in combination with other agents Therapeutic efficacy is, for example, LD 50 (the dose that is lethal to 50% of the population) and ED 50 (Collective in cell cultures or experimental animals to determine the dose that is therapeutically effective in 50% of cases The dose ratio between toxic and therapeutic effects can be determined by standard pharmaceutical procedures in the field. Number (LD 50 / ED 50 In certain embodiments, antibodies that exhibit high therapeutic indices are desired. The data obtained from these cell culture assays and animal studies support the need for The dosage of such compounds may be used to determine the range of dosages for use. Preferably, ED with little or no toxicity 50 The range of circulating concentrations includes: The dosage can vary within this range depending upon the dosage form and route of administration used.

[0180] In another embodiment, a composition comprising the IL-2 muteins disclosed herein is provided. The composition was determined according to the Physicians' Desk Reference 2003 (Thoms on Healthcare;57th edition (November 1, 2002) Accordingly, administer to the subject.

[0181] The mode of administration of the IL-2 muteins and compositions of the present invention may vary. These include oral, rectal, transmucosal, intestinal, parenteral, intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, and direct ventricular This includes intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, dermal, transdermal or intra-arterial. can be.

[0182] In a specific embodiment, the IL-2 muteins of the invention are administered via an invasive route, such as by injection. In additional embodiments, the IL-2 mutein or its pharmaceutical agent may be administered by intravenous route. The pharmaceutical composition is administered intravenously, subcutaneously, intrathecally, intramuscularly or intracerebrally. In another embodiment, the IL-2 mutein or pharmaceutical composition thereof is administered intravenously. In certain embodiments, the IL-2 mutein or pharmaceutical composition thereof is administered subcutaneously. do.

[0183] The compositions can be administered using medical devices known in the art. The pharmaceutical composition can be delivered, for example, via a prefilled syringe or a hypodermic needle, including an autoinjector. The drug may be administered by injection using

[0184] The pharmaceutical compositions disclosed herein may also be administered by injection.

[0185] The dosing regimen will depend on the serum or tissue turnover rate of the IL-2 mutein, the severity of symptoms, and the IL Immunogenicity of the -2 muteins and accessibility of target cells in biological matrices Preferably, the dosing regimen will minimize undesirable side effects. The objective of the present invention is to provide a method for reducing the amount of IL-2 mutein produced by the tumor while simultaneously delivering sufficient IL-2 mutein to result in amelioration of the targeted disease state. Thus, the amount of biological agent delivered will depend, in part, on the individual IL-2 muteins and Depends on the severity of the condition being treated. Guidance is available for selecting the appropriate dose. (See, for example, Wawrzynczak (1996) Antibody Therapy, B ios Scientific Pub.Ltd,Oxfordshire,UK;Kr esina (ed.) (1991) Monoclonal Antibodies, Cyto kines and Arthritis,Marcel Dekker,New Yo rk, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Disease Ses, Marcel Dekker, New York, NY; Baert et al. (200 3) New Engl. J. Med. 348:601-608; Milgrom et al. (19 99) New Engl. J. Med. 341: 1966-1973; Slamon et al. ( 2001)New Engl.J.Med.344:783-792;Beniamin ovitz et al. (2000) New Engl. J. Med. 342:613-619;G hosh et al. (2003) New Engl.J.Med.348:24-32;Lips See ky et al. (2000) New Engl. J. Med. 343:1594-1602 (I want to be).

[0186] Determining the appropriate dosage can be accomplished using techniques known in the art to, for example, affect treatment. The assessment is performed by the clinician using the parameter or factor under suspicion. In such cases, the dose should be started at a somewhat lower dose than the optimal dose and then gradually increased in comparison with the negative side effects. The dosage may be increased by small increments until the desired or optimum effect is achieved.

[0187] As previously described, the IL-2 muteins may be co-administered with one or more additional agents. The IL-2 mutein can be linked to an agent (e.g., a conjugate). In the latter case (separate administration), the IL-1 The mutein can be administered before, after, or simultaneously with the agent. may be co-administered with other known therapies.

[0188] kit

[0189] Also included herein is the product packaged in suitable packaging material. Kits containing the IL-2 muteins or compositions (e.g., pharmaceutical compositions) thereof provided herein. Provided herein are kits that, optionally, include a description of the components or Labels or instructions for in vitro, in vivo, or ex vivo use of the ingredients may contain a packaging insert.

[0190] The term "packaging material" refers to the physical structure that contains the components of the kit. Caging materials are capable of keeping ingredients sterile and are commonly used for such purposes. Materials to be used (e.g., paper, cardboard, glass, plastic, foil, ampoules, vials) , tube, etc.

[0191] The kits provided herein may include a label or an insert. The insert includes "printed matter," such as paper or cardboard, which is separate. or attached to the component, kit, or packaging material (e.g., box) Alternatively, for example, the kit may be accompanied by an ampoule, tube, or vial containing the kit components. The label or insert may further include a computer readable medium, such as a disk (e.g., hard disks, cards, memory disks), optical disks, e.g. CD- or DVD-ROM / RAM, DVD, MP3, magnetic tape, or electrical storage media, e.g. RAM and ROM, or a hybrid of these, e.g. magnetic / optical storage media, FLA This may include flash media or memory cards. Labels or inserts The data may include information identifying the manufacturer, lot number, manufacturer's location and date.

