JP7250790B2 - IL-2 muteins and uses thereof - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is based on U.S. Provisional Application No. 62/721,644, filed Aug. 23, 2018, and U.S. Provisional Application No. 62/675,972, filed May 24, 2018. , U.S. Provisional Application No. 62/595,357, filed Dec. 6, 2017; U.S. Present Application No. 16/109,875, filed Aug. 23, 2018; No. 16/109,897, filed on May 1, 2003, each of which is hereby incorporated by reference in its entirety.

Embodiments provided herein relate to proteins called IL-2 muteins, compositions comprising the same, and methods of using the same.

IL-2 binds to three transmembrane receptor subunits, IL-2Rβ and IL-2Rγ, and two other receptor subunits that together activate intracellular signaling events upon IL-2 binding. Binds CD25 (IL-2Rα), which serves to present IL-2. Signals transduced by IL-2Rβγ include PI3 kinase, Ras-MAP kinase, and STAT5 pathway signals.

T cells require expression of CD25 to respond to the low concentrations of IL-2 typically present in tissues. CD25-expressing T cells include both CD4 ⁺ FOXP3 ⁺ regulatory T cells (Treg cells), which are essential in suppressing autoimmune inflammation, and FOXP3 ⁻ T cells that have been activated to express CD25. be FOXP3 ⁻ CD4 ⁺ T effector cells (Teff) can be either CD4 ⁺ or CD8 ⁺ cells, both of which can be pro-inflammatory, as well as in autoimmune diseases, and when a subject's immune system is affected by an organ or other It can contribute to other diseases that attack tissue. IL-2-stimulated STAT5 signaling is critical for normal Treg cell proliferation and survival and high FOXP3 expression.

Due to the low affinity that IL-2 has for each of the three IL-2R chains, further decreases in affinity for IL-2Rβ and IL-2Rγ may be offset by increased affinity for CD25. be. Mutant variants of IL-2 have been generated. These IL-2 variants, sometimes called IL-2 muteins, have been found useful in the treatment of various diseases. However, there remains a need for additional IL-2 muteins that can be used in various applications and compositions. The present embodiments satisfy these and other needs.

In some embodiments, peptides are provided comprising the amino acid sequence of SEQ ID NO: 1 with mutations at positions 73, 76, 100, or 138.

In some embodiments, peptides are provided comprising the amino acid sequence of SEQ ID NO:2, including mutations at positions 53, 56, 80, or 118.

In some embodiments, peptides comprising the amino acid sequence of SEQ ID NO:43 are provided, wherein at least one of X ₁ , X ₂ , and X ₃ and X ₄ is I, and the rest are L or I.

Also provided are pharmaceutical compositions comprising nucleic acid molecules encoding the peptides and proteins described herein. Also provided herein are vectors containing nucleic acid molecules that encode the proteins described herein. In some embodiments, plasmids containing nucleic acids encoding the proteins described herein are provided. In some embodiments, cells are provided that contain nucleic acid molecules, vectors, or plasmids that encode the proteins described herein.

In some embodiments, methods of activating T regulatory cells are provided. In some embodiments, the method comprises contacting a T regulatory cell 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 method comprises administering a peptide (eg, a therapeutically effective amount of the 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 method comprises administering a peptide (eg, a therapeutically effective amount of the peptide) to the subject.

FIG. 1 shows non-limiting embodiments of IL-2 muteins provided herein.

Described herein are therapeutic agents that can modulate (eg, increase) Treg cell proliferation, survival, activation and/or function. In some embodiments, modulation is selective or specific for Treg cells.

As used herein, the term "selective" refers to therapeutic agents or proteins that modulate activity in Treg cells but have limited or lacking ability to promote activity in non-regulatory T cells. Point.

In some embodiments, the therapeutic agent is a variant of IL-2. Mutants of IL-2 may be referred to as IL-2 muteins. IL-2 can exist in two different forms, an immature form and a mature form. The mature form has the leader sequence removed. This is done during the post-translational process. The wild-type sequence of immature IL-2 is as follows:
MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 1)
The wild-type sequence of mature IL-2 is as follows:
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (mature IL-2 sequence) (SEQ ID NO: 2)

IL-2 mutein molecules can be produced by mutating one or more of the IL-2 residues. ＩＬ－２ムテインの非限定的な例は、ＷＯ２０１６／１６４９３７、ＵＳ９５８０４８６、ＵＳ７１０５６５３、ＵＳ９６１６１０５、ＵＳ９４２８５６７、ＵＳ２０１７／００５１０２９、ＵＳ２０１４／０２８６８９８Ａ１、ＷＯ２０１４１５３１１１Ａ２、ＷＯ２０１０／０８５４９５、ＷＯ２０１６０１４４２８Ａ２、ＷＯ２０１６０２５３８５Ａ１、およびＵＳ２００６０２６９５１５に見出すことができ、 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 a serine substituted for the cysteine at position 125 of the mature IL-2 sequence. Other combinations of mutations and substitutions that are IL-2 mutein molecules are described in US20060269515, which is incorporated by reference in its entirety. In some embodiments, cysteine at position 125 is also substituted with valine or alanine. In some embodiments, the IL-2 mutein molecule comprises a V91K substitution. In some embodiments, the IL-2 mutein molecule comprises an N88D substitution. In some embodiments, the IL-2 mutein molecule comprises an N88R substitution. In some embodiments, the IL-2 mutein molecule comprises substitutions of H16E, D84K, V91N, N88D, V91K, or V91R, any combination thereof. In some embodiments, these IL-2 mutein molecules also include a substitution at position 125 as described herein. In some embodiments, the IL-2 mutein molecule is 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, D84Q D84R, D84S, D84T, S87R, N88A, N88D, N88E, N88I, N88F, N88G, N88M, N88R, N88S, N88V, N88W, V91D, V91E, V9S1G, V9S , I92K, I92R, E95G, and Q126. In some embodiments, the amino acid sequence of the IL-2 mutein molecule has a C125A or C125S substitution as well as 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, The amino acid sequence differs from that set forth in the mature IL-2 sequence by a single substitution selected from V91G, V91S, I92K, I92R, E95G, Q126I, Q126L, and Q126F. In some embodiments, the IL-2 mutein molecule has a C125A or C125S substitution and a It differs from the amino acid sequence set forth in the mature IL-2 sequence by one substitution selected from I92R, L12K, L19D, L19N, L19T, N88D, N88R, N88S, V91D, V91G, V91K, and V91S. In some embodiments, the IL-2 mutein comprises N88R and/or D20H mutations.

In some embodiments, the IL-2 mutein molecule comprises a polypeptide sequence mutation at a position selected from the group consisting of amino acid 30, amino acid 31, amino acid 35, amino acid 69, and amino acid 74. In some embodiments, the position 30 mutation is N30S. In some embodiments, the position 31 mutation is Y31H. In some embodiments, the position 35 mutation 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 contain mutations at positions 30, 31, and/or 35.

In some embodiments, the IL-2 mutein molecule is from the group consisting of N88R, N88I, N88G, D20H, D109C, Q126L, Q126F, D84G, or D84I relative to the mature human IL-2 sequences provided above. Contains replacements to be selected. In some embodiments, the IL-2 mutein molecule comprises the D109C substitution and one or both of the N88R and C125S substitutions. In some embodiments, the cysteine at position 109 of the IL-2 mutein molecule is linked to a polyethylene glycol moiety having a molecular weight of about 5 to about 40 kDa. In some embodiments, the mutein does not contain mutations at positions 109, 126, or 84.

In some embodiments, any of the substitutions described herein are combined with the 125 substitution. Substitutions may be C125S, C125A, or C125V substitutions. In some embodiments, the mutein does not contain a mutation at position 125.

