CN118515780A

CN118515780A - IL-2 muteins and uses thereof

Info

Publication number: CN118515780A
Application number: CN202410591221.4A
Authority: CN
Inventors: N·希金森-斯科特; J·L·瓦伊尼; J·威斯维斯瓦拉亚; E·R·桑普森; K·L·奥蒂波比
Original assignee: Pandeon Operating Co
Current assignee: Pandeon Operating Co
Priority date: 2017-12-06
Filing date: 2018-11-28
Publication date: 2024-08-20
Also published as: CA3083941A1; EP3720470A4; AU2018378078A1; KR102687035B1; KR20200097275A; CN111432831A; SG11202003965VA; EP3720470A1; KR20240115352A; KR102684426B1; JP7250790B2; IL274844A; CN111432831B; AU2024216451A1; WO2019112854A1; MX2020005208A; BR112020011343A2; KR20240039201A; JP2021507690A; AU2018378078B2

Abstract

Described herein are therapeutic agents that can modulate (e.g., increase) T-reg cell proliferation, survival, activation, and/or function. In some embodiments, the modulation is selective or specific for T-reg cells. The application provides IL-2 muteins, compositions comprising the same, and methods of use thereof. In another aspect, embodiments of the application provide compositions, e.g., pharmaceutically acceptable compositions, comprising a therapeutic compound described herein (IL-2 mutein) formulated with a pharmaceutically acceptable carrier. Kits comprising the therapeutic compounds described herein are also within the scope of the application.

Description

IL-2 muteins and uses thereof

Technical Field

Embodiments provided herein relate to proteins known as IL-2 muteins, compositions comprising the same, and methods of use thereof.

Background

IL-2 binds to three transmembrane receptor subunits: IL-2Rβ and IL-2Rγ (which together activate intracellular signaling events upon IL-2 binding), and CD25 (IL-2Rα) (which is used to present IL-2 to the other 2 receptor subunits). Signals transmitted by IL-2Rβγ include signals of the PI 3-kinase, ras-MAP-kinase and STAT5 pathways.

T cells require expression of CD25 in response to low concentrations of IL-2 that are typically present in tissues. T cells expressing CD25 include CD4 ⁺FOXP3⁺ regulatory T cells (T-reg cells) -which are necessary for inhibiting autoimmune inflammation-and FOXP3 ^- T cells, which have been activated to express CD25.FOXP3 ^-CD4⁺ T effector cells (T-eff) may be CD4 ⁺ or CD8 ⁺ cells, both of which may be pro-inflammatory, and may contribute to autoimmunity and other diseases in which the subject's immune system attacks organs or other tissues. IL-2 stimulated STAT5 signaling is critical for normal T-reg cell growth and survival and for high FOXP3 expression.

Since IL-2 has a low affinity for each of the three IL-2R chains, further decreases in affinity for IL-2Rβ and IL-2Rγ can be offset by increases in affinity for CD 25. Mutant variants of IL-2 have been produced. These IL-2 mutants may be referred to as IL-2 muteins and have been found useful in the treatment of a variety of diseases. However, there remains a need for additional IL-2 muteins that can be used in a variety of applications and compositions. Embodiments of the present invention address these needs and others.

Disclosure of Invention

In some embodiments, a peptide comprising the amino acid sequence SEQ ID NO. 1 is provided, wherein the peptide comprises a mutation at position 73, 76, 100 or 138.

In some embodiments, a peptide comprising the amino acid sequence SEQ ID NO.2 is provided, wherein the peptide comprises a mutation at position 53, 56, 80 or 118.

In some embodiments, the peptide comprises the amino acid sequence SEQ ID NO:43, wherein at least one of X ₁、X₂, and X ₃ and X ₄ is I, and the remainder are L or I.

Pharmaceutical compositions comprising the peptides and nucleic acid molecules encoding the proteins described herein are also provided. Also provided herein are vectors comprising nucleic acid molecules encoding the proteins described herein. In some embodiments, plasmids comprising nucleic acids encoding the proteins described herein are provided. In some embodiments, cells comprising a nucleic acid molecule, vector, or plasmid encoding a protein described herein are provided.

In some embodiments, methods of activating T regulatory cells are provided. In some embodiments, the method comprises contacting the T regulatory cells with a peptide described herein or a pharmaceutical composition described herein.

In some embodiments, methods of treating an inflammatory disorder in a subject are provided. In some embodiments, the methods comprise administering a peptide (e.g., a therapeutically effective amount of a peptide) to a subject, including but not limited to a subject in need thereof.

In some embodiments, methods of promoting or stimulating STAT5 phosphorylation in T regulatory cells are provided. In some embodiments, the methods comprise administering a peptide (e.g., a therapeutically effective amount of a peptide) to a subject.

Drawings

FIG. 1 shows a non-limiting embodiment of an IL-2 mutein provided herein.

Detailed Description

Described herein are therapeutic agents that can modulate (e.g., increase) T-reg cell proliferation, survival, activation, and/or function. In some embodiments, the modulation is selective or specific for T-reg cells.

As used herein, the term "selective" refers to a therapeutic agent or protein that modulates activity in T-reg cells, but has limited or no ability to promote activity in non-regulatory T cells.

In some embodiments, the therapeutic agent is a mutant of IL-2. Mutants of IL-2 may be referred to as IL-2 muteins. IL-2 can exist in two different forms (immature and mature). The mature form is a form from which the leader sequence has been removed. This is done in a post-translational process. The wild-type sequence of immature IL-2 is as follows:

MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT FKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCE YADETATIVEFLNRWITFCQSIISTLT(SEQ ID NO:1).

the wild-type sequence of mature IL-2 is as follows:

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE ELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSI ISTLT( Mature IL-2 sequence) (SEQ ID NO: 2).

IL-2 mutein molecules can be prepared by mutating one or more residues of IL-2. Non-limiting examples of IL-2 muteins can be found in WO2016/164937、US9580486、US7105653、US9616105、US 9428567、US2017/0051029、US2014/0286898A1、WO2014153111A2、WO2010/085495、WO2016014428A2、WO2016025385A1 and US20060269515, each of which is incorporated by reference in its entirety.

In some embodiments, the alanine at position 1 of the above sequence (SEQ ID NO: 2) is deleted. In some embodiments, the IL-2 mutein molecule comprises serine that replaces the cysteine at position 125 of the mature IL-2 sequence. Other combinations of mutations and substitutions of IL-2 mutein molecules are described in US20060269515, which is incorporated by reference in its entirety. In some embodiments, the cysteine at position 125 is also substituted with valine or alanine. In some embodiments, the IL-2 mutein molecule comprises the V91K substitution. In some embodiments, the IL-2 mutein molecule comprises the N88D substitution. In some embodiments, the IL-2 mutein molecule comprises the N88R substitution. In some embodiments, the IL-2 mutein molecule comprises the H16E, D84K, V91N, N88D, V K or V91R substitution, any combination thereof. In some embodiments, these IL-2 mutein molecules further comprise a substitution at position 125 as described herein. In some embodiments, the IL-2 mutein molecule comprises one or more substitutions ：T3N、T3A、L12G、L12K、L12Q、L12S、Q13G、E15A、E15G、E15S、H16A、H16D、H16G、H16K、H16M、H16N、H16R、H16S、H16T、H16V、H16Y、L19A、L19D、L19E、L19G、L19N、L19R、L19S、L19T、L19V、D20A、D20E、D20H、D20I、D20Y、D20F、D20G、D20T、D20W、M23R、R81A、R81G、R81S、R81T、D84A、D84E、D84G、D84I、D84M、D84QD84R、D84S、D84T、S87R、N88A、N88D、N88E、N88I、N88F、N88G、N88M、N88R、N88S、N88V、N88W、V91D、V91E、V91G、V91S、I92K、I92R、E95G and Q126 selected from the group consisting of. In some embodiments, the amino acid sequence of the IL-2 mutein molecule differs from the amino acid sequence set forth in the mature IL-2 sequence in the C125A or C125S substitution and one substitution selected from T3N、T3A、L12G、L12K、L12Q、L12S、Q13G、E15A、E15G、E15S、H16A、H16D、H16G、H16K、H16M、H16N、H16R、H16S、H16T、H16V、H16Y、L19A、L19D、L19E、L19G、L19N、L19R、L19S、L19T、L19V、D20A、D20E、D20F、D20G、D20T、D20W、M23R、R81A、R81G、R81S、R81T、D84A、D84E、D84G、D84I、D84M、D84Q、D84R、D84S、D84T、S87R、N88A、N88D、N88E、N88F、N88I、N88G、N88M、N88R、N88S、N88V、N88W、V91D、V91E、V91G、V91S、I92K、I92R、E95G、Q126I、Q126L and Q126F. In some embodiments, the IL-2 mutein molecule differs from the amino acid sequence shown in the mature IL-2 sequence in the C125A or C125S substitution and one substitution selected from D20H、D20I、D20Y、D20E、D20G、D20W、D84A、D84S、H16D、H16G、H16K、H16R、H16T、H16V、I92K、I92R、L12K、L19D、L19N、L19T、N88D、N88R、N88S、V91D、V91G、V91K and V91S. In some embodiments, the IL-2 mutein comprises the N88R and/or D20H mutation.

In some embodiments, the IL-2 mutein molecule comprises a mutation at a position in the polypeptide sequence selected from the group consisting of amino acid position 30, amino acid position 31, amino acid position 35, amino acid position 69, and amino acid position 74, in some embodiments the mutation at position 30 is N30S. In some embodiments, the mutation at position 31 is Y31H. In some embodiments, the mutation at position 35 is K35R. In some embodiments, the mutation at position 69 is V69A. In some embodiments, the mutation at position 74 is Q74P. In some embodiments, the mutein does not comprise mutations at positions 30, 31 and/or 35.

In some embodiments, the IL-2 mutein molecule comprises a substitution selected from the group consisting of: N88R, N88I, N G, D20H, D109C, Q126L, Q F, D G or D84I. In some embodiments, the IL-2 mutein molecule comprises the D109C substitution and one or both of the N88R substitution and the C125S substitution. In some embodiments, the cysteine at position 109 in the IL-2 mutein molecule is linked to a polyethylene glycol moiety, wherein the molecular weight of the polyethylene glycol moiety is about 5 to about 40kDa. In some embodiments, the mutein does not comprise a mutation at position 109, 126 or 84.

In some embodiments, any permutation described herein is combined with a permutation at position 125. The substitution may be a C125S, C a or C125V substitution. In some embodiments, the mutein does not comprise a mutation at position 125.

Unless otherwise indicated, references herein to IL-2 muteins refer to the mature sequence. If the sequence or position is referenced to SEQ ID NO. 1, it is an immature sequence. However, in order to convert the position from the immature sequence (SEQ ID NO: 1) to the mature sequence (SEQ ID NO: 2), all that is required is to subtract 20 from the position referencing SEQ ID NO:1 to obtain the corresponding position in SEQ ID NO: 2.