[0192] The kits provided herein may further comprise other components. Each component of the kit may be individually The various containers may be enclosed within one or more containers, with all of the various containers being located within a single package. The kit may also be designed for refrigerated storage.

[0193] General method

[0194] Standard methods in molecular biology include those described by Sambrook, Fritsch and Man iatis (1982 & 1989 2 nd Edition, 2001 3 rd E dition)Molecular Cloning,A Laboratory Ma nual,Cold Spring Harbor Laboratory Press , Cold Spring Harbor, NY; Sambrook and Russell l(2001)Molecular Cloning,3 rd ed, Cold Spring ng Harbor Laboratory Press,Cold Spring H arbor,NY;Wu(1993)Recombinant DNA,Vol.217 ,Academic Press,San Diego,CA. Standards A typical method is described in Ausbel et al. (2001) Current Protocols in Molecular Biology,VoIs.1-4,John Wiley an and also described by MD Sons, Inc., New York, NY, which is a method for producing bacterial cells. Cloning and DNA mutagenesis in mammalian cells and yeast (Vol. 1) (Vol. 2), glycoconjugates and protein expression (Vol. 3), as well as bioinformatics (Vol. 4).

[0195] Protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation and crystallization Methods for preparation have been described (Coligan et al. (2000) Current Press, otocols in Protein Science,Vol.1,John Wi ley and Sons, Inc., New York). Chemical analysis, chemical modification, translation Post-modification, preparation of fusion proteins, glycosylation of proteins have been described (e.g., Coligan et al. (2000) Current Protocols in Prote in Science,Vol.2,John Wiley and Sons,Inc. .,New York;Ausubel et al. (2001)Current Protoco ls in Molecular Biology,Vol.3,John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich,Co.(2001)Products for Life Science Research, St. Louis, MO;pp.45-89;A mersham Pharmacia Biotech(2001)BioDirect (See, for example, P. Ory, Piscataway, NJ, pp. 384-391.) The production, purification and fragmentation of reclonal and monoclonal antibodies are described. (Coligan et al. (2001) Current Protocols in mmunology,Vol.1,John Wiley and Sons,Inc. , New York; Harlow and Lane (1999) Using Antib odies,Cold Spring Harbor Laboratory Pres. s, Cold Spring Harbor, NY; Harlow and Lane, op. cit. ). Standard techniques are available for characterizing ligand / receptor interactions (e.g., For example, Coligan et al. (2001) Current Protocols in Immunology Nology, Vol. 4, John Wiley, Inc., New York. (I want to be).

[0196] Methods for flow cytometry including fluorescence activated cell sorting and detection system (FACS) are available. It is possible (see, for example, Owens et al. (1994) Flow Cytometry Pri ciples for Clinical Laboratory Practice ,John Wiley and Sons,Hoboken,NJ;Givan(20 01) Flow Cytometry, 2 nd ed.;Wiley-Liss,Hob oken,NJ;Shapiro(2003)Practical Flow Cyto See Metry, John Wiley and Sons, Hoboken, NJ. (e.g., for use as diagnostic reagents), including nucleic acid primers and probes. Suitable fluorescent reagents for modifying nucleic acids, polypeptides, and antibodies are available (Mo lecular Probes(2003)Catalog,Molecular Probes, Inc., Eugene, OR; Sigma-Aldrich (2003 )Catalogue,St. Louis,MO).

[0197] Standard histological methods for the immune system have been described (e.g., Muller-Harme et al. link (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, PA ;Louis et al. (2002) Basic Histology:Text and At (See, e.g., McGraw-Hill, New York, NY).

[0198] For example, antigen fragments, leader sequences, protein folding, functional domains A software package for determining glycosylation sites and sequence alignments and databases are available (e.g., GenBank, Vector NTI (R) Suite (Informax, Inc., Bethesda, MD); GC G Wisconsin Package (Accelrys, Inc., San Di ego, CA); DeCypher (registered trademark) (TimeLogic Corp., C Menne et al. (2000) Bioinform atics 16:741-742;Menne et al. (2000)Bioinformat ics Applications Note 16:741-742;Wren et al.(2 002)Comput.Methods Programs Biomed.68:17 7-181;von Heijne(1983)Eur.J.Biochem.133: 17-21; von Heijne (1986) Nucleic Acids Res. 14:4683-4690).