Numbering referred to herein refers to the mature sequence unless otherwise indicated for the IL-2 mutein. If a sequence or position refers to SEQ ID NO: 1, this is an immature sequence. However, in order to replace a position from the immature sequence (SEQ ID NO:1) to the mature sequence (SEQ ID NO:2) all that needs to be done is subtract 20 from the mentioned position in SEQ ID NO:1 to the corresponding position in SEQ ID NO:2. It's just a matter of getting a position.

In addition to the substitutions or mutations described herein, in some embodiments the IL-2 mutein is at one or more of positions 73, 76, 100, or 138 corresponding to SEQ ID NO: 1 or Has a substitution/mutation at one or more of positions 53, 56, 80 or 118 corresponding to number 2. 73 and 100; 73 and 138; 76 and 100; 76 and 138; 100 and 138; 73, 100 and 138; 76, 100 and 138; or 73, 76, 100 and 138, respectively. 53 and 80; 53 and 118; 56 and 80; 56 and 118; 80 and 118; 53, 80 and 118; 56, 80 and 118; or 53, 56, 80 and 118, respectively. Because IL-2 can be fused or tethered to other proteins, as used herein terms referring to SEQ ID NO: 6 or 15 can be used using the NCBI website with the default settings of the alignment software. Refers to how the array is aligned. In some embodiments, the mutation is from leucine to isoleucine. Thus, the IL-2 mutein is 1 at positions 73, 76, 100, or 138, corresponding to SEQ ID NO:1, or at one or more positions 53, 56, 80, or 118, corresponding to SEQ ID NO:2. It may contain one or more isoleucines. In some embodiments, the mutein comprises a mutation in 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 in 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 from leucine to isoleucine. In some embodiments, the mutein also includes mutations at positions 69, 74, 88, 125 corresponding to SEQ ID NO:2, or any combination thereof in these muteins. In some embodiments the mutation is the V69A mutation. In some embodiments the mutation is the Q74P mutation. In some embodiments, the mutation is the N88D or N88R mutation. In some embodiments, the mutation is the C125A or C125S mutation.

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

In some embodiments, the IL-2 mutein is at positions 35, 36, 42, 104, 115, or 146, corresponding to SEQ ID NO: 1, or equivalent positions of SEQ ID NO: 2 (e.g., 15, 16, 22, 84 , 95, and 126). These mutations are the other leucine to isoleucine mutations described herein, or positions 73, 76, 100, or 138, corresponding to SEQ ID NO:1, or 53, 56, 80, corresponding to SEQ ID NO:2. , or in combination with mutations at one or more of positions 118. In some embodiments, the mutation is E35Q, H36N, Q42E, D104N, E115Q, or Q146E, or any combination thereof. In some embodiments, one or more of these substitutions are wild-type. In some embodiments, the mutein is at positions 35, 36, 42, 104, 115, or 146, corresponding to SEQ ID NO:1, or equivalent positions of SEQ ID NO:2 (e.g., 15, 16, 22, 84, 95, or position 126).

Mutations at these positions are at positions 73, 76, 100, or 138, corresponding to SEQ ID NO: 1, or at positions 53, 56, 80, or 118, corresponding to SEQ ID NO: 2, described herein and above. It may be combined with any of the other mutations described herein, including but not limited to substitutions at one or more positions. In some embodiments, the IL-2 mutein comprises the 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 the K55R mutation corresponding to SEQ ID NO:1. In some embodiments, the IL-2 mutein comprises the T57A mutation corresponding to SEQ ID NO:1. In some embodiments, the IL-2 mutein comprises the K68E mutation corresponding to SEQ ID NO:1. In some embodiments, the IL-2 mutein comprises the 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 the 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 each other. In some embodiments, the mutein includes each of these substitutions. In some embodiments, the mutein contains 1, 2, 3, 4, 5, 6, 7, or 8 of these mutations. In some embodiments, the mutein is at positions 35, 36, 42, 104, 115, or 146, corresponding to SEQ ID NO:1, or equivalent positions of SEQ ID NO:2 (e.g., 15, 16, 22, 84, 95, 126, and 126) contain wild-type residues at one or more.

In some embodiments, the IL-2 mutein comprises the 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 the K35R mutation corresponding to SEQ ID NO:2. In some embodiments, the IL-2 mutein comprises the T37A mutation corresponding to SEQ ID NO:2. In some embodiments, the IL-2 mutein comprises the K48E mutation corresponding to SEQ ID NO:2. In some embodiments, the IL-2 mutein comprises the V69A mutation corresponding to SEQ ID NO:2. In some embodiments, the IL-2 mutein comprises the N71R mutation corresponding to SEQ ID NO:2. In some embodiments, the IL-2 mutein comprises the Q74P mutation corresponding to SEQ ID NO:2. In some embodiments, the IL-2 mutein comprises the 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 each other. In some embodiments, the mutein contains 1, 2, 3, 4, 5, 6, 7, or 8 of these mutations. In some embodiments, the mutein includes each of these substitutions. In some embodiments, the mutein is at positions 35, 36, 42, 104, 115, or 146, corresponding to SEQ ID NO:1, or equivalent positions of SEQ ID NO:2 (e.g., 15, 16, 22, 84, 95, and 126) contain wild-type residues.

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

In some embodiments, the IL-2 mutein has the sequence:
MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATEIKHLQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 3)
including.

In some embodiments, the IL-2 mutein has the sequence:
MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHIQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 4)
including.

In some embodiments, the IL-2 mutein has the sequence:
MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHIRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 5)
including.

In some embodiments, the IL-2 mutein has the sequence:
MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFINRWITFSQSIISTLT (SEQ ID NO: 6)
including.

In some embodiments, the IL-2 mutein sequences described herein do not contain an IL-2 leader sequence. The IL-2 leader sequence can be represented by the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO:7). Thus, in some embodiments, the sequences exemplified above can also include peptides without leader sequences. A sequence with a unique mutation at one or more of positions 73, 76, 100 or 138 corresponding to SEQ ID NO: 1 or at positions 53, 56, 80 or 118 corresponding to SEQ ID NO: 2 Although numbers 3-6 are exemplified, peptides can contain 1, 2, 3, or 4 mutations at these positions. In some embodiments, each positional substitution is an isoleucine or other type of conservative amino acid substitution. In some embodiments, leucines at recited positions are 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 has one mutation selected from the group consisting of: V69A, Q74P, N88D, and C125S or C125A, and L53I, L56I, L80I, and L118I. Contains mutations. In some embodiments, the IL-2 protein comprises two mutations selected from the group consisting of L53I, L56I, L80I, and L118I. In some embodiments, the IL-2 protein comprises three or each mutation selected from the group consisting of L53I, L56I, L80I, and L118I. In some embodiments, the IL-2 protein is L53I and L56I, L53I and L80I, L53I and L118I, L56I and L80I, L56I and L118I, L80I and L118I, L53I, L56I, and L80I, L53I, L56I, and L118I , L56I, L80I, and L118I or L53I, L56I, L80I, and L118I. In some embodiments, the IL-2 mutein does not contain the L53I, L56I, L80I, or L118I mutations. In some embodiments the IL-2 mutein comprises a T3A mutation.

In some embodiments, the IL-2 protein of SEQ ID NO:2 has the following mutations: V69A, Q74P, N88D, and C125S or C125A and 45-55, 50-60, 52-57, 75 of SEQ ID NO:2. 85, 100-130, 115-125, including one or more mutations, including but not limited to conservative substitutions.