In addition to the substitutions or mutations described herein, in some embodiments, the IL-2 mutein has a substitution/mutation at one or more of positions 73, 76, 100 or 138 corresponding to SEQ ID NO. 1 or at one or more of positions 53, 56, 80 or 118 corresponding to SEQ ID NO. 2. In some embodiments, the IL-2 mutein comprises mutations at the following positions corresponding to SEQ ID NO: 1: 73 and 76;73 and 100;73 and 138;76 and 100;76 and 138;100 and 138; 73. 76 and 100; 73. 76 and 138; 73. 100 and 138; 76. 100 and 138; or 73, 76, 100 and 138. In some embodiments, the IL-2 mutein comprises mutations at the following positions corresponding to SEQ ID NO: 2: 53 and 56;53 and 80;53 and 118;56 and 80;56 and 118;80 and 118; 53. 56 and 80; 53. 56 and 118; 53. 80 and 118; 56. 80 and 118; or 53, 56, 80 and 118. Since IL-2 can be fused or tethered to other proteins (tethered), the term as used herein corresponds to reference to SEQ ID NO:6 or 15 to refer to how sequences are aligned to the default settings of the alignment software, such as may be used with the NCBI website. In some embodiments, the mutation is leucine to isoleucine. Thus, the IL-2 mutein comprises one or more isoleucine at position 73, 76, 100 or 138 corresponding to SEQ ID NO. 1 or at one or more of positions 53, 56, 80 or 118 corresponding to SEQ ID NO. 2. In some embodiments, the mutein comprises a mutation at L53 corresponding to SEQ ID NO. 2. In some embodiments, the mutein comprises a mutation at L56 corresponding to SEQ ID NO. 2. In some embodiments, the mutein comprises a mutation at L80 corresponding to SEQ ID NO. 2. In some embodiments, the mutein comprises a mutation at L118 corresponding to SEQ ID NO. 2. In some embodiments, the mutation is leucine to isoleucine. In some embodiments, the muteins further comprise mutations at positions 69, 74, 88, 125 of these muteins corresponding to SEQ ID NO. 2, or any combination thereof. In some embodiments, the mutation is a V69A mutation. In some embodiments, the mutation is a Q74P mutation. In some embodiments, the mutation is an N88D or N88R mutation. In some embodiments, the mutation is a C125A or C125S mutation.

In some embodiments, the IL-2 mutein comprises a mutation at one or more of positions 49, 51, 55, 57, 68, 89, 91, 94, 108 and 145 corresponding to SEQ ID NO. 1 or one or more of positions 29, 31, 35, 37, 48, 69, 71, 74, 88 and 125 corresponding to SEQ ID NO. 2. The substitutions may be used alone or in combination with one another. In some embodiments, the IL-2 mutein comprises mutations at positions 2,3,4,5,6, 7, 8, 9 or each of positions 49, 51, 55, 57, 68, 89, 91, 94, 108 and 145. Non-limiting examples of such combinations include, but are not limited to, mutations at the following positions: 49. 51, 55, 57, 68, 89, 91, 94, 108 and 145; 49. 51, 55, 57, 68, 89, 91, 94 and 108; 49. 51, 55, 57, 68, 89, 91 and 94; 49. 51, 55, 57, 68, 89 and 91; 49. 51, 55, 57, 68 and 89; 49. 51, 55, 57 and 68; 49. 51, 55 and 57; 49. 51 and 55;49 and 51; 51. 55, 57, 68, 89, 91, 94, 108 and 145; 51. 55, 57, 68, 89, 91, 94 and 108; 51. 55, 57, 68, 89, 91 and 94; 51. 55, 57, 68, 89 and 91; 51. 55, 57, 68 and 89; 55. 57 and 68;55 and 57; 55. 57, 68, 89, 91, 94, 108 and 145; 55. 57, 68, 89, 91, 94 and 108; 55. 57, 68, 89, 91 and 94; 55. 57, 68, 89, 91 and 94; 55. 57, 68, 89 and 91; 55. 57, 68 and 89; 55. 57 and 68;55 and 57; 57. 68, 89, 91, 94, 108 and 145; 57. 68, 89, 91, 94 and 108; 57. 68, 89, 91 and 94; 57. 68, 89 and 91; 57. 68 and 89;57 and 68; 68. 89, 91, 94, 108 and 145; 68. 89, 91, 94 and 108; 68. 89, 91 and 94; 68. 89 and 91;68 and 89; 89. 91, 94, 108 and 145; 89. 91, 94 and 108; 89. 91 and 94;89 and 91; 91. 94, 108 and 145; 91. 94 and 108;91 and 94; or 94 and 108. Each mutation may be combined with each other. The same substitutions can be made in SEQ ID NO. 2, but as will be clear from the disclosure, the numbering will be adjusted appropriately (20 fewer than the numbering of SEQ ID NO. 1, corresponding to the position in SEQ ID NO. 2).

In some embodiments, the IL-2 mutein comprises a mutation at one or more of positions corresponding to positions 35, 36, 42, 104, 115 or 146 of SEQ ID NO. 1 or at the equivalent position (e.g., positions 15, 16, 22, 84, 95 and 126) of SEQ ID NO. 2. These mutations may be combined with other leucine to isoleucine mutations described herein, either at positions 73, 76, 100 or 138 corresponding to SEQ ID NO. 1, or at one or more of positions 53, 56, 80 or 118 corresponding to SEQ ID NO. 2. In some embodiments, the mutation is E35Q, H N, Q42E, D N, E Q or Q146E, or any combination thereof. In some embodiments, one or more of these substitutions is wild-type. In some embodiments, the mutein comprises a wild-type residue at one or more of positions corresponding to positions 35, 36, 42, 104, 115 or 146 of SEQ ID No. 1 or at positions equivalent to positions 15, 16, 22, 84, 95 or 126 of SEQ ID No. 2.

Mutations at these positions may be combined with any other mutation described herein, including but not limited to substitutions at positions 73, 76, 100 or 138 corresponding to SEQ ID No.1 or at one or more of positions 53, 56, 80 or 118 corresponding to SEQ ID No. 2, as described herein and above. In some embodiments, the IL-2 mutein comprises an N49S mutation corresponding to SEQ ID NO. 1. In some embodiments, the IL-2 mutein comprises a Y51S or Y51H mutation corresponding to SEQ ID NO. 1. In some embodiments, the IL-2 mutein comprises a K55R mutation corresponding to SEQ ID NO. 1. In some embodiments, the IL-2 mutein comprises a T57A mutation corresponding to SEQ ID NO. 1. In some embodiments, the IL-2 mutein comprises a K68E mutation corresponding to SEQ ID NO. 1. In some embodiments, the IL-2 mutein comprises a V89A mutation corresponding to SEQ ID NO. 1. In some embodiments, the IL-2 mutein comprises the N91R mutation corresponding to SEQ ID NO. 1. In some embodiments, the IL-2 mutein comprises a Q94P mutation corresponding to SEQ ID NO. 1. In some embodiments, the IL-2 mutein comprises the N108D or N108R mutation corresponding to SEQ ID NO. 1. In some embodiments, the IL-2 mutein comprises a C145A or C145S mutation corresponding to SEQ ID NO. 1.

These substitutions may be used alone or in combination with one another. In some embodiments, the mutein comprises each of these substitutions. In some embodiments, the mutein comprises 1, 2,3, 4, 5, 6, 7, or 8 of these mutations. In some embodiments, the mutein comprises wild-type residues at one or more of the positions corresponding to positions 35, 36, 42, 104, 115 or 146 of SEQ ID NO. 1 or at positions equivalent to positions 15, 16, 22, 84, 95, 126 and 126 of SEQ ID NO. 2.

In some embodiments, the IL-2 mutein comprises an N29S mutation corresponding to SEQ ID NO. 2. In some embodiments, the IL-2 mutein comprises a Y31S or Y31H mutation corresponding to SEQ ID NO. 2. In some embodiments, the IL-2 mutein comprises a K35R mutation corresponding to SEQ ID NO. 2. In some embodiments, the IL-2 mutein comprises a T37A mutation corresponding to SEQ ID NO. 2. In some embodiments, the IL-2 mutein comprises a K48E mutation corresponding to SEQ ID NO. 2. In some embodiments, the IL-2 mutein comprises a V69A mutation corresponding to SEQ ID NO. 2. In some embodiments, the IL-2 mutein comprises a N71R mutation corresponding to SEQ ID NO. 2. In some embodiments, the IL-2 mutein comprises a Q74P mutation corresponding to SEQ ID NO. 2. In some embodiments, the IL-2 mutein comprises a N88D or N88R mutation corresponding to SEQ ID NO. 2. In some embodiments, the IL-2 mutein comprises a C125A or C125S mutation corresponding to SEQ ID NO. 2. These substitutions may be used alone or in combination with one another. In some embodiments, the mutein comprises 1, 2, 3, 4, 5, 6, 7, or 8 of these mutations. In some embodiments, the mutein comprises each of these substitutions. In some embodiments, the mutein comprises wild-type residues at one or more of the positions corresponding to positions 35, 36, 42, 104, 115 or 146 of SEQ ID NO. 1 or at the equivalent positions of SEQ ID NO. 2 (e.g., positions 15, 16, 22, 84, 95 and 126).

For any of the IL-2 muteins described herein, in some embodiments, one or more of the positions corresponding to positions 35, 36, 42, 104, 115, or 146 of SEQ ID NO.1 or at positions equivalent to SEQ ID NO.2 (e.g., positions 15, 16, 22, 84, 95, and 126) are wild-type (e.g., as shown in SEQ ID NO.1 or 2). In some embodiments, the 2, 3, 4, 5, 6 or each of positions corresponding to positions 35, 36, 42, 104, 115 or 146 of SEQ ID NO.1 or in equivalent positions (e.g., positions 15, 16, 22, 84, 95 and 126) of SEQ ID NO.2 are wild type.

In some embodiments, the IL-2 mutein comprises the following sequences:

MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLT FKFYMPEKATEIKHLQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT(SEQ ID NO:3)

In some embodiments, the IL-2 mutein comprises the following sequences:

MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLT FKFYMPEKATELKHIQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT(SEQ ID NO:4)

In some embodiments, the IL-2 mutein comprises the following sequences:

MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLT FKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHIRPRDLISDINVIVLELKGSETTFMCEY ADETATIVEFLNRWITFSQSIISTLT(SEQ ID NO:5)

In some embodiments, the IL-2 mutein comprises the following sequences:

MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLT FKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEY ADETATIVEFINRWITFSQSIISTLT(SEQ ID NO:6)

In some embodiments, the IL-2 mutein sequences described herein do not comprise an IL-2 leader sequence. The IL-2 leader sequence may be represented by sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7). Thus, in some embodiments, the sequences shown above may also encompass peptides without a leader sequence. Although shown as having mutations at only one of positions 73, 76, 100 or 138 corresponding to SEQ ID NO. 1 or one or more of positions 53, 56, 80 or 118 corresponding to SEQ ID NO. 2, the peptide may contain 1,2, 3 or 4 mutations at these positions. In some embodiments, the substitutions at each position are isoleucine or other types of conservative amino acid substitutions. In some embodiments, the leucine at the position is independently replaced with isoleucine, valine, methionine or glycine, alanine, glutamine or glutamic acid.

In some embodiments, the IL-2 protein of SEQ ID NO. 2 comprises the following mutations: V69A, Q74P, N D, and C125S or C125A, and one mutation selected from the group consisting of L53I, L I, L I and L118I. In some embodiments, the IL-2 protein comprises two mutations selected from the group consisting of L53I, L, I, L I and L118I. In some embodiments, the IL-2 protein comprises three mutations or each mutation selected from the group consisting of L53I, L, I, L I and L118I. In some embodiments, the IL-2 protein comprises L53I and L56I, L53I and L80I, L53I and L118I, L56I and L80I, L56I and L118I, L80I and L118I, L53I, L I and L80I, L53I, L56I and L118I, L56I, L I and L118I, or L53I, L56I, L I and L118I. In some embodiments, the IL-2 mutein does not comprise the L53I, L56I, L I or L118I mutation. In some embodiments, the IL-2 mutein comprises a T3A mutation.