[0199] Example 1. Cloning, expression and purification of IL-2 muteins

[0200] Polynucleotides encoding various IL-2 muteins were synthesized and expressed in the PTT5 plasmid. (Canadian National Research Council) The cells were incubated in suspension in Gibco ExpiCHO or Expi293 expression medium. ExpiCHO or Expi293 cells grown in 100% HO were incubated with the commercially available reagent Exp Using iFectami and the protocol (Thermo-Fisher), various Transient transfection with PTT5 constructs containing polynucleotides encoding IL-2 muteins Briefly, the cells were infected with Vi-Cell (Beckman-Coulter ) with >95% viability as measured using 1 μg of total DNA per mL of cells Cells were transfected on day 0 with PTT5 encoding an IL-2 mutein containing the Fc variant Fc-1 (SEQ ID NO: 13) The constructs were transfected. In some embodiments, monovalent IL-2 muteins were expressed. In the present invention, a gene encoding an IL-2 mutein containing the Fc variant Fc-2 (SEQ ID NO: 14) is and a second PTT5 construct encoding the Fc variant Fc-4 (SEQ ID NO: 17). Co-transfect the PTT5 constructs, the first PTT5 construct and the second PTT5 construct The ratio of monovalent IL-2 muteins to monovalent IL-2 muteins was 3:1. The first encodes an IL-2 mutein containing the Fc variant Fc-3 (SEQ ID NO: 15). PTT5 construct, and a second PTT encoding the Fc variant Fc-5 (SEQ ID NO: 18). 5 constructs were co-transfected and the ratio of the first PTT5 construct to the second PTT5 construct was The ratio is 3:1 for Expi293 cells on day 5 and for ExpiCHO cells on day 6. AK harvested cultures between days 8 and 12 depending on cell viability above 80%. Protein A chromatograph on a TA FPLC system (GE Healthcare) Using a mAbSelect Sure LX, GE Healthcare Monovalent or bivalent IL-2 muteins were purified from the clarified supernatant. After loading, the resin The column was washed with 20 column volumes of PBS and diluted with 20 mM sodium acetate (pH 3.5). The IL-2 muteins were eluted using protein A chromatography. Following precipitation, it was passed through an anion exchanger (Capto Q, GE Healthcare). This was typically sufficient to obtain a highly homogeneous protein. For the 2 muteins, the initial Protein A purification and anion exchange pass were followed by cation exchange. Using recombinant chromatography (Capto S), Fc variant homodimers, IL-2 The desired IL-2 mutein is isolated from contaminants such as mutein monomers and / or IL-2 mutein homodimers. Monovalent heterodimeric IL-2 muteins were isolated, yielding greater than 95% monovalent heterodimers Different mutants required different salt and pH conditions for their isolation. In some cases, size exclusion chromatography can be further used to isolate more than 95% monovalent heterodimers. Got a body.

[0201] Example 2. I for human IL2-Rα or IL2-Rβ by surface plasmon resonance Determination of binding affinity of L-2 muteins

[0202] Surface plasmon resonance (SPR) in a Biacore T200 (Cytiva) instrument ) assay to detect IL-2 muteins against the polyhistidine-tagged IL-2 receptor. The binding affinity of was determined using the Biacore T200 evaluation software. Each titration series was fitted to a 1:1 binding model or steady-state affinity. Degree constant (k on , M -1 s -1 ) and the dissociation rate constant (k off , s -1 ) Titration set The dissociation constants KD of each IL-2 mutein for each receptor were determined by using the determined values. k off / k on was calculated.

[0203] IL-2 Fc muteins and WT-IL-2 Fc fusion controls to the IL-2 receptor To measure the affinity of The IL-2 receptor was then captured as an analyte. Tein 86BCH, 43BGO, 44BGO, 47BJO, 48BJO, 49BJ O, 65BJO, 44BJP, 45BJP, 46BJP and 47BJP are univalent; On the other hand, 54BGO is a bivalent form of 43BGO, and 48BMP is a bivalent form of 49BJO. 21BMT is a bivalent form of 65BJO. 86BCH is a wild-type human IL-2 ( A substitution from T to A corresponding to position 3 of SEQ ID NO:39, and a substitution from C to S corresponding to position 125 of SEQ ID NO:39 The other exemplary IL-2 muteins contained substitutions of and the same T to A substitutions corresponding to positions 3 and 125, respectively, of wild-type human IL-2. and C to S substitutions. Table 1 shows the amino acid sequence introduced into each IL-2 mutein. The amino acid substitutions are summarized in Table 1. The positions of such substitutions are shown in the wild-type human IL-2 amino acid sequence. (SEQ ID NO: 39). Each of SEQ ID NOs: 1 and 2 is based on wild-type human IL- 2 amino acid sequence (SEQ ID NO: 39) does not contain the first amino acid found in the Note that such substitutions are different from those shown based on SEQ ID NO: 1 or 2. I want to be loved.

[0204] [Table 1]

[0205] As shown in Table 2, the binding affinity of the control 86BCH to human IL-2Rα The activity (average of four tests) was approximately 30 nM, whereas other The affinities of the exemplary IL-2 muteins ranged from 1.1 nM to 44 nM. On the other hand, the affinity of the control 86BCH for human IL-2Rβ (average of four tests) was approximately 1500 nM, whereas all other exemplary IL-2 muteins exhibited activity at 3 μM. When assayed, it showed no detectable binding to human IL-2Rβ.

[0206] [Table 2]

[0207] Example 3. Human peripheral blood mononuclear cell (PBMC) pSTAT5 assay

[0208] IL-2 muteins were prepared in serial dilutions and 50 μL was added in duplicate to U-bottom plates. 10 μL of PBMCs (approximately 500 kc / w) were added and the samples were incubated at 37 °C, 5% CO for 30 The mixture was allowed to stand for 1 min ("m"). Cold PBS was added, the samples were centrifuged, and the supernatant was removed. C Add Fixation Buffer (Invitrogen Cat 00-8222-49) and mix. The samples were then combined and incubated at room temperature for 20 minutes. The samples were then washed with FBS staining buffer. (BD Cat 554656) and then incubated with anti-CD40 on ice, covered. 3 antibody (Invitrogen Cat 47-0038-42) and anti-CD56 antibody The samples were stained with 100% Fibroblast Growth Protein (Biolegend Cat 318334) for 30 minutes. Wash twice with PBS and add pre-chilled Perm Buffer III (BD Cat The cells were permeabilized using 558050 (558050) for 30 minutes on ice, covered. The samples were washed twice with staining buffer and incubated covered on ice with anti-CD4 (Invitrogen) ogen Cat 46-0047-42), anti-CD8 (BD Cat 555634) , anti-CD25 (BD Cat 335807), anti-CD127 (Invitrogen Cat 12-1278-42), anti-pSTAT5 Y694 (BD Cat 6125 99) and anti-FoxP3 (Invitrogen Cat 48-4777-42). The samples were washed twice with staining buffer and resuspended in 130 μL Samples were then analyzed on a BD Symphony flow cytometer.