In some embodiments, the IL-2 mutein molecule is fused to an Fc region or other linker region described herein.そのような融合タンパク質の例は、ＵＳ９５８０４８６、ＵＳ７１０５６５３、ＵＳ９６１６１０５、ＵＳ ９４２８５６７、ＵＳ２０１７／００５１０２９、ＷＯ２０１６／１６４９３７、ＵＳ２０１４／０２８６８９８Ａ１、ＷＯ２０１４１５３１１１Ａ２、ＷＯ２０１０／０８５４９５、ＷＯ２０１６０１４４２８Ａ２、ＷＯ２０１６０２５３８５Ａ１、ＵＳ２０１７／００３７１０２、およびＵＳ２００６／０２６９５１５に見出す, each of which is incorporated by reference in its entirety.

In some embodiments, the Fc region contains what are known as LALA mutations. 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 contain a mutation at position G237 (EU numbering). Using Kabat numbering, this corresponds to L247A, L248A, and/or G250A. In some embodiments, the Fc region comprises the L234A, L235A, and/or G237A mutations using the EU numbering system. Regardless of the numbering system used, in some embodiments the Fc portion may contain mutations corresponding to one or more of these residues. In some embodiments, the Fc region comprises the N297G or N297A (kabat numbering) mutation. Kabat numbering is based on the full-length sequence, but will be used for fragments based on traditional alignments used by those skilled in the art for Fc regions (e.g., Kabat et al., "Sequence of "proteins of immunological interest", US Public Health Services, NIH Bethesda, Md., Publication No. 91. In some embodiments, the Fc region has the sequence:
DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSQVMHEALHLSTKS (SEQ ID NO:8)
including.
In some embodiments, the Fc region has the sequence:
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSQVMHEALHLSTPG (array number 1LSTPG)
including.

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 include the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 9), or may be or include the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 16). This is just a non-limiting example, linkers can have varying numbers 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. Linkers may be as described herein or as exemplified in the table below.

Thus, an IL-2/Fc fusion has the formula Z _IL-2M -L _gs -Z _Fc , where Z _IL-2M is an IL-2 mutein as described herein and L _gs is , is a linker sequence described herein (eg, a glycine/serine linker), and Z _Fc is an Fc region described herein or known to one of skill in the art. In some embodiments, the formula may be the reverse orientation Z _Fc -L _gs -Z _IL-2M .

In some embodiments, the IL-2/Fc fusion comprises the sequence:
MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATEIKHLQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG（配列番号１１）
including.

In some embodiments, the IL-2/Fc fusion comprises the sequence:
MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHIQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG（配列番号１２）
including.

In some embodiments, the IL-2/Fc fusion comprises the sequence:
MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHIRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG（配列番号１３）
including.

In some embodiments, the IL-2/Fc fusion comprises the sequence:
MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFINRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG（配列番号１４）
including.

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

The proteins described herein can also be fused to another protein such as an antibody or other type of therapeutic molecule.

In some embodiments, the sequences of IL-2 muteins or IL-2/Fc fusions are as shown in the table below:

Each of the proteins can also be considered to have the C125S and LALA and/or G237A mutations provided herein. The C125 substitution may also be C125A, described throughout this application.

In some embodiments, the sequences shown in the table or throughout the application contain or do not contain one or more mutations corresponding to positions L53, L56, L80, and L118. In some embodiments, the sequences shown in the tables or throughout the application have one or more mutations corresponding to positions L59I, L63I, I24L, L94I, L96I or L132I or other substitutions at the same positions. with or without In some embodiments, the mutation is from leucine to isoleucine. In some embodiments, the mutein does not contain additional mutations other than those shown or described herein. In some embodiments, the peptide is 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 lacks an Fc portion.

In some embodiments, a polypeptide comprising SEQ ID NO: 43, wherein at least one of _Xi , _X2 , _X3 , and _X4 is I and the rest are L or I is provided be. In some embodiments, X ₁ , X ₂ , and X ₃ are L and X ₄ is I. In some embodiments, X ₁ , X ₂ , and X ₄ are L and X ₃ is I. In some embodiments, X ₂ , X ₃ , and X ₄ are L and X ₁ is I. In some embodiments, X ₁ , X ₃ , and X ₄ are L and X ₂ is I. In some embodiments, X ₁ and X ₂ are 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 , X ₂ , and X ₃ are L and X ₄ is I. In some embodiments, X ₂ , X ₃ , and X ₄ are L and X ₁ is I. In some embodiments, X ₁ , X ₃ , and X ₄ are L and X ₂ is I. In some embodiments, X ₁ , X ₂ , and X ₄ are L and X ₃ is I.

In some embodiments, the IL-2 mutein may be in the format illustrated in FIG. However, as described herein, an IL-2 mutein may be used without an Fc domain in some embodiments, or an Fc domain is linked to the C-terminus of the IL-2 mutein. It is linked to the N-terminus of the IL-2 mutein as opposed to the Fc domain. The polypeptides described herein also encompass variants of the described peptides. In some embodiments, the IL-2 variant is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% a sequence provided herein , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% substantially similar amino acid sequences. 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, 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 table below:

The percent identity of two amino acid sequences or two nucleic acid sequences can be determined by visual inspection and mathematical calculations, or the comparison is made, for example, by comparing the sequence information using a computer program. An exemplary computer program is the Genetics Computer Group (GCG; Madison, Wis.) Wisconsin package version 10.0 program, GAP (Devereux et al. (1984) Nucleic Acids Res. 12:387-95). Preferred default parameters for the GAP program include (1) the GCG implementation of the unary comparison matrix for nucleotides (containing a value of 1 for identity and 0 for non-identity) and the Atlas of Polypeptide Sequence and Structure, Schwartz and Dayhoff Ed., National Biomedical Research Foundation, pp. 353-358 (1979), the weighted amino acid comparison matrix of Gribskov and Burgess ((1986) Nucleic Acids Res. 14:6745), or other equivalent comparison matrix; 2) a penalty of 8 for each gap and an additional penalty of 2 for each symbol in each gap for amino acid sequences, or a penalty of 50 for each gap and each symbol in each gap for nucleotide sequences; (3) no penalty for end gaps; and (4) no maximum penalty for long gaps. Other programs for sequence comparison used by those skilled in the art can also be used.

In some embodiments, the IL-2 muteins provided herein alter signaling through specific pathways that are activated by wild-type IL-2 via IL-2R, resulting in Included are proteins that provide preferential growth/survival/activation.

IL-2 muteins provided herein include those described in US Pat. No. 6,955,807 for the production of IL-2 variants, which are incorporated herein by reference. It can be made using any suitable method known in the art. Such methods include constructing DNA sequences encoding IL-2 variants and expressing those sequences in a suitably transformed host, such as a host cell. Utilization of these methods produces the recombinant proteins provided herein. Proteins can also be made synthetically, or synthetic fragments and fragments made recombinantly in cells can be combined to make the entire protein of interest.

In some embodiments, a nucleic acid molecule (eg, DNA or RNA) is produced by isolating or synthesizing a nucleic acid molecule that encodes a protein of interest. Alternatively, the IL-2 wild-type sequence can be isolated and mutated using routine techniques such as site-directed mutagenesis.

Another method of constructing DNA sequences encoding IL-2 variants would be chemical synthesis. This includes, for example, direct synthesis of peptides by chemical means of protein sequences encoding IL-2 variants exhibiting the properties described herein. This method can incorporate both natural and unnatural amino acids at various positions. Alternatively, nucleic acid molecules encoding the desired protein can be synthesized by chemical means using an oligonucleotide synthesizer. Oligonucleotides are designed based on the amino acid sequence of the desired protein, which may be selected by using codons that are favorable in the cell in which the recombinant variant is to be made. It is well recognized that the genetic code is degenerate, ie, one amino acid can 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 are many DNA degenerate sequences encoding IL-2 variants thereof. Accordingly, in some embodiments, nucleic acid molecules are provided that encode the proteins described herein. A nucleic acid molecule may be DNA or RNA.