In some embodiments, the IL-2 protein of SEQ ID NO. 2 comprises the following mutations: V69A, Q, P, N D, and C125S or C125A, and one or more mutations in the 45-55, 50-60, 52-57, 75-85, 100-130, 115-125 regions of SEQ ID NO. 2, such as, but not limited to, conservative substitutions.

In some embodiments, the IL-2 mutein molecule is fused to the Fc region or other linker region as described herein. Examples of such fusion proteins can be found in US9580486、US7105653、US9616105、US9428567、US2017/0051029、WO2016/164937、US2014/0286898A1、WO2014153111A2、WO2010/085495、WO2016014428A2、WO2016025385A1、US2017/0037102 and US2006/0269515, each of which is incorporated by reference in its entirety.

In some embodiments, the Fc region comprises a mutation known at the LALA mutation. In some embodiments, the Fc region comprises L234A and L235A mutations (EU numbering). In some embodiments, the Fc region comprises G237A (EU numbering). In some embodiments, the Fc region does not comprise a mutation at position G237 (EU numbering). Using Kabat numbering this would correspond to L247A, L248A and/or G250A. In some embodiments, the Fc region comprises an L234A mutation, an L235A mutation, and/or a G237A mutation using the EU numbering system. Regardless of the numbering system used, in some embodiments, the Fc portion may comprise mutations corresponding to one or more of these residues. In some embodiments, the Fc region comprises a N297G or N297A (kabat numbering) mutation. Kabat numbering is based on full length sequences, but conventional alignments based on use of Fc regions by those skilled in the art will be used in fragment form (see, e.g., Kabat et al.("Sequence of proteins of immunological interest,"US Public Health Services,NIH Bethesda,MD,Publication No.91,, incorporated herein by reference), which is incorporated herein by reference. In some embodiments, the Fc region comprises the following sequence:

DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.(SEQ ID NO:8)

In some embodiments, the Fc region comprises the following sequence:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:15)

In some embodiments, the IL-2 mutein is linked to the Fc region. A non-limiting example of a linker is a glycine/serine linker. For example, the glycine/serine linker may be or comprise the sequence GGGGSGGGGSGGGGGGSGGGGS (SEQ ID NO: 9), or may be or comprise the sequence GGGGSGGGGSGGGGGGS (SEQ ID NO: 16). This is just one non-limiting example, and the linker may have a different number of GGGGS (SEQ ID NO: 10) repeats. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 GGGGS (SEQ ID NO: 10) repeats.

In some embodiments, the IL-2 mutein is linked to the Fc region using a flexible, rigid or cleavable linker. The linker may be as described herein or as shown in the following table:

Type(s)	Sequence(s)
		Flexible and flexible	GGGGS
Flexible and flexible	(GGGGS)₃
		Flexible and flexible	(GGGGS)_n(n＝1,2,3,4)
Flexible and flexible	(Gly)₈
		Flexible and flexible	(Gly)₆
Rigidity of	(EAAAK)₃
		Rigidity of	(EAAK)_n(n＝1-3)
Rigidity of	A(EAAAK)₄ALEA(EAAAK)₄A
		Rigidity of	AEAAAKEAAAKA
Rigidity of	PAPAP
		Rigidity of	(Ala-Pro)_n(10-34aa)
Cuttable	Disulfide compounds
		Cuttable	VSQTSKLTRAETVFPDV
Cuttable	PLGLWA
		Cuttable	RVLAEA
Cuttable	EDVVCCSMSY
		Cuttable	GGIEGRGS
Cuttable	TRHRQPRGWE
		Cuttable	AGNRVRRSVG
Cuttable	RRRRRRRRR
		Cuttable	GFLG
Dipeptide	LE

Thus, an IL-2/Fc fusion may be represented by formula Z _IL-2M-L_gs-Z_Fc, wherein Z _IL-2M is an IL-2 mutein as described herein, L _gs is a linker sequence (e.g., glycine/serine linker) as described herein, and Z _Fc is an Fc region as described herein or known to those of skill in the art. In some embodiments, the formula may be a reverse orientation Z _Fc-L_gs-Z_IL-2M.

In some embodiments, the IL-2/Fc fusion comprises the following sequences:

MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATEIKHLQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:11)

In some embodiments, the IL-2/Fc fusion comprises the following sequences:

MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHIQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:12)

In some embodiments, the IL-2/Fc fusion comprises the following sequences:

MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHIRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:13)

In some embodiments, the IL-2/Fc fusion comprises the following sequences:

MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFINRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:14).

In some embodiments, the Fc region of SEQ ID NO. 8 is replaced with SEQ ID NO. 15.

The proteins described herein may also be fused to another protein (e.g., an antibody or other type of therapeutic molecule).

In some embodiments, the sequence of the IL-2 mutein or IL-2/Fc fusion is shown in the following table:

Each protein may also be considered to have C125S and LALA and/or G237A mutations as provided herein. The C125 substitution may also be C125A as described throughout the present application.

In some embodiments, the sequences shown in the table or throughout the present application comprise or do not comprise one or more mutations corresponding to positions L53, L56, L80 and L118. In some embodiments, the sequences shown in the table or throughout the present application comprise or do not comprise one or more mutations corresponding to positions L59I, L63, 63I, I24L, L94I, L I or L132I, or other substitutions at the same positions. In some embodiments, the mutation is leucine to isoleucine. In some embodiments, the mutein does not comprise another mutation than shown or described herein. In some embodiments, the peptide comprises the following sequence ：SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42 or SEQ ID NO 43.

In some embodiments, the Fc portion of the fusion is not included. In some embodiments, the peptide consists essentially of an IL-2 mutein provided herein. In some embodiments, the protein does not contain an Fc portion.

In some embodiments, a polypeptide comprising SEQ ID NO. 43 is provided, wherein at least one of X ₁、X₂、X₃ and X ₄ is I and the remainder are L or I. In some embodiments, X ₁、X₂ and X ₃ are L and X ₄ is I. In some embodiments, X ₁、X₂ and X ₄ are L and X ₃ is I. In some embodiments, X ₂、X₃ and X ₄ are L and X ₁ is I. In some embodiments, X ₁、X₃ and X ₄ are L and X ₂ is I. In some embodiments, X ₁、X₂ is L and X ₃ and X ₄ are I. In some embodiments, X ₁ and X ₃ are L and X ₂ and X ₄ are I. In some embodiments, X ₁ and X ₄ are L and X ₂ and X ₃ are I. In some embodiments, X ₂ and X ₃ are L and X ₁ and X ₄ are I. In some embodiments, X ₂ and X ₄ are L and X ₁ and X ₃ are I. In some embodiments, X ₃ and X ₄ are L and X ₁ and X ₂ are I. In some embodiments, X ₁、X₂ and X ₃ are L and X ₄ is I. In some embodiments, X ₂、X₃ and X ₄ are L and X ₁ is I. In some embodiments, X ₁、X₃ and X ₄ are L and X ₂ is I. In some embodiments, X ₁、X₂ and X ₄ are L and X ₃ is I.

In some embodiments, IL-2 mutant proteins can be in the form as shown in FIG. 1. However, as described herein, in some embodiments, IL-2 muteins without an Fc domain may be used, or the Fc domain may be linked to the N-terminus of the IL-2 mutein, as opposed to the C-terminus of the Fc domain to the IL-2 mutein. The polypeptides described herein also encompass variants of the peptides. In some embodiments, the IL-2 variant comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% substantially similar to the sequences provided herein. Variants include those described herein, with various substitutions described herein and above. In some embodiments, the variant has 1,2,3,4, or 5 additional substitutions. In some embodiments, the substitutions are G to A, L to I, G to S, K to R, or other types of conservative substitutions. In some embodiments, conservative substitutions are selected based on the following table:

The percent identity of two amino acid or two nucleic acid sequences can be determined by visual inspection and mathematical calculation, or, for example, by comparing sequence information using a computer program. Exemplary computer programs are preferred default parameters for Genetics Computer Group(GCG;Madison,Wis.)Wisconsin package version 10.0program,GAP(Devereux et al.(1984),Nucleic Acids Res.12:387-95).GAP programs including: (1) A unitary comparison matrix of nucleotides (containing a value of 1 for identity and 0 for non-identity) was implemented by GCG, and a weighted amino acid comparison matrix of Gribskov and Burgess ((1986) Nucleic Acids Res. 14:6745), as described in Atlas of Polypeptide Sequence and Structure,Schwartz and Dayhoff,eds.,National Biomedical Research Foundation,pp.353-358(1979), or other comparable comparison matrix; (2) For amino acid sequences, each gap is penalized 8 and each symbol in each gap is additionally penalized 2, or for nucleotide sequences, each gap is penalized 50 and each symbol in each gap is additionally penalized 3; (3) no penalty for end gaps; and (4) no maximum penalty for long gaps. Other procedures used by those skilled in the art of sequence comparison may also be used.

In some embodiments, the IL-2 muteins provided herein include proteins that have altered signaling through certain pathways of wild-type IL-2 activation via IL-2R and result in preferential proliferation/survival/activation of T-reg.

The IL-2 muteins provided herein can be produced using any suitable method known in the art, including those methods described in U.S. Pat. No. 6,955,807 for producing IL-2 variants, which are incorporated herein by reference. Such methods include constructing DNA sequences encoding IL-2 variants and expressing these sequences in a suitably transformed host, such as a host cell. Recombinant proteins as provided herein will be produced using these methods. The proteins may also be produced synthetically, or a combination of fragments may be produced synthetically and recombinantly in cells, and then the fragments combined to produce the complete protein of interest.

In some embodiments, the nucleic acid molecule (e.g., DNA or RNA) is prepared by isolating or synthesizing a nucleic acid molecule encoding the protein of interest. Alternatively, the wild-type sequence of IL-2 may be isolated and mutated using conventional techniques, such as site-specific mutagenesis.

Another method of constructing a DNA sequence encoding an IL-2 variant would be chemical synthesis. This includes, for example, direct synthesis by chemical means of peptides encoding protein sequences of IL-2 variants exhibiting the properties described herein. The method can introduce natural and unnatural amino acids at different positions. Alternatively, the nucleic acid molecule encoding the desired protein may be synthesized by chemical means using an oligonucleotide synthesizer. Oligonucleotides are designed based on the amino acid sequence of the desired protein, which can also be selected by using codons that are advantageous in the cell in which the recombinant variant is to be produced. It is recognized that the genetic code is degenerate-amino acids may be encoded by more than one codon. Thus, it will be appreciated that for a given DNA sequence encoding a particular IL-2 protein, there will be many degenerate sequences of DNA that will encode a variant of that IL-2. Thus, in some embodiments, nucleic acid molecules encoding the proteins described herein are provided. The nucleic acid molecule may be DNA or RNA.

In some embodiments, the nucleic acid molecule will encode a signal sequence. The signal sequence may be selected based on the cell in which it is to be expressed. In some embodiments, if the host cell is prokaryotic, the nucleic acid molecule does not comprise a signal sequence. In some embodiments, if the host cell is a eukaryotic cell, a signal sequence may be used. In some embodiments, the signal sequence is an IL-2 signal sequence.

As referred to herein, a nucleic acid molecule "encodes" a protein if the nucleic acid molecule or its complement comprises codons encoding the protein.

"Recombinant" when applied to a polypeptide or protein means that the production of the protein depends on at least one step in which nucleic acids that may or may not encode the protein are introduced into cells in which they are not naturally occurring.