[0209] The FCS files were imported into FlowJo or FCSExpress. Lymphocytes were gated using forward scatter area versus side scatter. Then, forward scatter height vs. forward scatter width were used to gate single cells. From this, CD3-negative cells expressing CD56 were gated on NK cells. The CD3+ cells were then cultured to evaluate pSTAT5 expression in CD4+ cells. or subgated on CD8 positive cells (pSTAT The expression of CD25 and FoxP3 was used to further classify CD4+ cells. The cells that were CD4 positive but double negative for CD25 and FoxP3 were classified as Tc These cells were assessed for pSTAT5 expression. and FoxP3 double positive CD4+ cells were identified as regulatory T cells. (These cells were used to assess pSTAT5 expression.) Untreated samples were used to assess pSTAT5 expression. Gating was conducted on AT5 positive cells.

[0210] PBMCs were stimulated with serial dilutions of various exemplary IL-2 muteins, including Fc variants. 86BCH or 99BHY was used as a control. The difference between these is that the Fc mutant in 99BHY lacks its C-terminal lysine residue. The percentage of positive pSTAT5 at the highest concentration was taken as 100%, and the untreated control was taken as 0%. and other exemplary IL-2 muteins were used to assess the pSTAT5 response of 86BCH or Normalized to 99BHY. Log(agonist) vs. response - variable slope (4 parameters) Dose-response curves were generated using a vector fit and EC50s were calculated.

[0211] The ability of such IL-2 muteins to activate pSTAT5 was examined by CD4 + CD25 + Foxp3 + Regulatory (Treg) cells and effector T cell populations (CD8 + T cells and CD4 + CD25 - Foxp3 - Gating on T cells Therefore, we evaluated Treg cells in a mixed population of PBMCs from multiple donors. D8 + T cells and CD4 + CD25 - Foxp3 - Compared with T cells, This indicates that the STAT5 EC50 response of STAT5 to Treg cells is lower than that of STAT5. Affinity for the expressed trimeric IL-2 receptor complex (IL-2R α / β / γ) However, CD8 + T cells and CD4 + CD25 - Foxp3 - Expressed on T cells Higher affinity for the dimeric IL-2 receptor complex (IL-2R β / γ) This is consistent with the results shown in Figs. 1A-1C for 86BCH and 1C for 99BHY. 2A-2C). In contrast, stimulation of PBMCs with other exemplary IL-2 muteins At concentrations up to 1 μM, CD8 + T cells and CD4 + CD25 - Foxp3- T Selectively inhibits STAT5 phosphorylation in Tregs without inducing STAT5 phosphorylation in cells. T5 phosphorylation was induced (Figures 1A-1C and 2A-2C). CD4 + CD25 + Foxp3 + The preferential activity of these muteins against the Treg cell compartment Consistently, induction of STAT5 phosphorylation at doses higher than 0.1 μM was significantly higher than in controls. t, CD8 + T cells and CD4 + CD25 - Foxp3 - In T cells, These responses were observed in multiple donors (n = 100). These results were consistent across a range of IL-2 muteins. D8 + T effector cells or CD4 + CD25 - Foxp3 - Activate T cells They demonstrate that the antibody selectively activates primary human Treg cells without activating Treg cells.

[0212] The EC50 values ​​of exemplary IL-2 muteins for activating primary human Treg cells are given below. This is summarized in Table 3A below.

[0213] [Table 3A]

[0214] As shown in Table 3B below, additional studies were performed, again using 99BHY as a control. The same experiment as above was carried out for the exemplary IL-2 muteins.

[0215] [Table 3B]

[0216] DNB558 is monovalent and is similar to 65BJO (also monovalent) and 21BMT (bivalent). DNB558 and 65BJO have the same IL-2 mutations. DNB557 is monovalent and has an additional mutation in 49BJO (monovalent) and 48BMP (bivalent). 49BJO differs from 9BJO in that it has additional mutations in the Fc region.

[0217] Similarly, Treg cells are CD8 + T cells and CD4 + CD25 - Foxp3 - T cells showed a lower pSTAT5 EC50 response relative to the control. This suggests that the trimeric IL-2 receptor complex (IL-2R α) expressed on Treg cells / β / γ) affinity to CD8 + T cells and CD4 + CD25 - F oxp3 - The dimeric IL-2 receptor complex (IL-2R β / γ) expressed on T cells This is consistent with the higher affinity for 99BHY (Figure 3A for 99BHY). Specifically, stimulation of PBMCs with additional exemplary IL-2 muteins evaluated demonstrated up to 1 μM At concentration, CD8 + T cells and CD4 + CD25 - Foxp3 - In T cells Selective STAT5 phosphorylation in Tregs without induction of STAT5 phosphorylation (Figures 3A-3C). CD4 + CD25+ Foxp3 + Treg cell conjugate Consistent with the preferential activity of these muteins for the 10-fold compartment, doses of >0.1 μM Induction of STAT5 phosphorylation in CD8 cells was significantly greater than in controls. + T cells and CD4 4 + CD25 - Foxp3 - It was minimal in T cells (Figures 3B-3C). These responses were consistent across multiple donors (n=7). In accordance with the results in, various exemplary IL-2 muteins inhibited CD8 + T Effector -cells or CD4 + CD25 - Foxp3 - Without activating T cells, primary It has been demonstrated that human Treg cells were selectively activated.