In some embodiments, the nucleic acid molecule will encode a signal sequence. Signal sequences can also be chosen based on the cells in which they are to be expressed. In some embodiments, if the host cell is prokaryotic, the nucleic acid molecule does not contain a signal sequence. In some embodiments, if the host cell is eukaryotic, a signal sequence may be used. In some embodiments, the signal sequence is the IL-2 signal sequence.

A nucleic acid molecule "encodes" a protein as meant herein if the nucleic acid molecule or its complement contains codons that encode the protein.

"Recombinant", when applied to a polypeptide or protein, is the process by which nucleic acids, which may or may not encode the protein, are introduced into cells in which they are not found in nature. means that it depends on

A variety of hosts (animals or cell lines) can be used to produce the proteins described herein. Examples of suitable host cells include, but are not limited to, bacterial, fungal (including yeast), plant, insect, mammalian, or other suitable animal cells or cell lines, and transgenic animals or plants. not. In some embodiments, these hosts include well-known eukaryotic and prokaryotic hosts such as E. coli, Pseudomonas, Bacilli, Streptomyces, fungi, yeast, insect (such as Spodoptera frugiperda (Sf9)) cells. , animal cells (such as Chinese hamster ovary (CHO) cells and mouse cells such as NS/O, African green monkey cells such as COS 1, COS 7, BSC 1, BSC 40, and BNT 10, and human cells), and tissue culture Plant cell lines such as those listed below may be included. For animal cell expression, CHO cells and COS 7 cells in culture, especially CHO cell lines CHO (DHFR-) or HKB lines can be used.

Of course, it should be understood that not all vectors and expression control sequences will function equally well to express the DNA sequences described herein. Also, not all hosts function equally well with the same expression system. However, one of ordinary skill in the art can make a selection among these vectors, expression control sequences and hosts without undue experimentation. For example, in choosing a vector, the host must be considered since the vector must replicate in the host. The vector copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered. For example, preferred vectors for use in the present invention include those that allow the DNA encoding the IL-2 variant to be copy number amplified. Such amplifiable vectors are well known in the art.

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

Host cells that can be used herein as described herein

Pharmaceutical Compositions In another aspect, this embodiment provides a composition comprising a therapeutic compound (IL-2 mutein) described herein formulated with a pharmaceutically acceptable carrier, such as , provides pharmaceutically acceptable compositions. As used herein, "pharmaceutically acceptable carrier" includes any physiologically compatible solvents, dispersion media, isotonic and absorption delaying agents and the like. The carrier may be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, local, topical, spinal or cutaneous administration (eg, by injection or infusion).

The compositions of the invention may be in various forms. These include, for example, liquid, semisolid and solid dosage forms such as liquid solutions (eg, 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 (eg, 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 locally, eg, by injection or topical application, to the target site.

The phrases "parenteral administration" and "administered parenterally" as used herein refer to modes of administration other than enteral and topical administration, usually by injection, including intravenous, intramuscular , intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injections and Including but not limited to infusion.

Therapeutic compositions typically should be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high therapeutic concentration of the molecule. Sterile injectable solutions are prepared by incorporating the active compound (i.e., therapeutic molecule) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. can be done. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the manufacture of sterile injectable solutions, the preferred methods of manufacture are vacuum drying and lyophilization, which yields a powder of the active ingredient plus any additional desired ingredient from a solution thereof that has been previously sterile filtered. . Proper fluidity of the solution can be maintained by use of a coating such as lecithin, maintenance of required particle size in the case of dispersions, and use of surfactants. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate and gelatin.

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

In certain embodiments, therapeutic compounds may be administered orally, eg, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) can also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds can be incorporated 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 a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with a material that protects it from inactivation, or to co-administer the compound with a material that protects it from inactivation. A therapeutic composition can also be administered with medical devices known in the art.

Dosage regimens are adjusted to provide the optimum desired response (eg, a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. . It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated, each unit in association with the required pharmaceutical carrier. It contains a predetermined amount of active compound calculated to produce the desired therapeutic effect. The dosage unit form specifications of the present invention include (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) techniques for formulating such active compound for the treatment of susceptibility in individuals. It is dictated and directly influenced by the limitations inherent in the field.

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

Pharmaceutical compositions of the invention may comprise a “therapeutically effective amount” or a “prophylactically effective amount” of a therapeutic molecule of the invention. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of a therapeutic molecule may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic compound to elicit the desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic molecule are outweighed by the therapeutically beneficial effects. A "therapeutically effective dose" preferably reduces a measurable parameter, e.g., immune attack, by at least about 20%, more preferably by at least about 40%, and even more preferably by at least Inhibit about 60%, and even more preferably at least about 80%. Measurable parameters, such as the ability of a compound to inhibit immune attack, may be evaluated in animal model systems predictive of efficacy in graft rejection or autoimmune disorders. Alternatively, this property of a composition can be evaluated by examining such inhibition in vitro, the ability of a compound to inhibit, by assays known to those skilled in the art.

A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, a prophylactic dose will be used in a subject prior to or at an earlier stage of the disease, so the prophylactically effective amount will be less than the therapeutically effective amount.

Kits containing the therapeutic compounds described herein are also within the scope of the invention. Kits may include instructions for use; other reagents, such as labels, therapeutic agents, or agents useful for chelating or otherwise coupling therapeutic molecules to labels or other therapeutic agents, or radioprotective compositions. one or more other elements, including an object; a device or other material for manufacturing a therapeutic molecule for administration; a pharmaceutically acceptable carrier; and a device or other material for administration to a subject. may include

Combinations The proteins described herein can also be administered in combination with other agents useful for treating the condition with which the patient is afflicted. Examples of such agents include both proteinaceous and non-proteinaceous drugs. When multiple therapeutic agents are co-administered, dosages may be adjusted accordingly as recognized in the relevant art. "Co-administration" and combination therapy are not limited to simultaneous administration, but are therapeutic regimens in which a Treg-selective IL-2 protein is administered at least once during a course of treatment that includes administration of at least one other therapeutic agent to the patient. Also includes

In some embodiments, the Treg-selective IL-2 protein is administered in combination with an inhibitor of the PI3-K/AKT/mTOR pathway, eg, rapamycin (rapamune, sirolimus). Inhibitors of this pathway in combination with IL-2 favor Treg enrichment. In some embodiments, the IL-2 protein is administered without another therapeutic agent that is not directly fused or conjugated to the IL-2 protein.

Therapeutic Methods "Treatment" of any disease referred to herein includes alleviating at least one symptom of the disease, reducing the severity of the disease, or optionally treating the disease or at least one other disease. It includes delaying or preventing disease progression to more severe symptoms that may accompany the disease. Treatment need not mean that the disease is completely cured. Useful therapeutic agents reduce the severity of the disease, reduce the severity of the disease or symptoms associated with its treatment, or reduce the severity of more severe symptoms or more severe disease that may occur with a certain frequency after treatment of the condition. It is only necessary to delay the onset. For example, if the disease is inflammatory bowel disease, the therapeutic agent reduces the number of overt inflammatory sites in the bowel, the total extent of affected bowel, reduces pain and/or swelling, reduces diarrhea, constipation, or It may reduce symptoms such as vomiting and/or prevent bowel perforation. Patient status can be assessed by standard procedures such as x-rays, endoscopies, colonoscopies, and/or biopsies performed after a barium enema or hyperbolic enema. Appropriate procedures vary according to the patient's condition and symptoms.