Various hosts (animal or cellular systems) can be used to produce the proteins described herein. Examples of suitable host cells include, but are not limited to, bacteria, fungi (including yeast), plants, insects, mammals, or other suitable animal cells or cell lines, as well as transgenic animals or plants. In some embodiments, these hosts may include well-known eukaryotic and prokaryotic hosts in tissue culture, such as E.coli strains, pseudomonas strains, bacillus strains, streptomyces strains, fungi, yeasts, insect cells such as Spodoptera frugiperda (Sf 9), animal cells such as Chinese Hamster Ovary (CHO) and mouse cells such as NS/O, african green monkey cells such as COS1, COS 7, BSC 1, BSC 40, and BNT 10, and human cells, as well as plant cells. For animal cell expression, CHO cells and COS 7 cells in culture can be used, and in particular CHO cell lines CHO (DHFR-) or HKB lines.

Of course, it should be understood that not all vectors and expression control sequences will be equally well capable of expressing the DNA sequences described herein. Nor do all hosts function equally well with the same expression system. However, one skilled in the art can select among these vectors, expression control sequences and hosts without undue experimentation. For example, in selecting a vector, the host must be considered, as the vector must replicate therein. Vector copy number, the ability to control that copy number, and the expression of any other protein encoded by the vector (e.g., antibiotic markers) should also be considered. For example, preferred vectors for use in the present invention include those that allow for copy number amplification of DNA encoding IL-2 variants. Such amplifiable vectors are well known in the art.

Vectors and host cells

Thus, in some embodiments, vectors encoding the proteins described herein are provided, as well as host cells transformed with such vectors. Any nucleic acid encoding a protein described herein may be contained in a vector, which may, for example, contain a selectable marker and an origin of replication for propagation in a host. In some embodiments, the vector further comprises suitable transcriptional or translational regulatory sequences, such as those derived from mammalian, microbial, viral, or insect genes, operably linked to the nucleic acid molecule encoding the protein. Examples of such regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosome binding sites, and appropriate sequences which control transcription and translation. Nucleotide sequences are operably linked when the regulatory sequences are functionally related to the DNA encoding the target protein. Thus, a promoter nucleotide sequence is operably linked to a nucleic acid molecule if the promoter nucleotide sequence directs the transcription of the nucleic acid molecule.

Described herein are host cells that can be used herein.

Pharmaceutical composition

In another aspect, embodiments of the invention provide compositions, e.g., pharmaceutically acceptable compositions, comprising a therapeutic compound described herein (IL-2 mutein) formulated with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, topical, spinal or epidermal administration (e.g., by injection or infusion).

The compositions of the present invention may be in various forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical compositions are in the form of injectable or infusible solutions. In one embodiment, the mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In one embodiment, the therapeutic molecule is administered by intravenous infusion or injection. In another embodiment, the therapeutic molecule is administered by intramuscular or subcutaneous injection. In another embodiment, the therapeutic molecule is administered topically, e.g., by injection or topically, to the target site.

The phrases "parenteral administration" and "parenteral administration" as used herein refer to modes of administration other than enteral and topical administration, typically by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular (subcuticular), intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

The therapeutic composition should generally be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, dispersions, liposomes or other ordered structures suitable for high therapeutic molecule concentrations. Sterile injectable solutions may be prepared by incorporating the active compound (i.e., the therapeutic molecule) in the required amount in the appropriate solvent with one or more enumerated ingredients, or a combination thereof, as required, followed by filtered sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. For example, by using a coating such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants, proper fluidity of the solution may be maintained. Prolonged absorption of the injectable compositions can be brought about by the inclusion in the composition of agents which delay absorption, for example, monostearates and gelatins.

As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired result. In certain embodiments, the active compounds may be prepared with carriers that will protect the compound from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Many methods of preparing such formulations have been patented or are well known to those skilled in the art. See, for example ,Sustained and Controlled Release Drug Delivery Systems,J.R.Robinson,ed.,Marcel Dekker,Inc.,New York,1978.

In certain embodiments, the therapeutic compound may be administered orally, e.g., with an inert diluent or an assimilable edible carrier. The compounds (and other ingredients, if desired) may also be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or introduced directly into the subject's diet. For oral therapeutic administration, the compounds may be combined with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. In order to administer the compounds of the present invention by means other than parenteral administration, it may be desirable to coat the compound with a material or co-administer the compound with the material to prevent its inactivation. The therapeutic composition may also be administered with medical devices known in the art.

The dosage regimen is adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus may be administered, several separate doses may be administered over time, or the doses may be proportionally reduced or increased as indicated by the emergency of the treatment situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for subjects to be treated; each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect, and the desired pharmaceutical carrier. The specification of the dosage unit form of the invention is determined by (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such active compounds for the treatment of sensitivity in an individual, and is directly dependent on (a) and (b).

An exemplary non-limiting range of a therapeutically or prophylactically effective amount of the therapeutic compound is 0.1-30mg/kg, more preferably 1-25mg/kg. The dosage of the therapeutic compound and the treatment regimen can be determined by the skilled artisan. In certain embodiments, the therapeutic compound is administered by injection (e.g., subcutaneously or intravenously) at a dose of about 1 to 40mg/kg, e.g., 1 to 30mg/kg, e.g., about 5 to 25mg/kg, about 10 to 20mg/kg, about 1 to 5mg/kg,1 to 10mg/kg,5 to 15mg/kg,10 to 20mg/kg,15 to 25mg/kg, or about 3 mg/kg. The dosing regimen may vary from, for example, once a week to once every 2, 3, or 4 weeks, or in some embodiments, the dosing regimen may be once a month, once every 2 months, once every 3 months, or once every 6 months. In one embodiment, the therapeutic compound is administered at a dose of about 10 to 20mg/kg every two weeks. The therapeutic compound may be administered by intravenous infusion at a rate in excess of 20mg/min, such as 20-40mg/min, and typically greater than or equal to 40mg/min, to achieve a dose of about 35 to 440mg/m2, typically about 70 to 310mg/m2, and more typically about 110 to 130mg/m 2. In embodiments, an infusion rate of about 110 to 130mg/m2 achieves a level of about 3 mg/kg. In other embodiments, the therapeutic compound may be administered at a rate of less than 10mg/min, such as less than or equal to 5mg/min, by intravenous infusion to achieve a dose of about 1 to 100mg/m2, such as about 5 to 50mg/m2, about 7 to 25mg/m2, or about 10mg/m 2. In some embodiments, the therapeutic compound is infused over a period of about 30 minutes. It should be noted that the dosage value may vary with the type and severity of the condition to be alleviated. It will further be appreciated that for any particular subject, the particular dosage regimen should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

The pharmaceutical compositions of the invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of a therapeutic molecule of the invention. By "therapeutically effective amount" is meant an amount effective to achieve the desired therapeutic result at the necessary dosage and for the period of time. The therapeutically effective amount of the therapeutic molecule may vary depending on factors such as the disease state, age, sex and weight of the individual, and the ability of the therapeutic compound to elicit a desired response in the individual. A therapeutically effective amount is also an amount by which any toxic or detrimental effects of the therapeutic molecule are exceeded by the therapeutically beneficial effects. The "therapeutically effective dose" preferably inhibits a measurable parameter (e.g., immune challenge) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and still more preferably at least about 80% relative to an untreated subject. The ability of a compound to inhibit a measurable parameter (e.g., immune attack) can be evaluated in an animal model system that predicts efficacy in transplant rejection or autoimmune disorders. Alternatively, such properties of the composition may be assessed by examining the ability of the compound to inhibit such inhibition in vitro using assays known to the skilled practitioner.

By "prophylactically effective amount" is meant an amount effective to achieve the desired prophylactic result at the necessary dosage and for the period of time. Typically, since a prophylactic dose is used in a subject prior to or at an early stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

Kits comprising the therapeutic compounds described herein are also within the scope of the invention. The kit may include one or more other elements including: instructions for use; other agents, such as labels, therapeutic agents, or agents for chelating or otherwise coupling therapeutic molecules to labels or other therapeutic agents, or radioprotective compositions; preparing a device or other material of the therapeutic molecule for administration; a pharmaceutically acceptable carrier; and devices or other materials for administration to a subject.

Combination of

The proteins described herein may also be administered in combination with other agents for treating conditions in a patient. Examples of such agents include proteinaceous and non-proteinaceous drugs. When multiple therapeutic agents are co-administered, the dosage may be adjusted accordingly as recognized in the relevant art. "co-administration" and combination therapy are not limited to simultaneous administration, but also include treatment regimens in which the T-reg-selective IL-2 protein is administered at least once during a course of treatment involving administration of at least one other therapeutic agent to a patient.

In some embodiments, the T-reg-selective IL-2 protein is administered in combination with an inhibitor of the PI3-K/AKT/mTOR pathway, such as rapamycin (rapamune), sirolimus (sirolimus)). Inhibitors of this pathway in combination with IL-2 facilitate T-reg enrichment. In some embodiments, the IL-2 protein is administered without administration of another therapeutic agent that is not directly fused or attached to the IL-2 protein.

Therapeutic method

"Treating" of any disease as referred to herein includes alleviating at least one symptom of the disease, reducing the severity of the disease, or delaying or preventing the progression of the disease to a more severe symptom that may in some cases be accompanied by the disease or to at least one other disease. Treatment need not mean complete cure of the disease. Useful therapeutic agents need only reduce the severity of the disease, reduce the severity of one or more symptoms associated with the disease or treatment thereof, or delay the onset of more severe symptoms or more severe disease that may occur at a frequency after the treated condition. For example, if the disease is an inflammatory bowel disease, the therapeutic agent may reduce the number of distinct sites of inflammation in the intestine, the overall extent of the affected intestine, reduce pain and/or swelling, reduce symptoms such as diarrhea, constipation, or vomiting, and/or prevent perforation of the intestine. The condition of the patient may be assessed by standard techniques, such as X-ray, endoscopy, colonoscopy and/or biopsy performed after barium enema or enema. Suitable procedures vary depending on the condition and symptoms of the patient.

In some embodiments, the protein is used to treat an inflammatory disorder. In some embodiments, the inflammatory disorder is inflammation, autoimmune disease, atopic disease, paraneoplastic autoimmune disease, cartilage inflammation, arthritis, rheumatoid arthritis (e.g., mobility), juvenile arthritis, juvenile rheumatoid arthritis, polyarthritis juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathy arthritis, juvenile reactive arthritis, juvenile rette syndrome, SEA syndrome (serum negative, end disease, joint disease syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, Juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, juvenile rheumatoid arthritis, polyarthritis rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathy arthritis, reactive arthritis, rette syndrome, SEA syndrome (seronegative, terminal disease, joint syndrome), dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myositis, multiple myositis, polyarteritis nodosa, wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosing, primary biliary sclerosis, sclerosing cholangitis, sjogren's syndrome, psoriasis, plaque psoriasis, sarcoidosis, psoriasis, and rheumatoid arthritis, Trichome psoriasis, psoriasis pigmentosum, impetigo psoriasis, erythroderma psoriasis, dermatitis, atopic dermatitis, dermatitis herpetiformis, behcet's disease (including but not limited to effects on the skin), alopecia areata, alopecia totalis, atherosclerosis, lupus, still's disease, systemic Lupus Erythematosus (SLE) (e.g., mobility), myasthenia gravis, inflammatory Bowel Disease (IBD), crohn's disease, ulcerative colitis, celiac disease, multiple Sclerosis (MS), asthma, COPD, sinusitis, nasal polyps, eosinophilic esophagitis, eosinophilic bronchitis, inflammatory Bowel Disease (IBD), Guillain-Barre disease (Guillain-Barre disease), type I diabetes mellitus, thyroiditis (e.g., graves 'disease), addison's disease, raynaud's phenomenon (Raynaud's phenomenon), autoimmune hepatitis, graft-versus-host disease, steroid refractory chronic graft-versus-host disease, graft rejection (e.g., kidney, lung, heart, skin, etc.), kidney injury, hepatitis C-induced vasculitis, spontaneous loss of pregnancy (spontaneous loss of pregnancy), Alopecia, vitiligo, focal Segmental Glomerulosclerosis (FSGS), micromanipulation disease (MINIMAL CHANGE DISEASE), membranous nephropathy, ANCA-related glomerulonephritis, membranous proliferative glomerulonephritis, igA nephropathy, lupus nephritis, etc. In some embodiments, the protein is used to treat steroid-refractory chronic graft versus host disease. In some embodiments, the protein is used to treat active systemic lupus erythematosus. In some embodiments, the protein is used to treat active rheumatoid arthritis.