[0218] Example 4. Rhesus monkey whole blood pSTAT5 assay

[0219] Serial dilutions of IL-2 muteins were prepared and 25 μL was plated in duplicate on deep well plates. The plate was then sealed and incubated at 37°C, 5% CO 2 The whole blood of rhesus monkeys was left at room temperature for 30 minutes. The samples were incubated at 37°C, 5% CO 2 Incubated for 20 min at The cells were incubated with anti-CD3 (BD ​​Cat 557917) and anti-CD127 (Invitro gen Cat 12-1278-42) antibody, anti-CD25 (Invitrogen C at 25-0257-42) antibody and anti-NKG2A (Miltenyi Cat 1 30-113-565) antibody and incubated at room temperature for 2 h with gentle agitation, covered. The cells were then incubated for 10 min. The cells were then lysed and fixed with 1× Lyse / Fix. Fix using Fix Buffer (BD Cat 558049) and mix thoroughly. The cells were incubated covered at room temperature for 10 min. The plates were washed twice with BD Cat 554656 and transferred to a 96-well plate. Add Perm Buffer III (BD Cat 558050) to the sample. The samples were then washed with staining buffer, mixed and incubated covered on ice for 30 minutes. The cells were washed twice with chlorine. The intracellular antibody anti-FoxP3 (Invitrogen Cat 48-4777-42), anti-pSTAT5 Y694 (BD Cat 612599), Anti-CD4 (BD Cat 552838) and anti-CD8 (BD Cat 563795 ) was added to the samples and incubated covered at room temperature for 1 hour. The samples were washed twice with color buffer and resuspended in 130 μL. ny flow cytometer.

[0220] The FCS file was imported into FlowJo. Forward and side scatter were used to calculate the Lymphocytes were gated. Single cells were identified using forward scatter area versus forward scatter height. From this, NKG2A-expressing CD3-negative cells were gated on NK cells. These cells were then gated on for pSTAT5 expression. The data were sub-gated on CD4+ or CD8+ cells (these CD8+ cells were We assessed pSTAT5 expression using CD25 and FoxP3 expression. The cells were further dissected and found to be CD4 positive but double negative for CD25 and FoxP3. These cells were identified as Tconv cells (pSTAT5 expression was assessed from these cells). CD4 positive cells were further sub-gated for CD25 and CD127. Cells that were positive and CD127 negative were assessed for FoxP3 and identified as regulatory T cells. These cells were assessed for pSTAT5 expression. Gating was guided using FMO for STAT5 and pSTAT5.

[0221] The whole blood was stimulated with serial dilutions of various exemplary IL-2 muteins, including Fc variants. 99BHY was used as a control. The percentage of positive pSTAT5 at the highest concentration was 100%. The untreated control was used as 0%, and the pST of other exemplary IL-2 muteins was used as 0%. AT5 responses were normalized to 99BHY. Log(agonist) vs. response - variable slope Dose-response curves were generated using a four-parameter fit and EC50s were calculated.

[0222] The ability of such IL-2 muteins to activate pSTAT5 was examined by CD4 + CD12 7 - CD25 + Foxp3 + Regulatory (Treg) and effector T cell populations Group (CD8 + T cells and CD4 + CD25 - Foxp3 - (including T cells) Gating was used to assess the activity of whole blood matrices from multiple rhesus donors. Treg cells are CD8 + T cells and CD4 + CD25 - Foxp3 - T The cells showed a lower pSTAT5 EC50 response to the control compared to the control. The trimeric IL-2 receptor complex (IL-2R α / β / IL-2R α / β) expressed on Treg cells γ) is CD8 + T cells and CD4 + CD25 - Foxp 3 - IL-2R β / γ, a dimeric IL-2 receptor complex expressed on T cells This is consistent with the higher affinity of the 99BHY complex compared to that of the 99BHY complex (Figures 3A-3C for 99BHY). In contrast, stimulation of whole blood with other exemplary IL-2 muteins at concentrations up to 3 μM resulted in a C D8 + T cells and CD4 + CD25 - Foxp3 - STAT5 in T cells Selectively induced STAT5 phosphorylation in Tregs without inducing STAT5 phosphorylation (Figures 4A-4C). CD4 + CD25 + Foxp3 + Treg cell compartment Consistent with the preferential activity of these muteins against ST at doses greater than 1 μM, Induction of AT5 phosphorylation was significantly greater in CD8 cells compared to controls. + T cells and CD4 + CD25 - Foxp3 - The responses were minimal in T cells (Figures 4B-4C). These results were consistent across multiple donors (n=2). -2 mutein, CD8 + T effector cells or CD4 + CD25 - Foxp 3 - Selectively activated primary rhesus Treg cells without activating T cells This proves that.

[0223] EC50 of exemplary IL-2 muteins for activating primary rhesus Treg cells The values ​​are summarized below in Table 4A.

[0224] [Table 4A]

[0225] As shown in Table 4B below, additional studies were performed, again using 99BHY as a control. For the exemplary IL-2 muteins, the same experiment as above was performed. DNB558 is monovalent and , with the same IL-2 mutations as 65BJO (also monovalent) and 21BMT (bivalent). DNB557 is monovalent and has the same IL as 49BJO (monovalent) and 48BMP (bivalent). -2 mutation.