In some embodiments, proteins are used to treat inflammatory disorders. In some embodiments, the inflammatory disorder is inflammation, autoimmune disease, atopic disease, paraneoplastic autoimmune disease, cartilage inflammation, arthritis, rheumatoid arthritis (e.g., active), juvenile arthritis, juvenile rheumatoid arthritis , small articular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronegative, enthesopathy, arthritic syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, small joint rheumatoid arthritis, polyarthritis Rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome (seronegative, enthesopathy, arthritic syndrome), dermatomyositis, psoriatic arthritis, scleroderma vasculitis, myositis, polymyositis, dermatomyositis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatoid arthritis, sarcoidosis, sclerosis, primary biliary sclerosis, bile sclerosing inflammation, Sjögren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythroderma psoriasis, dermatitis, atopic dermatitis, dermatitis herpetiformis, Behcet's disease (including effects on the skin) , but not limited to), alopecia, alopecia areata, alopecia totalis, atherosclerosis, lupus, Still's disease, systemic lupus erythematosus (SLE) (e.g. active), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, sinusitis, sinusitis with polyps, eosinophilic esophagitis, eosinophilic Bronchitis, Guillain-Barré disease, type I diabetes, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, graft-versus-host disease, steroid-resistant chronic graft-versus-host disease, transplant rejection (e.g., kidney, lung, heart, skin, etc.), kidney injury, hepatitis C-induced vasculitis, spontaneous pregnancy loss, alopecia, vitiligo, focal segmental glomerulosclerosis (FSGS), minimal changes, membranes nephropathy, ANCA-associated glomerulonephropathy, membranous proliferative glomerulonephropathy, IgA nephropathy, lupus nephritis, and the like. In some embodiments, the protein is used to treat steroid-resistant 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 can be administered in the form of compositions (eg, pharmaceutical compositions) described herein. For example, a composition may comprise an IL-2 protein as described herein plus a buffer, an antioxidant such as ascorbic acid, a low molecular weight polypeptide (such as having less than 10 amino acids), a protein, an amino acid, Carbohydrates such as glucose, sucrose, or dextrin may be included, chelating agents such as EDTA, glutathione, and/or other stabilizers, excipients, and/or preservatives. Compositions may be formulated as liquids or lyophilizates. Further examples of ingredients that can be used in pharmaceutical formulations are found in Remington's Pharmaceutical Sciences, 16. sup. th Ed. , Mack Publishing Company, Easton, Pa. , (1980) and other references cited herein.

Compositions containing therapeutic molecules described herein to treat diseases of interest include parenteral, topical, oral, nasal, vaginal, rectal, or pulmonary (by inhalation) administration, although may be administered by any suitable method, including but not limited to. If injected, the composition may be administered intraarticularly, intravenously, intraarterially, intramuscularly, intraperitoneally, or subcutaneously by bolus injection or continuous infusion. Topical administration (ie, at the site of disease) is contemplated as transdermal delivery and sustained release from depots, 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. Administration via suppositories inserted into body cavities can be accomplished, for example, by inserting the solid composition into the body cavity of choice and allowing it to dissolve. Other alternatives include eye drops, oral agents such as pills, troches, syrups, and chewing gums, and topical agents such as lotions, gels, sprays, and ointments. In most cases, therapeutic molecules that are polypeptides can 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 dosage, frequency, and duration that can be effective to treat the condition under treatment. Dosages will depend on the molecular nature of the therapeutic molecule and the nature of the disorder being treated. Treatment may be continued as long as necessary to achieve the desired result. The therapeutic molecules of the invention may be administered as a single dose or, among other possible dosing regimens, several times a day, once a day, every other day, twice a week, three times a week, once a week, It may be administered as a series of doses given on a regular basis, including every other week and monthly doses. The periodicity of treatment may or may not be constant throughout the treatment period. For example, treatment may initially occur at weekly intervals and then at weekly intervals. Treatments having a duration of days, weeks, months or years are encompassed by the present invention. Treatment may be discontinued and then resumed. A 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 as milligrams per square meter of skin surface (mg/m ² ), or as a fixed dose regardless of height or weight. All of these are standard dosage units in the art. A person's skin surface area is calculated from height and weight 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 same.

As used herein, the phrase "in need of" means that a subject (animal or mammal) has been identified as in need of a particular method or treatment. In some embodiments, identification may be by any means of diagnosis. The animal or mammal may be in need of any of the methods and treatments described herein. In some embodiments, the animal or mammal is in or moves 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.

As used herein, the terms "a" or "an" mean "at least one" or "one or more" unless the context clearly dictates otherwise.

As used herein, the term "about" means that the numerical value is approximate and small variations do not materially affect the practice of the disclosed embodiments. When numerical limitations are used, unless the context dictates otherwise, "about" means that the numerical values can vary ±10% and remain within the disclosed embodiments.

As used herein, the terms "individual" or "subject" or "patient" used interchangeably include mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep. , horses, or any animal including mammals such as primates such as humans.

As used herein, the terms “comprising” (and any form of including, such as “comprise,” “comprises,” and “comprised”), “having” (and having) any form such as “have” and “has”), “including” (and any form of including such as “includes” and “include”), or “containing” (and (eg, “contains” and “contains”) is inclusive or non-limiting and does not exclude additional, unlisted elements or method steps. Any process or composition that uses the transitional phrases "comprise" or "comprising" is also said to describe the same for the transitional phrases "consisting of" or "consists." can.

As used herein, the term "contacting" means to bring two elements together in an in vitro or in vivo system. For example, "contacting" a Treg cell or an individual or patient or cell with a peptide or composition described herein includes administration of the compound to an individual, such as a human, or patient, and, for example, Treg cells. Including introducing the compound into a sample containing cell preparation or purified preparation containing the compound.

As used herein, the terms "fused" or "linked" when used in reference to proteins having different domains or heterologous sequences refer to peptide bonds or other covalent bonds are part of the same peptide chain. The domains or sections 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 are still considered fused or linked to each other. It will be. In some embodiments, the various domains or proteins provided herein are directly linked or fused to each other, or linker sequences such as the glycine/serine sequences described herein connect the two domains.