In some embodiments, the method comprises administering to the subject a pharmaceutical composition comprising a protein described herein. In some embodiments, the subject is a subject in need thereof. Any of the above therapeutic proteins may be administered in the form of a composition (e.g., a pharmaceutical composition) as described herein. For example, the composition may comprise an IL-2 protein as described herein plus a buffer, an antioxidant such as ascorbic acid, low molecular weight polypeptides (such as those having less than 10 amino acids), proteins, amino acids, carbohydrates such as glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione, and/or other stabilizers, excipients, and/or preservatives. The composition may be formulated as a liquid or as a lyophilisate. Additional examples of components that may be used in pharmaceutical formulations are set forth in Remington's Pharmaceutical Sciences,16.sup.th Ed, mack Publishing Company, easton, pa., (1980) and elsewhere as described herein.

For the treatment of a disease of interest, the composition comprising the therapeutic molecules described herein may be administered by any suitable method, including, but not limited to, parenteral, topical, oral, nasal, vaginal, rectal, or pulmonary (by inhalation) administration. If injected, one or more compositions may be administered intra-articular, intravenous, intra-arterial, intramuscular, intraperitoneal, or subcutaneous by bolus injection or continuous infusion. Topical application, i.e. application at the site of disease, is envisaged as transdermal delivery and sustained release from implants, skin patches or suppositories. Delivery by inhalation includes, for example, nasal or oral inhalation, use of a nebulizer, inhalation in aerosol form, and the like. Suppository administration by insertion into a body cavity may be accomplished, for example, by inserting the solid form of the composition into the selected body cavity and dissolving it. Other alternatives include eye drops, oral preparations such as pills, lozenges, syrups and chewing gums, and external preparations such as lotions, gels, sprays and ointments. In most cases, the therapeutic molecule as a polypeptide may be administered topically or by injection or inhalation.

In practicing the methods of treatment, the therapeutic molecules described above can be administered as described herein and above. For example, the composition may be administered at any dose, frequency, and duration effective to treat the condition being treated. The dosage will depend on the molecular nature of the therapeutic molecule and the nature of the condition being treated. Treatment may be continued as long as needed to achieve the desired result. The therapeutic molecules of the invention may be administered as a single dose or as a series of doses administered periodically, including multiple times per day, every other day, twice per week, three times per week, weekly, every other week, and monthly, among other possible dosing regimens. The periodicity of the treatment may or may not be constant throughout the treatment period. For example, treatment may be initially performed at weekly intervals, and then at weekly intervals. The invention includes treatments that last days, weeks, months or years. The treatment may be discontinued and then restarted. The maintenance dose may or may not be administered after the initial treatment.

Dosages may be measured as milligrams per kilogram of body weight (mg/kg) or milligrams per square meter of skin surface (mg/m ²) or as fixed dosages, independent of height or weight. All of these are standard dosage units in the art. The skin surface area of a person is calculated from the height and weight of the person using standard formulas.

Also provided herein are methods of promoting or stimulating STAT5 phosphorylation in T regulatory cells. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a peptide described herein or a pharmaceutical composition comprising the same.

As used herein, the phrase "in need of" means that the subject (animal or mammal) has been identified as in need of a particular method or treatment. In some embodiments, the identification may be by any diagnostic means. In any of the methods and treatments described herein, an animal or mammal may be in need thereof. In some embodiments, the animal or mammal is in or will travel to an environment in which a particular disease, disorder, or condition is prevalent.

Unless defined otherwise, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed embodiments belong.

The terms "a" or "an" as used herein mean "at least one" or "one or more" unless the context clearly indicates otherwise.

As used herein, the term "about" means that the values are approximate, and that small variations do not significantly affect the practice of the disclosed embodiments. Where numerical limits are used, unless the context indicates otherwise, "about" means that the value may vary by + -10% and remain within the scope of the disclosed embodiments.

As used herein, the term "individual" or "subject" or "patient" is used interchangeably to refer to any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, or primates, such as humans.

As used herein, the terms "comprises," "comprising," "including," and any form of comprising, such as "comprises," "containing," and "being contained," are inclusive or open-ended, and do not exclude additional unrecited elements or method steps. Any step or composition using the transitional phrase "comprising" or "containing" may also be considered as describing the same as "consisting of … …" or "consisting of.

As used herein, the term "contacting" refers to bringing together two elements in an in vitro system or an in vivo system. For example, "contacting" a peptide or composition described herein with a T-reg cell or with an individual or patient or cell includes administering a compound to the individual or patient, e.g., a human, and, e.g., introducing the compound into a sample containing the cell or into a purified preparation containing the T-reg cell.

As used herein, the term "fused" or "linked" when used in reference to proteins having different domains or heterologous sequences "

Refers to protein domains that are part of the same peptide chain linked to each other by peptide bonds or other covalent bonds. The domains or portions may be directly linked or fused to each other, or another domain or peptide sequence may be between the two domains or sequences, and such sequences will still be considered fused or linked to each other. In some embodiments, the various domains or proteins provided herein are directly linked or fused to each other, or a linker sequence, such as the glycine/serine sequences described herein, links the two domains together.

In some embodiments, embodiments provided herein further include, but are not limited to:

1. A peptide comprising the amino acid sequence SEQ ID No. 1, wherein said peptide comprises a mutation at position 73, 76, 100 or 138.

2. The peptide of embodiment 1, wherein the mutation is an L to I mutation at position 73, 76, 100 or 138.

3. The peptide of embodiment 1, wherein the peptide further comprises mutations at one or more of positions 49, 51, 55, 57, 68, 89, 91, 94, 108 and 145.

4. The peptide of embodiment 1, further comprising a mutation at one or more of positions E35, H36, Q42, D104, E115, or Q146, and 1, 2,3, 4, 5, or each of E35, H36, Q42, D104, E115, or Q146 is wild-type.

5. The peptide of embodiment 4, wherein the mutation is one or more of E35Q, H N, Q42E, D104N, E Q or Q146E.

6. The peptide of any one of embodiments 1-5, wherein the peptide comprises an N49S mutation.

7. The peptide of any one of embodiments 1-6, wherein the peptide comprises a Y51S or Y51H mutation.

8. The peptide of any one of embodiments 1-7, wherein the peptide comprises a K55R mutation.

9. The peptide of any one of embodiments 1-8, wherein the peptide comprises a T57A mutation.

10. The peptide of any one of embodiments 1-9, wherein the peptide comprises a K68E mutation.

11. The peptide of any one of embodiments 1-10, wherein the peptide comprises a V89A mutation.

12. The peptide of any one of embodiments 1-11, wherein the peptide comprises an N91R mutation.

13. The peptide of any one of embodiments 1-12, wherein the peptide comprises a Q94P mutation.

14. The peptide of any one of embodiments 1-13, wherein the peptide comprises an N108D or N108R mutation.

15. The peptide of any one of embodiments 1-14, wherein the peptide comprises a C145A or C145S mutation.

15.1. The peptide of any one of embodiments 1-15, wherein the peptide comprises V89A, Q94P, N R or N108D, and one or more of the L73I, L76I, L100I, L118I mutations, and optionally a C145A or C145S mutation.

16. A peptide comprising the amino acid sequence SEQ ID No. 2, wherein said peptide comprises a mutation at position 53, 56, 80 or 118.

17. The peptide of embodiment 16, wherein the mutation is an L to I mutation at position 53, 56, 80 or 118.

18. The peptide of embodiment 16 or 17, wherein the peptide further comprises a mutation at one or more of positions 29, 31, 35, 37, 48, 69, 71, 74, 88 and 125.

19. The peptide of embodiment 16, further comprising a mutation at one or more of positions E15, H16, Q22, D84, E95, or Q126, or 1,2, 3,4, 5, or each of positions E15, H16, Q22, D84, E95, or Q126 is wild-type.

20. The peptide of embodiment 19, wherein the mutation is one or more of E15Q, H16N, Q22E, D84N, E Q or Q126E.

21. The peptide of any one of embodiments 16-20, wherein the peptide comprises an N29S mutation.

22. The peptide of any one of embodiments 16-21, wherein the peptide comprises a Y31S or Y51H mutation.

23. The peptide of any one of embodiments 16-22, wherein the peptide comprises a K35R mutation.

24. The peptide of any one of embodiments 16-23, wherein the peptide comprises a T37A mutation.

25. The peptide of any one of embodiments 16-24, wherein the peptide comprises a K48E mutation.

26. The peptide of any one of embodiments 16-25, wherein the peptide comprises a V69A mutation.

27. The peptide of any one of embodiments 16-26, wherein the peptide comprises an N71R mutation.

28. The peptide of any one of embodiments 16-27, wherein the peptide comprises a Q74P mutation.

29. The peptide of any one of embodiments 16-28, wherein the peptide comprises an N88D or N88R mutation.

30. The peptide of any one of embodiments 16-29, wherein the peptide comprises a C125A or C125S mutation.

31. The peptide of any one of embodiments 16-30, wherein the peptide comprises one or more of V69A, Q74P, N R or N88D, and a L53I, L56I, L80I, L118I mutation, and optionally a C125A or C125S mutation.

32. The peptide according to any of the preceding embodiments, wherein the peptide does not comprise a mutation corresponding to position 30, 31 and/or 35.

33. The peptide of any one of the preceding embodiments, further comprising an Fc peptide.

The peptide of embodiment 33, wherein the Fc peptide comprises mutations at positions L247, L248 and G250 (using Kabat numbering) or at positions L234, L235 and G237 (using EU numbering).

The peptide of embodiment 33, wherein the Fc peptide comprises the following mutations: L247A, L A and G250A (Kabat numbering) or L234A, L235A and G237A (EU numbering).

34. The peptide of embodiment 33, wherein the Fc peptide comprises the sequence SEQ ID NO 8 or SEQ ID NO 15.

35. The peptide of any one of the preceding embodiments, further comprising a linker peptide linking the peptide of SEQ ID No. 1 or SEQ ID No. 2 and the Fc peptide.

36. The peptide of embodiment 35, wherein the linker comprises the sequence ggggsggggsggggsgggsggggs or GGGGSGGGGSGGGGS.

37. The peptide according to any of the preceding embodiments, wherein the peptide comprises the sequence SEQ ID NO 17-43.

38. A peptide comprising the amino acid sequence seq id No. 27.

39. The peptide of embodiment 38, further comprising an N-terminal leader peptide having the sequence SEQ ID NO. 7.

40. The peptide of embodiment 1, further comprising a linker peptide at the C-terminus.

41. The peptide of embodiment 40, wherein the linker peptide comprises the sequence (GGGGS) _n, wherein n is 1,2, 3, or 4.