[0226] [Table 4B]

[0227] Similarly, Treg cells are CD8 + T cells and CD4 + CD25 - Foxp3 - T cells showed a lower pSTAT5 EC50 response relative to the control. This suggests that the trimeric IL-2 receptor complex (IL-2R α) expressed on Treg cells / β / γ) affinity to CD8 + T cells and CD4 + CD25 - F oxp3 - The dimeric IL-2 receptor complex (IL-2R β / γ) expressed on T cells This is consistent with the higher affinity for 99BHY (Figure 5A for 99BHY). Specifically, stimulation of PBMCs with additional exemplary IL-2 muteins evaluated demonstrated up to 1 μM At concentration, CD8 + T cells and CD4 + CD25 - Foxp3 - In T cells Selective STAT5 phosphorylation in Tregs without induction of STAT5 phosphorylation (Figures 5A-5C). CD4 + CD25 + Foxp3 + Treg cell conjugate Consistent with the preferential activity of these muteins for the 10-fold compartment, doses of >0.1 μM Induction of STAT5 phosphorylation in CD8 cells was significantly greater than in controls. + T cells and CD4 4 + CD25 - Foxp3 - It was minimal in T cells (Figures 5B-5C). These responses were consistent across multiple donors (n=7). In accordance with the results in, various exemplary IL-2 muteins inhibited CD8 + T Effector -cells or CD4 + CD25 - Foxp3 - Without activating T cells, primary It has been demonstrated that human Treg cells were selectively activated.

[0228] Example 5. Expansion and activation of human Tregs in a xeno-GvHD model

[0229] Xeno-GvHD (Graft-versus-host disease) model using huPBMC-NSG mice The muteins were evaluated for their ability to expand and activate human Tregs. NSG mice were purchased from Jackson Laboratory and were inoculated with human PMBCs. 14-17 days after human PBMC (peripheral blood mononuclear cell) transplantation, the mice were Exemplary IL-2 muteins evaluated in this example were The results are shown in Table 5 below. Splenocytes from the mice were analyzed by FACS (fluorescence-activated cell sorting). Depending on the analysis, samples were profiled daily for five days or, in some studies, eight days. Human Tregs in splenocytes express human CD45 + CD56 - CD3 + CD4 + CD12 7 low Foxp3 + All muteins evaluated were identified as human CD4 T cells. 4 + FoxP3 + and enhanced Treg expression (Figures 6A-6D).

[0230] Surface expression of CD25 on human Tregs was quantified by FACS analysis. The IL-2 mutein enhanced CD25 expression on the surface of Tregs (Figures 7A-7D).

[0231] [Table 5]

[0232] Example 6. PF / PK Profiles of Monovalent and Bivalent Forms of Muteins in Rhesus Monkeys R

[0233] PK / PD Profiles of Certain Exemplary IL-2 Muteins in Bivalent and Monovalent Forms The study design is shown in Table 6 below. Male monkeys were used in the study. Three animals were used per group. Days 0 and 14 The animals were administered the solution via the subcutaneous route at 1 mL / kg. The study period was 2 It lasted for 8 days.

[0234] [Table 6]

[0235] Pharmacokinetic (PK) serum (7 aliquots of 100 μL each) was obtained as follows: Day 0, 1st dose: Pre-dose, 15 min, 2 hr, 6 hr; Day 1, Day 2, Day 3, Day 7 Day 14, Day 2 Dose: Pre-dose, 15 min, 2 hr, 6 hr; Day 15, Day 16 Days 1, 2, 3, and 4. Whole blood (2 Aliquots were collected and immunophenotyped (Treg, Tco) for pharmacodynamic (PD) analysis. nv, CD8, NK cells) were performed as follows.

[0236] Panel A: Day -5, Day 0, 1st Dose: Pre-dose; Day 1, Day 3, Day 4, Day 7, Day Day 9; Day 14 Second dose: before administration; Days 15, 17, 18, 21, and 23 Sunday, 28th day.

[0237] Panel B: Day -5; Day 0, 1st dose: Pre-dose, 2 hours; Day 1, Day 2, Day 3, Day Day 7, Day 9; Day 14, 2nd dose: Pre-dose, 2 hours; Day 15, Day 16, Day 17, Day 21st, 23rd, 28th.

[0238] Serum chemistry and CRP (c-reactive protein) were assessed as follows: day -5; Day 0 1st Dose: Pre-dose, 2 hours; Day 1, Day 7; Day 14 2nd Dose: Pre-dose, 2 hours Days 15, 16, 17, 21, 23, and 28. Hematology was as follows: The following were evaluated: Day -5, Day 0; First Dose: Pre-dose; Day 1, Day 3, Day 4, Day 7, Day 8, Day 9, Day 10, Day 11, Day 12, Day 13, Day 14, Day 15, Day 16, Day 17, Day 18, Day 19, Day 20, Day 21, Day 22, Day 23, Day 24, Day 25, Day Day 9; Day 14 2nd Dose: Pre-dose; Day 15, Day 17, Day 18, Day 21, Day 23 Sunday, 28th day.

[0239] The PK / PD results after the first dose were as follows for both monovalent and bivalent muteins: (see Tables 7A and 7B, respectively) and shown in Figures 8A-8D.