In some embodiments, embodiments provided herein also include, but are not limited to:
1. A peptide comprising the amino acid sequence of SEQ ID NO: 1 containing a mutation at position 73, 76, 100 or 138.
2. 2. The peptide of embodiment 1, wherein the mutation is an L to I mutation at positions 73, 76, 100, or 138.
3. 2. The peptide of embodiment 1, further comprising mutations at one or more of positions 49, 51, 55, 57, 68, 89, 91, 94, 108, and 145.
4. further comprising a mutation at one or more of positions E35, H36, Q42, D104, E115, or Q146, or one, two, three, four of E35, H36, Q42, D104, E115, or Q146; 2. The peptide of embodiment 1, wherein 5 or each is wild type.
5. 5. The peptide of embodiment 4, wherein the mutations are one or more of E35Q, H36N, Q42E, D104N, E115Q, or Q146E.
6. 6. The peptide of any one of embodiments 1-5, comprising the N49S mutation.
7. 7. The peptide of any one of embodiments 1-6, comprising a Y51S or Y51H mutation.
8. A peptide according to any one of embodiments 1-7, comprising a K55R mutation.
9. A peptide according to any one of embodiments 1-8, comprising the T57A mutation.
10. 10. The peptide of any one of embodiments 1-9, comprising the K68E mutation.
11. 11. The peptide of any one of embodiments 1-10, comprising the V89A mutation.
12. 12. The peptide of any one of embodiments 1-11, comprising the N91R mutation.
13. 13. The peptide of any one of embodiments 1-12, comprising the Q94P mutation.
14. 14. The peptide of any one of embodiments 1-13, comprising the N108D or N108R mutation.
15. 15. The peptide of any one of embodiments 1-14, comprising a C145A or C145S mutation.
15.1. 16. The peptide of any one of embodiments 1-15, comprising one or more of V89A, Q94P, N108R or N108D, and L73I, L76I, L100I, L118I mutations, and optionally C145A or C145S mutations.
16. A peptide comprising the amino acid sequence of SEQ ID NO:2 containing a mutation at positions 53, 56, 80, or 118.
17. 17. The peptide of embodiment 16, wherein the mutation is an L to I mutation at positions 53, 56, 80, or 118.
18. 18. The peptide of embodiment 16 or 17, further comprising mutations at one or more of positions 29, 31, 35, 37, 48, 69, 71, 74, 88, and 125.
19. further comprising a mutation at one or more of positions E15, H16, Q22, D84, E95, or Q126, or one, two, three, four of positions E15, H16, Q22, D84, E95, or Q126 , 5, or each is wild-type.
20. 20. The peptide of embodiment 19, wherein the mutations are one or more of E15Q, H16N, Q22E, D84N, E95Q, or Q126E.
21. 21. A peptide according to any one of embodiments 16-20, comprising the N29S mutation.
22. 22. A peptide according to any one of embodiments 16-21, comprising a Y31S or Y51H mutation.
23. 23. A peptide according to any one of embodiments 16-22, comprising the K35R mutation.
24. 24. The peptide of any one of embodiments 16-23, comprising the T37A mutation.
25. 25. The peptide of any one of embodiments 16-24, comprising the K48E mutation.
26. 26. A peptide according to any one of embodiments 16-25, comprising a V69A mutation.
27. 27. The peptide of any one of embodiments 16-26, comprising the N71R mutation.
28. 28. The peptide of any one of embodiments 16-27, comprising the Q74P mutation.
29. 29. The peptide of any one of embodiments 16-28, comprising the N88D or N88R mutation.
30. 30. The peptide of any one of embodiments 16-29, comprising a C125A or C125S mutation.
31. 31. The peptide of any one of embodiments 16-30, comprising one or more of V69A, Q74P, N88R or N88D, and L53I, L56I, L80I, L118I mutations, and optionally C125A or C125S mutations.
32. 32. The peptide according to any one of embodiments 1-31, which does not contain mutations corresponding to positions 30, 31 and/or 35.
33. 33. The peptide of any one of embodiments 1-32, further comprising an Fc peptide.
33A. 34. 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.
33B. 34. The peptide of embodiment 33, wherein the Fc peptide comprises the following mutations: L247A, L248A and G250A (Kabat numbering) or L234A L235A and G237A (EU numbering).
34. 34. The peptide of embodiment 33, wherein the Fc peptide comprises the sequence of SEQ ID NO:8 or SEQ ID NO:15.
35. 35. The peptide of any one of embodiments 1-34, further comprising a linker peptide connecting the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 and the Fc peptide.
36. 36. The peptide of embodiment 35, wherein the linker comprises the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 9) or GGGGSGGGGSGGGGGS (SEQ ID NO: 16).
37. 37. The peptide according to any one of embodiments 1-36, comprising the sequences of SEQ ID NOs: 17-43.
38. A peptide comprising the amino acid sequence of SEQ ID NO:27.
39. 39. The peptide of embodiment 38, further comprising an N-terminal leader peptide having the sequence of SEQ ID NO:7.
40. 2. The peptide of embodiment 1, further comprising a linker peptide at the C-terminus.
41. 41. The peptide of embodiment 40, wherein the linker peptide comprises the sequence (GGGGS) _n (SEQ ID NO: 10), where n is 1, 2, 3, or 4.
42. 42. The peptide of embodiment 41, wherein n is 1.
43. 42. The peptide of embodiment 41, wherein n is two.
44. 42. The peptide of embodiment 41, wherein n is 3.
45. 42. The peptide of embodiment 41, wherein n is 4.
46. 46. The peptide according to any one of embodiments 48-45, further comprising an Fc peptide.
47. 47. The peptide of embodiment 46, wherein the Fc peptide comprises the sequence of SEQ ID NO:8 or SEQ ID NO:15.
48. 48. The peptide of embodiment 47, further comprising a leader peptide having the sequence of SEQ ID NO:7 at its N-terminus.
49. 47. The peptide of embodiment 46, wherein the Fc peptide is C-terminal to the peptide comprising SEQ ID NO:27.
50. 47. The peptide of embodiment 46, wherein the Fc peptide is N-terminal to the peptide comprising SEQ ID NO:27.
51. 2. The peptide of embodiment 1, further comprising a linker peptide connecting the peptide of SEQ ID NO:27 and the Fc peptide.
52. 52. The peptide of embodiment 51, wherein the linker peptide is (GGGGS) _n (SEQ ID NO: 10), where n is 1, 2, 3, or 4.
53. 53. The peptide of embodiment 52, wherein n is 4.
54. 54. The peptide of embodiments 51-53, wherein the Fc peptide comprises the sequence of SEQ ID NO:8 or SEQ ID NO:15.
55. 55. The peptide according to embodiments 51-54, wherein the Fc peptide is C-terminal to the peptide comprising SEQ ID NO:27.
56. 55. The peptide according to embodiments 51-54, wherein the Fc peptide is N-terminal to the peptide comprising SEQ ID NO:27.
57. 15. The peptide of embodiment 14, comprising a peptide comprising the sequence of SEQ ID NO:37, 38, 39, or 40.
58. A pharmaceutical composition comprising a peptide according to any one of embodiments 1-57.
59. A method of activating a T regulatory cell comprising contacting the T regulatory cell with a peptide according to any one of embodiments 1-57 or a pharmaceutical composition according to embodiment 58. .
60. A method of treating an inflammatory disorder in a subject, wherein a peptide according to any one of embodiments 1-57 or a pharmaceutical composition according to embodiment 58 is therapeutically effective in a subject in need thereof. amount.
61. Inflammatory disorders include inflammation, autoimmune diseases, atopic diseases, paraneoplastic autoimmune diseases, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, small articular juvenile rheumatoid arthritis, polyarticular Juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome) ), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, small joint rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spine inflammation, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome (seronegative, enthesopathy, arthrosis syndrome), dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myositis, polymyositis, skin Myositis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatoid arthritis, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjögren's syndrome, psoriasis, psoriasis vulgaris, drops psoriasis, inverse psoriasis, pustular psoriasis, erythroderma psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, sinusitis, sinusitis with polyps, eosinophilic esophagitis, eosinophilic Bronchitis, Guillain-Barré disease, type I diabetes, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, graft-versus-host disease (GVHD), steroid-resistant chronic graft-versus-host disease, Transplant rejection (e.g. kidney, lung, heart, skin, etc.), kidney injury, hepatitis C-induced vasculitis, spontaneous pregnancy loss, alopecia, vitiligo, focal segmental glomerulosclerosis (FSGS), minimal changes 61. The method of embodiment 60, which is a group, membranous nephropathy, ANCA-associated glomerulonephropathy, membranous proliferative glomerulonephropathy, IgA nephropathy, lupus nephritis, and the like.
62. A method of promoting or stimulating STAT5 phosphorylation in a T regulatory cell comprising administering to a subject in need thereof a peptide according to any one of embodiments 1-57 or a pharmaceutical composition according to embodiment 58 The above method of administering a therapeutically effective amount of
63. Use of a peptide according to any one of embodiments 1-57 or a pharmaceutical composition according to embodiment 58 in the manufacture of a medicament for the treatment of an inflammatory disorder.
64. Inflammatory disorders include inflammation, autoimmune diseases, atopic diseases, paraneoplastic autoimmune diseases, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, small articular juvenile rheumatoid arthritis, polyarticular Juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome) ), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, small joint rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spine inflammation, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome (seronegative, enthesopathy, arthrosis syndrome), dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myositis, polymyositis, skin Myositis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatoid arthritis, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjögren's syndrome, psoriasis, psoriasis vulgaris, drops psoriasis, inverse psoriasis, pustular psoriasis, erythroderma psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, sinusitis, sinusitis with polyps, eosinophilic esophagitis, eosinophilic Bronchitis, Guillain-Barré disease, type I diabetes, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, graft-versus-host disease (GVHD), steroid-resistant chronic graft-versus-host disease, Transplant rejection (e.g. kidney, lung, heart, skin, etc.), kidney injury, hepatitis C-induced vasculitis, spontaneous pregnancy loss, alopecia, vitiligo, focal segmental glomerulosclerosis (FSGS), minimal changes 64. Use according to embodiment 63, which is a group, membranous nephropathy, ANCA-associated glomerulonephropathy, membranous proliferative glomerulonephropathy, IgA nephropathy, lupus nephritis, and the like.
65. A nucleic acid molecule encoding a peptide according to 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 containing the plasmid of embodiment 67.
70. A cell comprising the vector of embodiment 66.
71. A cell comprising or expressing a peptide according to any one of embodiments 1-57 or a peptide described herein.