42. The peptide of embodiment 41, wherein n is 1.

43. The peptide of embodiment 41, wherein n is 2.

44. The peptide of embodiment 41, wherein n is 3.

45. The peptide of embodiment 41, wherein n is 4.

46. The peptide of any one of embodiments 48-45, further comprising an Fc peptide.

47. The peptide of embodiment 46, wherein the Fc peptide comprises the sequence SEQ ID NO 8 or SEQ ID NO 15.

48. The peptide of embodiment 47, further comprising a leader peptide having the sequence SEQ ID No. 7 at the N-terminus.

49. The peptide of embodiment 46, wherein the Fc peptide is at the C-terminus of the peptide comprising SEQ ID NO. 27.

50. The peptide of embodiment 46, wherein the Fc peptide is at the N-terminus of the peptide comprising SEQ ID NO. 27.

51. The peptide of embodiment 1, further comprising a linker peptide linking the peptide of SEQ ID NO. 27 and the Fc peptide.

52. The peptide of embodiment 51, wherein the linker peptide is (GGGGS) _n, wherein n is 1, 2,3, or 4.

53. The peptide of embodiment 52, wherein n is 4.

54. The peptide of embodiments 51-53, wherein the Fc peptide comprises the sequence SEQ ID NO 8 or SEQ ID NO 15.

55. The peptide of embodiments 51-54, wherein the Fc peptide is at the C-terminus of the peptide comprising SEQ ID NO 27.

56. The peptide of embodiments 51-54, wherein the Fc peptide is at the N-terminus of the peptide comprising SEQ ID NO 27.

57. The peptide of embodiment 14, wherein the peptide comprises a peptide comprising the sequence SEQ ID NO 37, 38, 39 or 40.

58. A pharmaceutical composition comprising a peptide according to any one of the preceding embodiments.

59. A method of activating a T regulatory cell, the method comprising contacting the T regulatory cell with the peptide of any one of embodiments 1-57 or the pharmaceutical composition of embodiment 58.

60. A method of treating an inflammatory disorder in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the peptide of any one of embodiments 1-57 or the pharmaceutical composition of embodiment 58.

61. According to the method of embodiment 60, wherein the inflammatory condition is inflammation, autoimmune disease, atopic disease, paraneoplastic autoimmune disease, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, polyarthritic rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathy arthritis, juvenile reactive arthritis, juvenile lister syndrome, SEA syndrome (seronegative, terminal disease, joint disease syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, juvenile rheumatoid arthritis, polyarthritis rheumatoid arthritis, systemic sclerosis, rheumatoid arthritis, juvenile reactive arthritis, juvenile rette systemic onset rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, lisi syndrome, SEA syndrome (seronegative, terminal disease, joint disease syndrome), dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myositis, polymyositis, polyarteritis nodosa, wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, cirrhosis, primary biliary sclerosis, sclerosing cholangitis, sjogren's syndrome, psoriasis, plaque psoriasis, trichome psoriasis, psoriasis pigskin, pustule psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus, stethosis, systemic Lupus Erythematosus (SLE), myasthenia gravis, inflammatory Bowel Disease (IBD), crohn's disease, ulcerative colitis, celiac disease, multiple Sclerosis (MS), asthma, COPD, sinusitis with nasal polyps, eosinophilic esophagitis, eosinophilic bronchitis, guillain-barre disease, type I diabetes mellitus, thyroiditis (e.g., graves ' disease), addison's disease, raynaud's phenomenon, autoimmune hepatitis, graft Versus Host Disease (GVHD), steroid refractory chronic graft versus host disease, graft rejection (e.g., kidney, lung, heart, skin, etc.), kidney injury, hepatitis c induced vasculitis, spontaneous loss of pregnancy, alopecia, vitiligo, focal Segmental Glomerulosclerosis (FSGS), morbid diseases, membranous nephropathy, ANCA-related glomerulonephritis, igA nephropathy, lupus nephritis, and the like.

62. A method of promoting or stimulating STAT5 phosphorylation in a T regulatory cell, the method comprising administering to a subject in need thereof a therapeutically effective amount of the peptide of any one of embodiments 1-57 or the pharmaceutical composition of embodiment 58.

63. Use of the peptide of any one of embodiments 1-57 or the pharmaceutical composition of embodiment 58 in the manufacture of a medicament for treating an inflammatory disorder.

64. According to the use of embodiment 63, wherein the inflammatory condition is inflammation, autoimmune disease, atopic disease, paraneoplastic autoimmune disease, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, polyarthritic rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathy arthritis, juvenile reactive arthritis, juvenile lister syndrome, SEA syndrome (seronegative, terminal disease, joint disease syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, juvenile rheumatoid arthritis, polyarthritis rheumatoid arthritis, systemic sclerosis, rheumatoid arthritis, juvenile reactive arthritis, juvenile rette systemic onset rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, lisi syndrome, SEA syndrome (seronegative, terminal disease, joint disease syndrome), dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myositis, polymyositis, polyarteritis nodosa, wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, cirrhosis, primary biliary sclerosis, sclerosing cholangitis, sjogren's syndrome, psoriasis, plaque psoriasis, trichome psoriasis, psoriasis pigskin, pustule psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus, stethosis, systemic Lupus Erythematosus (SLE), myasthenia gravis, inflammatory Bowel Disease (IBD), crohn's disease, ulcerative colitis, celiac disease, multiple Sclerosis (MS), asthma, COPD, sinusitis with nasal polyps, eosinophilic esophagitis, eosinophilic bronchitis, guillain-barre disease, type I diabetes mellitus, thyroiditis (e.g., graves ' disease), addison's disease, raynaud's phenomenon, autoimmune hepatitis, graft Versus Host Disease (GVHD), steroid refractory chronic graft versus host disease, graft rejection (e.g., kidney, lung, heart, skin, etc.), kidney injury, hepatitis c induced vasculitis, spontaneous loss of pregnancy, alopecia, vitiligo, focal Segmental Glomerulosclerosis (FSGS), morbid diseases, membranous nephropathy, ANCA-related glomerulonephritis, igA nephropathy, lupus nephritis, and the like.

65. A nucleic acid molecule encoding the peptide of any one of embodiments 1-57.

66. A vector comprising the nucleic acid molecule of embodiment 65.

67. A plasmid comprising the nucleic acid molecule of embodiment 65.

68. A cell comprising the nucleic acid molecule of embodiment 65.

69. A cell comprising the plasmid of embodiment 67.

70. A cell comprising the vector of embodiment 66.

71. A cell comprising or expressing the peptide of any one of embodiments 1-57 or a peptide as described herein.

The following examples are illustrative of the compounds, compositions, and methods described herein and are not intended to be limiting. Other suitable modifications and adaptations known to those skilled in the art are within the scope of the following embodiments.

Examples

Example 1:

a therapeutic composition comprising a protein of SEQ ID NO:11, 12, 13 or 14 is administered to a subject suffering from IBD. The immune system of the subject is down-regulated and the symptoms of IBD are reduced.

Example 2:

A therapeutic composition comprising a protein of SEQ ID NO 3,4, 5 or 6 with or without a leader sequence is administered to a subject suffering from IBD. The immune system of the subject is down-regulated and the symptoms of IBD are reduced.

Example 3: IL-mutein production

PTT5 vector containing a single gene encoding a human IL-2M polypeptide fused to the N-terminus (SEQ ID NO: 40) or C-terminus (SEQ ID NO: 41) of the human IgG1 Fc domain was transfected into HEK293 Expi cells. After 5-7 days, cell culture supernatants expressing IL-2M were harvested and clarified by centrifugation and filtered through a 0.22 μm filter device. IL-2M was captured on proA resin. The resin was washed with PBS pH7.4 and the captured protein was eluted with 0.25% acetic acid pH3.5 and neutralized with one tenth of the volume of 1M Tris pH 8.0. Proteins were buffer exchanged to 30mM HEPES150mM NaCl at pH7 and analyzed by size exclusion chromatography on a Superdex 200.2/300 column. 5 μg of purified material was analyzed by reducing and non-reducing SDS-PAGE on Bis-Tris4-12% gels. IL-2M was expressed at more than 10mg/L and was more than 95% monodisperse after purification as indicated by size exclusion chromatography and reducing/non-reducing SDS-PAGE.

Example 4: IL-2M molecules can bind CD25

The immunosorbent plate was coated with CD25 at a concentration of 0.5. Mu.g/mL in PBS pH7.4, 75. Mu.l/well, and incubated overnight at 4 ℃. Wells were washed three times with PBS (wash buffer) at ph7.4 containing 0.05% Tween-20 and then blocked with 200 μl/well of PBS (blocking buffer) at ph7.4 containing 1% bsa for two hours at room temperature. After three washes with wash buffer, IL-2M molecules were diluted to 11-2 fold serial dilutions in PBS (assay buffer) containing 1% BSA and 0.05% Tween-20, with 2nM being the highest concentration. The diluted material was added to the CD25 coated plate at 75 μl/well for 1 hour at room temperature. After three washes with wash buffer, sheep biotinylated anti-IL-2 polyclonal antibody diluted to 0.05. Mu.g/mL in assay buffer was added to the plate at 75. Mu.l/well for 1 hour at room temperature. After three washes with wash buffer, streptavidin HRP diluted 1:5000 in assay buffer was added to the plate at 75 μl/well for 15 min at room temperature. After three washes with wash buffer and 1 wash with wash buffer (without tween-20), the assay was developed with TMB and quenched with 1N HCl. OD 450nm was measured. The experiments included suitable controls for non-specific binding of the IL-2M molecule to the plate/block in the absence of CD25 and negative control molecules that were unable to bind CD 25.

The results indicate that IL-2M molecules are capable of binding CD25 with a subnanomolar EC50 at a concentration of 2nM-1.9 pM. In addition, when CD25 was not present on the plate surface, no compound was detected at any of the test concentrations, indicating no non-specific interaction of the test compound with the plate surface (data not shown).

Example 5: in vitro P-STAT5 assays to determine potency and selectivity of IL-2M molecules. Peripheral Blood Mononuclear Cells (PBMC) were prepared from freshly isolated heparinized human whole blood using FICOLL-PAQUE Premium and Sepmate tubes. PBMC were incubated in 10% fetal bovine serum RPMI medium for 20min in the presence of wild-type IL-2 or IL-2M from example 12 and then fixed with BD Cytofix for 10 min.

The fixed cells were permeabilized sequentially with BD perm III and then with BioLegend FOXP3 permeabilization buffer. After blocking with human serum for 10 min, cells were stained with antibodies to phospho-STAT 5 FITC, CD25 PE, FOXP3 AF647 and CD4 PerCP cy5.5 for 30 min and then obtained on Attune NXT with a plate reader. IL-2M of SEQ ID NO. 23 effectively and selectively induces STAT5 phosphorylation in tregs but not Teff.

Example 6: immunogenicity of IL-2 muteins

IL-2 mutein mutant sequences were analyzed using NETMHCIIPAN 3.2.2 software, which can be found on www.cbs.dtu.dk/services/NETMHCIIPAN/respectively. Artificial neural networks were used to determine the affinity of peptides for MHC class II alleles. In this analysis, peptides with 9 residues potentially directly interacting with MHC class II molecules are considered to be binding cores. Residues adjacent to the binding core (which have the potential to indirectly affect binding) were also tested as masking residues. Peptides comprising binding core and masking residues are labeled as strong binders when their predicted K _D for MHC class II molecules is below 50 nM. Strong conjugates have a greater chance of introducing T cell immunogenicity.