[0240] [Table 7A]

[0241] [Table 7B]

[0242] Table 8 also shows the results of rhesus monkey STAT5 and human STAT5, as well as the results of evaluation and examples. The PK / PD results for various IL-2 muteins are shown in Table 8 below. Results for the rhesus monkey and human STAT5 assays were calculated using geometric means.

[0243] [Table 8]

[0244] All references cited herein are incorporated by reference in their entirety, including their respective publications, database entries, and / or publication names. - (e.g., GenBank sequence or GeneID entry), patent application or patent as if specifically and individually indicated to be incorporated by reference herein. This statement of incorporation by reference is hereby incorporated by reference. Unless such citation immediately precedes or follows a statement of incorporation by reference, However, in accordance with 37 CFR § 1.57(b)(1), each individual publication, database, Source entries (e.g., Genbank sequences or GeneID entries), patent issues application or patent [each of which is expressly set forth in accordance with 37 CFR § 1.57(b)(2)] The applicant intends that the invention be directed to a "specifically clearly identified" If a statement of incorporation by reference is included herein, it is understood that the Nothing in this document should be construed as intended to dilute the general statement of incorporation by reference thereto. Citation of a reference herein is not an admission that the reference is relevant prior art. nor does it make any admission as to the contents or dates of these publications or documents. It is not something that is done.

[0245] The present invention is to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein may be made. Such modifications will become apparent to those skilled in the art from the following detailed description and accompanying drawings. It is intended to be within the scope of the claims.

[0246] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. and are within the scope of the appended claims.

[0247] Table 9 below summarizes all the sequences disclosed herein.

[0248]

Table 9

Claims

1. IL comprising a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2. -2 mutein, in which D at position 19 of SEQ ID NO: 1 or 2 is replaced with N. and wherein P at position 33 of SEQ ID NO: 1 or 2 is replaced with R, wherein the polypeptide comprises: Optionally, it may contain one or more additional amino acid substitutions relative to SEQ ID NO: 1 or 2. Good, said IL-2 muteins.

2. IL comprising a first polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2. -2 mutein, in which D at position 19 of SEQ ID NO: 1 or 2 is replaced with N. and replacing E at position 67 of SEQ ID NO: 1 or 2 with S, wherein the polypeptide comprises: Optionally, it may contain one or more additional amino acid substitutions relative to SEQ ID NO: 1 or 2. Good, said IL-2 muteins.

3. The first polypeptide further comprises an E to S substitution at position 67 of SEQ ID NO: 1 or 2. Item 2. An IL-2 mutein according to item 1.

4. The first polypeptide comprises a substitution of V to A at position 68, N to The invention according to any one of claims 1 to 3, comprising a substitution of R or a substitution of Q at position 73 to P. IL-2 mutein.

5. The first polypeptide comprises the following three substitutions: V at position 68 of SEQ ID NO: 1 or 2 of substitutions of N at position 70 to A, N at position 70 to R, or Q at position 73 to P The IL-2 mutein of any one of claims 1 to 3, further comprising any two of the following:

6. The first polypeptide comprises a substitution of V to A at position 68, N to Any one of claims 1 to 3, further comprising a substitution of R and a substitution of Q to P at position 73. The described IL-2 muteins.

7. Amino acids set forth in SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 The IL-2 mutein comprises a first polypeptide comprising an amino acid sequence.

8. The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 13, 14 or 15. The IL-2 mutein of any one of claims 1 to 7, further comprising

9. 9. The method of claim 8, wherein the first polypeptide further comprises a linker as set forth in SEQ ID NO:

16. The IL-2 muteins described above.

10. further comprising a second polypeptide, (1) The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14, and the second polypeptide comprises the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:17; or (2) The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 15, and the second 10. The polypeptide of claim 8 or 9, comprising the amino acid sequence set forth in SEQ ID NO:

18. The described IL-2 muteins.

11. comprising a first polypeptide and a second polypeptide, The first polypeptide is selected from SEQ ID NOs: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 7 or 28, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17 The amino acid sequence described in IL-2 muteins.

12. The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:23, and the second polypeptide The IL-1 peptide of claim 11, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO:

17. 2 Mutein.

13. The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:24, and the second polypeptide The IL-1 peptide of claim 11, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO:

17. 2 Mutein.

14. comprising a first polypeptide and a second polypeptide, The first polypeptide is selected from SEQ ID NOs: 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 7 or 38, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 18 The amino acid sequence described in IL-2 muteins.

15. The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:33, and the second polypeptide The IL-1 peptide of claim 14, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO:

18. 2 Mutein.

16. The first polypeptide comprises the amino acid sequence set forth in SEQ ID NO:34, and the second polypeptide The IL-1 peptide of claim 14, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO:

18. 2 Mutein.

17. comprising a first polypeptide and a second polypeptide, The first and second polypeptides are each represented by SEQ ID NOs: 45, 46, 47, The same comprising the amino acid sequence as set forth in 48, 49, 50, 51, 52, 53 or 54 containing the same amino acid sequence as IL-2 muteins.

18. The first polypeptide comprises the amino acid sequence of SEQ ID NO:45 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

18. The IL-2 mutein of claim 17, comprising the amino acid sequence set forth in No.

45.