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

A therapeutic composition comprising the protein of SEQ ID NO: 11, 12, 13, or 14 was administered to a subject suffering from IBD. The subject's immune system is downregulated and the symptoms of IBD are alleviated.

A therapeutic composition comprising the protein of SEQ ID NO: 3, 4, 5, or 6 with or without a leader sequence was administered to a subject suffering from IBD. The subject's immune system is downregulated and the symptoms of IBD are alleviated.

Generation of IL-muteins A pTT5 vector containing a single gene encoding a human IL-2M polypeptide N-terminally (SEQ ID NO:40) or C-terminally (SEQ ID NO:41) fused to a human IgG1 Fc domain was injected into HEK293 Expi cells. transfected. After 5-7 days, cell culture supernatants expressing IL-2M were harvested, clarified by centrifugation and filtered through a 0.22 um filtration device. IL-2M was captured on protein A resin. The resin was washed with PBS pH 7.4 and captured protein was eluted using 0.25% acetic acid, pH 3.5 while neutralizing using 1/10 volume of 1 M Tris pH 8.0. Proteins were buffer exchanged into 30 mM HEPES 150 mM NaCl pH 7 and analyzed by size exclusion chromatography on a Superdex 200 3.2/300 column. 5 ug of purified material was analyzed by reducing and non-reducing SDS-PAGE on Bis-Tris 4-12% gels. IL-2M was expressed at >10 mg/L after purification and was >95% monodisperse as shown by size exclusion chromatography and reducing/non-reducing SDS-PAGE.

IL-2M Molecules Can Bind CD25 Immunosorbent plates were coated with 75 ul/well of CD25 at a concentration of 0.5 μg/mL in PBS pH 7.4 and incubated overnight at 4°C. Wells were washed three times with PBS pH 7.4 containing 0.05% Tween-20 (wash buffer) and then with 200 ul/well 1% BSA in PBS pH 7.4 (block buffer) at room temperature. Blocked for 2 hours. After washing three times with wash buffer, IL-2M molecules were diluted in 12-fold serial dilutions in PBS containing 1% BSA and 0.05% Tween-20 (assay buffer) with a top concentration of 2 nM. Diluted material was added to CD25 coated plates at 75 ul/well for 1 hour at room temperature. After washing three times with wash buffer, goat biotinylated anti-IL-2 polyclonal antibody diluted to 0.05 μg/mL in assay buffer was added to the plate at 75 ul/well for 1 hour at room temperature. After washing three times with wash buffer, streptavidin HRP diluted 1:5000 in assay buffer was added to the plate at 75 ul/well for 15 minutes at room temperature. After washing three times with wash buffer and once with wash buffer (without tween-20), the assay was developed with TMB and stopped with 1N HCL. OD450nm was measured. Experiments included appropriate controls for non-specific binding of IL-2M molecules to the plate/block in the absence of CD25, and a negative control molecule unable to bind CD25.

The results showed that at concentrations from 2 nM to 1.9 pM, IL-2M molecules were able to bind CD25 with sub-nanomolar EC50s. Furthermore, when CD25 was not present on the plate surface, no compound was detected at any concentration tested, indicating that the test compound was not non-specifically interacting with the plate surface (data not shown). .

In Vitro P-STAT5 Assay to Determine Potency and Selectivity of IL-2M Molecules Peripheral Blood Mononuclear Cells (PBMC) were obtained from freshly isolated heparinized human whole blood using FICOLL-PAQUE Premium and Sepmate tubes. manufactured. PBMCs were cultured in 10% fetal bovine serum RPMI media for 20 minutes in the presence of wild-type IL-2 or IL-2M of Example 12, then fixed with BD Cytofix for 10 minutes.

Fixed cells were permeabilized sequentially with BD Perm III and then BioLegend FOXP3 permeabilization buffer. After blocking with human serum for 10 min, cells were stained with antibodies against phospho-STAT5 FITC, CD25 PE, FOXP3 AF647 and CD4 PerCP Cy5.5 for 30 min followed by acquisition on an Attune NXT with plate reader. . IL-2M of SEQ ID NO:23 potently and selectively induced STAT5 phosphorylation in Tregs, whereas Teff did not.

Immunogenicity of IL-2 muteins IL-2 mutein variant sequences were analyzed using NetMHCIIPan 3.2 software which can be found at www 'dot' cbs 'dot' dtu 'dot' dk/services/NetMHCIIpan/ bottom. Peptide affinities for MHC class II alleles were determined using artificial neural networks. Within that analysis, a 9-residue peptide that might have direct interactions with MHC class II molecules was recognized as the binding core. Residues flanking the binding core that might indirectly affect binding were also investigated as masking residues. Peptides containing both binding core and masking residues were marked as strong binders if the predicted KD for MHC class II molecules was lower than 50 nM. Strong binders are more likely to induce T cell immunogenicity.

A total of 9 MHCII alleles highly represented in North America and Europe were included in the computational analysis. The panel of IL-2M (IL-2 mutein) molecules tested included IL-2 muteins with L53I, L56I, L80I, or L118I mutations. Only the MHCII alleles DRB1_1101, DRB1_1501, DRB1_0701, and DRB1_0101 yielded hits with any of the molecules evaluated. Peptide hits to DRB — 1501 were identical among all constructs tested, including wild-type IL-2 with the C125S mutation. Addition of L80I removed one T-cell epitope for DRB1_0101 [ALNLAPSKNFHLRPR (SEQ ID NO:60)] and reduced the affinity of two other T-cell epitopes [EEALNLAPSKNFHLR (SEQ ID NO:61) and EALNLAPSKNFHLRP (SEQ ID NO:62)]. sexuality decreased slightly. For the MHCII allele DRB1 — 0701, L80I removed one T-cell epitope [EEALNLAPSKNFHLR (SEQ ID NO: 63)]. Therefore, the data indicated that IL-2 muteins containing the L80I mutation should be less immunogenic. This was a surprising and unexpected result from computational analysis.

Generation of Additional IL-2 Muteins Human IL-2M or single IL-2M with IL-2M fused to the N-terminus of a human IgG1 Fc domain (IL-2 mutein) SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 HEK293 Expi cells were transfected with a pTT5 vector containing a single gene encoding SEQ ID NO: 40 (and IL-2M control; SEQ ID NO: 34) polypeptide. 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, clarified by centrifugation and 0. Filtered through a 22um filtration device. 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 captured on protein A resin. The resin was washed with PBS pH 7.4 and captured protein was eluted using 0.25% acetic acid, pH 3.5 while neutralizing using 1/10 volume of 1 M Tris pH 8.0. Proteins were buffer exchanged into 30 mM HEPES 150 mM NaCl pH 7 and analyzed by size exclusion chromatography on a Superdex 200 3.2/300 column. 5 ug of purified material was analyzed by reducing and non-reducing SDS-PAGE on Bis-Tris 4-12% gels.