A total of 9 mhc ii alleles highly expressed in north america and europe were included in the computer analysis. The set of IL-2M (IL-2 muteins) molecules tested included IL-2 muteins with the L53I, L, I, L I or L118I mutation. Only the MHCII alleles drb1_1101, drb1_1501, drb1_0701 and drb1_0101 produce hits with any evaluated molecules. The peptide hit of drb_1501 was identical between all test constructs including wild-type IL-2 with C125S mutation. The addition of L80I removes 1T cell epitope of DRB1-0101[ ALNLAPSKNFHLRPR ] and moderately reduces the affinity of two other T cell epitopes [ EEALNLAPSKNFHLR and EALNLAPSKNFHLRP ]. For the MHCII allele DRB1-0701, L80I removes the 1T cell epitope [ EEALNLAPSKNFHLR ]. Thus, the data indicate that IL-2 muteins comprising the L80I mutation should have a lower immunogenicity, which is a surprising and unexpected result based on in silico analysis.

Example 7: production of additional IL-2 muteins

PTT5 vector containing a single gene encoding one of IL-2M (IL-2 mutein) SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40 (vs. IL-2M; SEQ ID NO: 34) polypeptide and human IL-2M or IL-2M fused to the N-terminus of the human IgG1 Fc domain was transfected into HEK293 Expi cells. After 5-7 days, cell culture supernatants expressing SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 (and IL-2M control; SEQ ID NO: 34) were harvested and clarified by centrifugation and filtration through a 0.22. Mu.M filter device. SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40 (against IL-2M control; SEQ ID NO. 34) were captured on proA resin. The resin was washed with PBS pH7.4 and the captured protein was eluted using 0.25% acetic acid pH3.5, wherein it was neutralized with one tenth of the volume of 1M Tris pH 8.0. Proteins were buffer exchanged to 30mM HEPES150 mM NaCl at pH7 and analyzed by size exclusion chromatography on a Superdex 200.2/300 column. 5 μg of purified material was analyzed by reducing and non-reducing SDS-PAGE on Bis-Tris4-12% gels.

IL-2M SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 (vs. IL-2M control; SEQ ID NO: 34) was expressed at more than 45mg/L and was more than 95% monodisperse after purification as shown by size exclusion chromatography and reducing/non-reducing SDS-PAGE.

Example 8: IL-2M can bind CD25

The immunosorbent plate was coated with CD25 at a concentration of 0.5. Mu.g/mL in PBS pH7.4, 75. Mu.l/well, and incubated overnight at 4 ℃. Wells were washed three times with PBS (wash buffer) at ph7.4 containing 0.05% Tween-20 and then blocked with 200 μl/well of PBS (blocking buffer) at ph7.4 containing 1% bsa for two hours at room temperature. After three washes with wash buffer, IL-2M SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 were diluted to 11-2 fold serial dilutions in PBS (assay buffer) containing 1% BSA and 0.05% Tween-20, with 2nM being the highest concentration. The diluted material was added to the CD25 coated plate at 75 μl/well for 1 hour at room temperature. After three washes with wash buffer, sheep biotinylated anti-IL-2 polyclonal antibody diluted to 0.05. Mu.g/mL in assay buffer was added to the plate at 75. Mu.l/well for 1 hour at room temperature. After three washes with wash buffer, high sensitivity streptavidin HRP diluted 1:5000 in assay buffer was added to the plate at 75 μl/well for 15 min at room temperature. After three washes with wash buffer and 1 wash with wash buffer (without tween-20), the assay was developed with TMB and quenched with 1N HCl. OD450 nm was measured. The experiments included appropriate controls for nonspecific binding of molecules to the plate/block in the absence of CD25. The results indicate that the mutein of example 7 is capable of binding CD25 with a subnanomolar EC50 at a concentration of 2nM-1.9 pM. In addition, when CD25 was not present on the plate surface, no compound was detected at any of the test concentrations, indicating that no test compound interacted non-specifically with the plate surface. Thus, the mutein of example 7 can bind CD25.

Example 9: the IL-2 muteins of example 7 are potent and selective

Peripheral Blood Mononuclear Cells (PBMC) were prepared from freshly isolated heparinized human whole blood using FICOLL-PAQUE Premium and Sepmate tubes. PBMCs were incubated in 10% fetal bovine serum RPMI medium for 20 min in the presence of wild-type IL-2 or the mutein of example 7 and then fixed with BD Cytofix for 10 min. The fixed cells were permeabilized sequentially with BD perm III and then with BioLegend FOXP3 permeabilization buffer. After blocking with human serum for 10 min, cells were stained with antibodies to phospho-STAT 5 FITC (CST), CD25 PE, FOXP3 AF647 and CD4 PerCP cy5.5 (all BD) for 30min and then obtained on Attune NXT with a plate reader. The IL-2 mutein of example 7 was found to be potent and selective for tregs relative to Teff. Mutant proteins comprising the L118I mutation were found to have increased activity and selectivity compared to other mutant proteins.

Example 10: IL-2 muteins expand Treg in humanized mice

NSG mice humanized with human CD34+ hematopoietic stem cells were purchased from Jackson Labs. On days 0 and 7, mice were subcutaneously dosed with 1 μg of IL-2 mutein SEQ ID NO 34 or other IL-2 mutein SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39 or SEQ ID NO 40. On day 7, mice were euthanized and whole blood and spleen were collected. Whole blood was aliquoted into 96-well deep well plates and fixed for 10 minutes using BD Fix Lyse. Spleen cells were isolated using a 70 μm filter (BD) and erythrocytes were lysed using RBC lysis buffer from BioLegend. After washing with 2% fetal bovine serum PBS, spleen cells were labeled with near infrared live-dead stain (Invitrogen) for 20 minutes and then fixed using Biolegend fixation buffer for 20 minutes. Whole blood cells and spleen cells were then permeabilized using a BioLegend FOXP3 permeabilization buffer, blocked with human serum, and stained with antibodies against human CD8a FITC (BL), human CD25 PE (BD), human FOXP3 AF647 (BD) CD4 Percp cy5.5 (BD), human Siglec-8 PE Cy7 (BL), human CD3BV421 (BL), human CD45 BV605 (BL), human CD56 BV785 (BL), and mouse CD45 (BV 711) for 30 minutes and obtained on Attune NXT with a loading plate (plate loader).

IL-2M SEQ ID NO:37 and SEQ ID NO:38 and SEQ ID NO:39 and SEQ ID NO:40 selectively induced human Treg in mouse spleen and whole blood of humanized mice compared to vector controls. The frequency of human CD56pos NK cells, CD3pos T cells, CD8pos cytotoxic T lymphocytes, CD4pos helper T cells or CD25lo/FOXP3neg T effectors did not change significantly. These results demonstrate that IL-2 muteins increase the frequency of regulatory T cells.

Example 11: persistence of IL-2 mutein-induced signaling

Peripheral Blood Mononuclear Cells (PBMC) were prepared from freshly isolated heparinized human whole blood using FICOLL-PAQUE Premium and Sepmate tubes. PBMCs were incubated in 10% fetal bovine serum RPMI medium for 60 min in the presence of IL-2M. The cells were then washed 3 times and incubated for an additional 3 hours. Cells were then fixed with BD Cytofix for 10 min. The fixed cells were sequentially permeabilized with BD perm III and then BioLegend FOXP3 permeabilization buffer. After blocking with human serum for 10min, cells were stained with antibodies to phospho-STAT 5FITC, CD25 PE, FOXP3 AF647 and CD4 PerCP cy5.5 for 30min and then obtained on Attune NXT with a plate reader. In contrast to the control, all four IL-2 muteins of example 19 induced durable signaling in Treg but not in Teff. SEQ ID NO. 40 is superior to SEQ ID NO. 39, SEQ ID NO. 38 or SEQ ID NO. 37. These results demonstrate that IL-2 can induce durable and selective signaling in tregs, which will lead to greater Treg expansion in vivo and allow for less frequent dosing to achieve Treg expansion.

The examples provided herein demonstrate the surprising and unexpected result that IL-2 muteins can act to selectively and effectively activate tregs relative to Teff, demonstrating that the molecules can be used to treat or ameliorate the conditions described herein. IL-2 muteins as provided herein may also be produced and used with or without fusion to an Fc domain or linker as provided herein.

The implementations have been described with reference to specific examples. These examples are not meant to limit the embodiments in any way. It should be understood that various changes and modifications may be made for the purposes of this disclosure that are well within the scope of this disclosure. Many other variations are possible which will be readily apparent to those skilled in the art and are encompassed within the spirit of the invention disclosed herein and as defined by the appended claims.

Example 12: muteins display overall POI and lower aggregation.

Expression of an IL-2 mutein having the mutations V69A, Q74P, N88D and C125S and one of the following mutations in the pTT5 vector was achieved by transfection of the vector into HEK293 Expi cells: L3I, L56I, L I or L118I, which is linked to an Fc region comprising L234A, L235A and G237A mutations as provided herein. IL-2 muteins were linked to the N-terminus of the Fc region using 4 GGGGS repeats. After 5-7 days, cell culture supernatants expressing the different IL-2 muteins were harvested and clarified by centrifugation and filtration through a 0.22 μm filter device. IL-2 muteins are captured on proA resin. The resin was washed with PBS pH7.4 and the captured protein was eluted with 0.25% acetic acid pH3.5 and neutralized with one tenth volume of 1MTris pH 8.0. Proteins were buffer exchanged into 30mM HEPES150 mM NaCl at pH7 and analyzed for percent peak of interest (POI) by size exclusion chromatography on AdvanceBio SEC column. The results demonstrate that different muteins are expressed at more than 60 mg/L. However, it was surprisingly found that the muteins with the L80I or L118I mutation were more than 90% monodisperse, whereas the muteins with the L53I or L56I mutation were not shown by size exclusion chromatography. Thus, muteins with either the L80I or L118I substitutions have lower aggregation. Due to the type of mutation generated, the difference in aggregation between the four molecules (IL-2 muteins comprising L80I, L118I, L I and L56I) was surprising. Thus, a mutein having a mutation of L80I or L118I has a surprising advantage over other muteins in that it aggregates less than other muteins.

Example 13: IL-2 muteins have unexpected increased potency

The efficacy of the muteins described in example 12 was analyzed in an in vitro assay. Briefly, PBMCs were isolated from heparinized human whole blood and stimulated with different muteins at 37C for 30min at a certain concentration. Stimulation was terminated by fixation. After permeabilization, PBMC staining was used for intracellular FoxP3 and phospho-STAT 5 levels and surface CD4 and CD25 expression and analyzed by flow cytometry. Regulatory T cells (tregs) and effector T cells (teffs) are gated as cd4+cd4+cd hiFoxP3+ or cd4+cd loFoxP3-, respectively. The percentage of cells positive for phospho-STAT 5 staining is shown. This assay measures the ability of the mutein to specifically stimulate Treg but not Teff. The EC50 values were calculated using the best fit dose-response curve for each test article.

Surprisingly, muteins with the L118I, L80I, L I or L53I mutation have increased potency (Treg stimulation) compared to IL-2 muteins without any of these mutations. The IL-2 mutein without the L118I, L80I, L I or L53I mutation, but with the V69A, Q P and N88D mutations was about 51pM. EC ₅₀ comprising a mutein of one of L118I, L, I, L I or L53I has respective EC ₅₀ of about 30, 40, 41 and 45, respectively. The differences in EC50 for Treg stimulation (no change in Teff stimulation) are surprising and would not be predicted for a mutein with one of the mutations described in this example. The data can also be assessed by comparing the ratio of the parent IL-2 mutein (comprising V69A, Q74P, N88D and C125S) to a mutein further comprising one of the L118I, L80I, L I or L53I mutations. This ratio was used to normalize the different cell populations used for the different experiments. Using this ratio, L118I increased efficacy by about 25% on average (standard error of 0.16 on average) compared to the parental control, while other mutations decreased activity compared to the parental control using this ratio.