19. The first polypeptide comprises the amino acid sequence of SEQ ID NO:46 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:

18. The IL-2 mutein of claim 17, comprising a sequence of 46 amino acids.

20. A pharmaceutical composition comprising an IL-2 mutein according to any one of claims 1 to 19 and a pharma- ceutically acceptable carrier. A pharmaceutical composition comprising:

21. A therapeutically effective amount of an IL-2 mutein according to any one of claims 1 to 19 or claim 2 A method for treating an IL-2 mediated disease in a subject, comprising administering to the subject the pharmaceutical composition according to any one of claims 1 to 5. Treatment method.

22. 22. The method of claim 21, wherein the IL-2 mediated disease is an autoimmune disease.

23. IL-2 mediated diseases include rheumatoid arthritis, Crohn's disease, psoriasis, psoriatic arthritis, and multiple sclerosis. , systemic lupus erythematosus (SLE), cutaneous lupus erythematosus (CLE), lupus nephritis , ankylosing spondylitis, type I diabetes, Sjogren's syndrome, ulcerative colitis, neuromyelitis optica, parsley Acne, scleroderma, temporal arteritis, atopic dermatitis, alopecia areata, graft-versus-host disease (GV 2. The patient is suffering from a chronic inflammatory disease (CIHD), autoimmune hepatitis, primary sclerosing cholangitis or inflammatory myopathy.

1. The method described in 1.

24. Selecting Regulatory T Cells Without Activating CD8+ T Cells in Subjects in Need Thereof A method for selectively activating an IL-1 receptor comprising administering a therapeutically effective amount of an IL-1 receptor according to any one of claims 1 to 19.

21. The method of claim 20, further comprising administering to a subject a -2 mutein or a pharmaceutical composition according to claim 20. method.

25. Activating cells expressing the IL-2 receptor α subunit in a subject in need thereof. A method for selectively activating cells expressing the IL-2 receptor β subunit without A therapeutically effective amount of an IL-2 mutein or a compound according to any one of claims 1 to 19.

21. The method of claim 20, further comprising administering the pharmaceutical composition of claim 20 to a subject. method.

26. (a) a first polypeptide of an IL-2 mutein according to any one of claims 1 to 19, (b) a second polypeptide of an IL-2 mutein according to any one of claims 10 to 19, or (c) a first polypeptide of an IL-2 mutein according to any one of claims 10 to 19; and and a second polypeptide An isolated nucleic acid comprising a nucleotide sequence encoding

27. 27. An expression vector comprising the isolated nucleic acid of claim 26.

28. A host cell comprising the isolated nucleic acid of claim 26 or the expression vector of claim 27. 。

29. Under conditions in which the IL-2 mutein is expressed, (a) culturing the host cell of claim 28; (b) expressing the expression vector of claim 27; or (c) expressing the isolated nucleic acid of claim 26. A method for producing an IL-2 mutein comprising the steps of:

30. A method according to any one of claims 1 to 19 for treating an IL-2 mediated disease in a subject. Use of the IL-2 mutein described above or the pharmaceutical composition according to claim 20.

31. A method according to any one of claims 1 to 19 for the treatment of an IL-2 mediated disease in a subject. or the pharmaceutical composition according to claim 20.

32. A method for producing a medicament for treating an IL-2 mediated disease in a subject, comprising administering to said subject a compound according to claims 1 to 4.

21. Use of an IL-2 mutein according to any one of claims 17 or a pharmaceutical composition according to claim 20.

33. The use according to any one of claims 30 to 32, wherein the IL-2 mediated disease is an autoimmune disease.

34. IL-2 mediated diseases include rheumatoid arthritis, Crohn's disease, psoriasis, psoriatic arthritis, and multiple sclerosis. , systemic lupus erythematosus (SLE), cutaneous lupus erythematosus (CLE), lupus nephritis , ankylosing spondylitis, type I diabetes, Sjogren's syndrome, ulcerative colitis, neuromyelitis optica, parsley Acne, scleroderma, temporal arteritis, atopic dermatitis, alopecia areata, graft-versus-host disease (GV 3. The disease is selected from the group consisting of hypercalcaemia, hypercalcaemia, and hypercalcaemia, and is selected from the group consisting of hypercalcaemia, hypercalcaemia, and hypercalcaemia.

33. The use according to any one of claims 0 to 32.

35. The method of claim 23 or the method of claim 34, wherein the IL-2 mediated disease is GVHD. use.

36. The method of claim 23 or the use of claim 34, wherein the IL-2 mediated disease is SLE. For.

37. The method of claim 23 or the use of claim 34, wherein the IL-2 mediated disease is CLE. For.

38. The method of claim 23 or claim 34, wherein the IL-2 mediated disease is multiple sclerosis. Use of the above.

39. The method of claim 23 or claim 34, wherein the IL-2 mediated disease is ulcerative colitis. Use of the above.

40. The method of claim 23 or claim 34, wherein the IL-2 mediated disease is Crohn's disease. Use of.

41. 20. A pharmaceutical composition comprising the IL-2 mutein of claim 18 and a pharma- ceutically acceptable carrier.

42. 20. A pharmaceutical composition comprising the IL-2 mutein of claim 19 and a pharma- ceutically acceptable carrier.

43. A therapeutically effective amount of the IL-2 mutein according to claim 18 or the pharmaceutical composition according to claim 41.

23. A method of treating an IL-2 mediated disease in a subject comprising administering to the subject.

44. A therapeutically effective amount of the IL-2 mutein according to claim 19 or the pharmaceutical composition according to claim 42. A method of treating an IL-2 mediated disease in a subject comprising administering to the subject.

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