In vitro data for muteins with one of the L118I, L80I, L I or L53I mutations were confirmed in vivo. L118I was found to be more potent in vivo than a mutant protein without the L118I mutation. Briefly, 1 microgram of the indicated test preparation or vector was subcutaneously injected into Nod-Scid-IL-2rγ -deficient (NSG) mice reconstituted with human cd34+ hematopoietic stem cells on days 0 and 7. On day 11, mice were sacrificed and blood was collected into heparin-containing tubes by cardiac puncture. Peripheral Blood Leukocytes (PBLs) were isolated by lysing erythrocytes and stained with antibodies reactive with the human markers CD45, CD3, CD8, CD4, foxP3, CD25 and CD 56. The percentages of human regulatory T cells (Treg, cd45+cd3+cd4+cd25+foxp3+), activated effector T cells (act Teff, cd45+cd3+cd4+cd25+foxp3-) and NK cells (cd45+cd56+) were determined by flow cytometry. The frequencies of total cd4+ and total cd8+ cells were unchanged. Similar results were observed in the spleens of mice. The in vivo potency of the IL-2 mutein with the L80I mutation as determined in this assay is slightly increased compared to the mutein without the L80I mutation, and the in vivo potency of the mutein with the L56I or L53I mutation as determined in this assay is about the same as the mutein without said mutation. The muteins were N-terminally linked to the Fc region as described herein using a 20 amino acid (GGGGS) ₄ linker. Which is a linker linking the C-terminus of the IL-2 mutein with the N-terminus of the Fc region.

Example 14: n-terminal Fc orientation with a 20 amino acid linker was most effective in stimulating Treg. IL-2 muteins with V69A, Q P and N88D were fused to the mutated Fc region comprising the L234A, L235A and G237A mutations using GGGGS repeats of different lengths. IL2 mutein molecules were tested by fusing the c-terminus of mutein to the n-terminus of human IgG1 Fc with a linker comprising 3 and 4 GGGGS repeats to determine if the length of the linker affected the potency of the IL-2 mutein. Muteins were also tested by fusing their n-terminus with the c-terminus of human IgG1 Fc with one GGGGS repeat. Briefly, on day 0, nod-Scid-IL-2rγ -deficient (NSG) mice reconstituted with human cd34+ hematopoietic stem cells were subcutaneously injected with 1 microgram of different IL-2 muteins or vectors with different linker lengths. On day 7, mice were sacrificed and blood was collected into heparin-containing tubes by cardiac puncture. Peripheral Blood Leukocytes (PBLs) were isolated by lysing erythrocytes and stained with antibodies reactive with the human markers CD45, CD3, CD8, CD4, foxP3, CD25 and CD 56. The percentages of human regulatory T cells (Treg, cd45+cd3+cd4+cd25+foxp3+), activated effector T cells (act Teff, cd45+cd3+cd4+cd25+foxp3-) and NK cells (cd45+cd56+) were determined by flow cytometry. The frequencies of total cd4+ and total cd8+ cells were unchanged. Similar results were observed in the spleens of mice.

It was found that a mutein fused at the N-terminus of human IgG1 Fc with a linker comprising 4 GGGGS repeats was most potent compared to a mutein having only 3 GGGGS repeats or to a mutein fused at the c-terminus of human IgG1 Fc with a single GGGGS repeat. In addition, while proteins with 4 GGGGS repeats are more efficient in expanding tregs, the configuration does not trigger any differential expansion of cd56+ NK cells. Proteins with muteins fused with longer linkers to the N-terminal Fc would not be predicted to be the most potent and would not trigger any differential expansion of cd56+ NK cells.

Example 15: treating patients suffering from active rheumatoid arthritis.

A pharmaceutical composition comprising an IL-2 mutein protein comprising the sequence SEQ ID No. 37, 38, 39 or 40 is administered to a patient suffering from active rheumatoid arthritis. IL-2 muteins were found to be effective in treating active rheumatoid arthritis in patients.

Example 16: patients and subjects with active systemic lupus erythematosus are treated.

A pharmaceutical composition comprising an IL-2 mutein protein comprising the sequence SEQ ID No. 37, 38, 39 or 40 is administered to a patient suffering from active systemic lupus erythematosus. IL-2 muteins were found to be effective in the treatment of active systemic lupus erythematosus.

Example 17: patients and subjects suffering from steroid refractory chronic graft versus host disease are treated.

A pharmaceutical composition comprising an IL-2 mutein protein comprising the sequence SEQ ID NO:37, 38, 39 or 40 is administered to a patient suffering from steroid refractory chronic graft-versus-host disease. IL-2 muteins were found to be effective in the treatment of steroid-refractory chronic graft-versus-host disease.

Example 18: IL-2 muteins induced pSTAT5 in human tregs. Purified PBMC from heparinized whole blood from six healthy donors were treated with serial dilutions of IL-2 mutein proteins comprising the sequence of SEQ ID NO:39 or 40 at 37C for 30 min. Cells were fixed, washed, permeabilized and washed. Cells were stained with antibodies that detected surface markers and intracellular/nuclear markers (pSTAT 5 and FOXP 3). Data were collected on ATTUNE NxT cytometer. Tregs are gated as monokaryons, monoweights, CD3pos, CD4pos, CD25hi, foxP3pos. The% of gated tregs expressing phosphorylated STAT5 was determined. A best fit curve of dose-response fitted to pSTAT5 and EC50 values was determined. For IL-2 of SEQ ID NO39 (37.26.+ -. 7.30; n=16) and IL-2 of SEQ ID NO 40 (23.11.+ -. 5.35; n=15), the average EC50 value of all 6 donors was determined. The data demonstrate that IL-2 muteins can induce pSTAT5 in human tregs. IL-2 comprising the sequence of SEQ ID NO. 40 is more potent than IL-2 comprising the sequence of SEQ ID NO. 39, but both are active in multiple cell populations.

Example 19: IL-2 muteins induced pSTAT5 in monkey PBMC in vitro. Purified PBMC from heparinized whole blood from three healthy monkeys were treated with serial dilutions of IL-2 mutein proteins comprising the sequence of SEQ ID NO:39 or 40 at 37C for 60 min. Fluorescent dye (Fluorochrome) conjugated anti-CD 25 and anti-CD 4 were added during the last 30min of IL-2 mutein treatment. Cells were fixed, washed, permeabilized and washed. Cells were stained with the remaining antibodies to detect surface markers and intracellular/nuclear markers (pSTAT 5 and FOXP 3). Data were collected on ATTUNE NxT cytometer. Tregs are gated as monokaryons, monoweights, CD4pos, CD25hi, foxP3pos. The% of gated tregs expressing phosphorylated STAT5 was determined. The IL-2 mutein was found to induce pSTAT5 in monkeys.

Example 20: IL-2 muteins induce expansion of Treg cells and induce proliferation of tregs in vivo. Venous whole blood from the monkeys (cynomolgus monkeys) was collected in K2EDTA tubes prior to administration with the IL-2 muteins of SEQ ID NO 39 or 40 (2 time points/cynomolgus monkey, 5 cynomolgus monkeys) and after administration with either SEQ ID NO 39 (5 time points/cynomolgus monkey, 2 cynomolgus monkeys) or SEQ ID NO 40 (5 time points/cynomolgus monkey, 3 cynomolgus monkeys). The samples were split into two and stained for two FACS sets, respectively. One is the "Treg group" and one is the general immunophenotype group. RBCs are lysed and after fixation and permeabilization, the surface of the cells and intracellular markers are stained. For FACs analysis, total cell number/. Mu.l was determined by ADVIA. The number of cells per μl of a given subpopulation was then calculated using the total number per μl and% of the total number. For each monkey, the average number of given cell types per μl of pre-dose hemorrhage was averaged and used to normalize post-dose hemorrhage to determine the "fold change from pre-dose". For analysis of serum cytokines and chemokines, plasma from K2EDTA whole blood was frozen until the end of the study. The amounts of chemokines and cytokines were quantified by multiplex MSD assay using serial dilutions of standard controls. The mean and range of MCP-1 and IP-10 in pre-dosing bleeding was determined. Both muteins were found to amplify tregs and induce Treg proliferation in monkeys. These results demonstrate that IL-2 muteins function in vivo animal models similar to humans. It was also found that neither molecule significantly expanded Tconv cells, CD4 cells (T naive (Tnaive)) or CD8 cells (cytotoxic T), NK cells in monkeys (non-human primate). It was also found that neither molecule significantly induced serum chemokines. This data demonstrates that IL-2 muteins can expand and induce Treg cell proliferation without unwanted expansion or activation of other pathways. Thus, IL-2 muteins have a surprising efficacy, efficacy and selectivity for Treg expansion and proliferation.

In summary, the embodiments and examples provided herein demonstrate that IL-2 muteins can function as intended and can be used to treat the diseases and conditions described herein.

This specification contains a great deal of citations for patents, patent applications and publications. Each is incorporated by reference herein for all purposes.

Claims

1. A dimer for activating regulatory T cells, wherein the dimer comprises two peptides, each peptide independently consisting of the sequence of SEQ ID No. 40.

2. A pharmaceutical composition comprising the dimer of claim 1 and a pharmaceutically acceptable carrier.

3. Use of a dimer according to claim 1 in the manufacture of a medicament for activating T regulatory cells.

4. Use of a dimer according to claim 1 in the manufacture of a medicament for treating an inflammatory disorder in a subject.

5. The use according to claim 4, wherein the inflammatory condition is alopecia areata, atopic dermatitis, crohn's disease, ulcerative colitis, vitiligo, and Systemic Lupus Erythematosus (SLE).

6. Use of a dimer according to claim 1 in the manufacture of a medicament for promoting or stimulating STAT5 phosphorylation in T regulatory cells.

7. Use of a pharmaceutical composition according to claim 2 in the manufacture of a medicament for activating T regulatory cells.

8. Use of a pharmaceutical composition according to claim 2 in the manufacture of a medicament for treating an inflammatory disorder in a subject.

9. The use of claim 8, wherein the inflammatory disorder is alopecia areata, atopic dermatitis, crohn's disease, ulcerative colitis, vitiligo, and Systemic Lupus Erythematosus (SLE).

10. Use of a pharmaceutical composition according to claim 2 in the manufacture of a medicament for promoting or stimulating STAT5 phosphorylation in T regulatory cells.

11. The use according to claim 4, wherein the inflammatory condition is alopecia areata.

12. The use according to claim 4, wherein the inflammatory condition is atopic dermatitis.

13. The use of claim 4, wherein the inflammatory disorder is crohn's disease.

14. The use of claim 4, wherein the inflammatory disorder is ulcerative colitis.

15. The use according to claim 4, wherein the inflammatory condition is vitiligo.

16. The use of claim 4, wherein the inflammatory disorder is Systemic Lupus Erythematosus (SLE).

17. The use of claim 8, wherein the inflammatory disorder is alopecia areata.

18. The use of claim 8, wherein the inflammatory disorder is atopic dermatitis.

19. The use of claim 8, wherein the inflammatory disorder is crohn's disease.

20. The use of claim 8, wherein the inflammatory disorder is ulcerative colitis.

21. The use according to claim 8, wherein the inflammatory condition is vitiligo.

22. The use of claim 8, wherein the inflammatory disorder is Systemic Lupus Erythematosus (SLE).