IL-2M 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: It was expressed at >45 mg/L after purification and was >95% monodispersed.

IL-2M Can Bind CD25 Immunosorbent plates were coated with 75 ul/well of CD25 at a concentration of 0.5 μg/mL in PBS pH 7.4 and incubated overnight at 4°C. Wells were washed three times with PBS pH 7.4 containing 0.05% Tween-20 (wash buffer) and then with 200 ul/well 1% BSA in PBS pH 7.4 (block buffer) at room temperature. Blocked for 2 hours. After washing three times with wash buffer, IL-2M SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 in PBS containing 1% BSA and 0.05% Tween-20 (assay buffer) , diluted in 12-fold serial dilutions with a top concentration of 2 nM. Diluted material was added to CD25 coated plates at 75 ul/well for 1 hour at room temperature. After washing three times with wash buffer, goat biotinylated anti-IL-2 polyclonal antibody diluted to 0.05 μg/mL in assay buffer was added to the plate at 75 ul/well for 1 hour at room temperature. After washing three times with wash buffer, highly sensitive streptavidin HRP diluted 1:5000 in assay buffer was added to the plate at 75 ul/well for 15 minutes at room temperature. After washing three times with wash buffer and once with wash buffer (without tween-20), the assay was developed with TMB and stopped with 1N HCL. OD450nm was measured. Experiments included appropriate controls for non-specific binding of molecules to plates/blocks in the absence of CD25. The results showed that from 2 nM to 1.9 pM, the mutein of Example 7 was able to bind CD25 with a sub-nanomolar EC50. Furthermore, when CD25 was not present on the plate surface, no compound was detected at any concentration tested, indicating that the test compound was not non-specifically interacting with the plate surface. Therefore, the mutein of Example 7 can bind to CD25.

IL-2 Muteins of Example 7 Are Potent and Selective Peripheral blood mononuclear cells (PBMC) were produced from freshly isolated heparinized human whole blood using FICOLL-PAQUE Premium and Sepmate tubes. PBMCs were cultured in 10% fetal bovine serum RPMI media for 20 minutes in the presence of wild-type IL-2 or the mutein of Example 7, then fixed with BD Cytofix for 10 minutes. Fixed cells were permeabilized sequentially with BD Perm III and then BioLegend FOXP3 permeabilization buffer. After blocking with human serum for 10 minutes, cells were stained with antibodies against phospho-STAT5 FITC (CST), CD25 PE, FOXP3 AF647 and CD4 PerCP Cy5.5 (all BD) for 30 minutes and then equipped with a plate reader. Acquisition was performed on an Attune NXT. The IL-2 mutein of Example 7 was found to be potent and selective for Tregs over Teff. A mutein with the L118I mutation was found to have increased activity and selectivity compared to other muteins.

IL-2 Muteins Expand Tregs in Humanized Mice NSG mice humanized with human CD34+ hematopoietic stem cells were purchased from Jackson Labs. On days 0 and 7, mice were administered 1 ug IL-2 mutein SEQ ID NO:34 or other IL-2 muteins SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, or SEQ ID NO:40 subcutaneously. Mice were euthanized on day 7 and whole blood and spleens were collected. Whole blood was aliquoted into 96-well deep-well plates and fixed using BD Fix Lyse for 10 minutes. Splenocytes were isolated using a 70um filter (BD) and red blood cells were lysed using RBC lysis buffer from BioLegend. After washing with 2% fetal bovine serum in PBS, splenocytes were labeled with a near-infrared viability stain (Invitrogen) for 20 minutes and then fixed using BioLegend fixation buffer for 20 minutes. Both whole blood and splenocytes were then permeabilized using BioLegend FOXP3 permeabilization buffer, blocked with human serum, human CD8a FITC (BL), human CD25 PE (BD), human FOXP3 AF647 ( BD), CD4 PerCP Cy5.5 (BD), human Siglec-8 PE Cy7 (BL), human CD3 BV421 (BL), human CD45 BV605 (BL), human CD56 BV785 (BL) and mouse CD45 (BV711) for 30 minutes and acquisition was performed on an Attune NXT equipped with a plate reader.

IL-2M SEQ ID NO:37 and SEQ ID NO:38 and SEQ ID NO:39 and SEQ ID NO:40 selectively induced human Tregs in mouse spleen and whole blood of humanized mice compared to vehicle controls. There were no significant changes in the frequencies of human CD56pos NK cells, CD3pos T cells, CD8pos cytotoxic T lymphocytes, CD4pos helper T cells or CD25lo/FOXP3neg T effectors. These results indicate that IL-2 muteins increase the frequency of regulatory T cells.

Persistence of Signaling Induced by IL-2 Muteins Peripheral blood mononuclear cells (PBMCs) were produced from freshly isolated heparinized human whole blood using FICOLL-PAQUE Premium and Sepmate tubes. PBMC were cultured in 10% fetal bovine serum RPMI medium for 60 minutes in the presence of IL-2M. Cells were then washed three times and incubated for an additional 3 hours. Cells were then fixed with BD Cytofix for 10 minutes. Fixed cells were permeabilized sequentially with BD Perm III and then BioLegend FOXP3 permeabilization buffer. After blocking with human serum for 10 min, cells were stained with antibodies against phospho-STAT5 FITC, CD25 PE, FOXP3 AF647 and CD4 PerCP Cy5.5 for 30 min followed by acquisition on an Attune NXT with plate reader. . All four IL-2 muteins of Example 19, but not Teff, induced persistent signaling in Tregs compared to controls. SEQ ID NO:40 was superior to SEQ ID NO:39, SEQ ID NO:38 or SEQ ID NO:37. These results indicate that IL-2 can induce persistent and selective signaling in Tregs that should lead to greater Treg expansion in vivo, allowing low-frequency dosing to achieve Treg expansion. showing.

The examples provided herein demonstrate the surprising and unexpected results that IL-2 muteins can function to selectively and potently activate Tregs over Teff. This indicates that the molecule can be used to treat or ameliorate the conditions described herein. The IL-2 muteins provided herein can be produced and used fused or unfused to an Fc domain or linker provided herein.

Embodiments have been described with reference to specific examples. These examples are not meant to limit the embodiments in any way. It is understood that for the purposes of this disclosure, various changes and modifications may be made which are fully within the scope of this disclosure. Many other modifications may be made which will readily occur to those skilled in the art and which are within the spirit of the invention disclosed herein and defined in the appended claims.

Muteins Show Overall POI and Lower Aggregation IL-2 muteins with one of L80I, L118I, or L118I were transfected into HEK293 Expi cells with the pTT5 vector and expressed in the vector. An IL-2 mutein was ligated to the N-terminus of the Fc region with 4GGGGS (SEQ ID NO: 10) repeats. After 5-7 days, cell culture supernatants expressing different IL-2 muteins were harvested, clarified by centrifugation and filtered through a 0.22um filtration device. IL-2 muteins were captured on protein A resin. The resin was washed with PBS pH 7.4 and captured protein was eluted using 0.25% acetic acid, pH 3.5 while neutralizing using 1/10 volume of 1 M Tris pH 8.0. Proteins were buffer exchanged into 30 mM HEPES 150 mM NaCl pH 7 and analyzed by size exclusion chromatography on an AdvanceBio SEC column for percent peak of interest (POI). Results show that various muteins were expressed at 60 mg/L and above. However, it was surprisingly found that over 90% of the muteins with the L80I or L118I mutations were monodisperse, but not the muteins with the L53I or L56I mutations, as shown by size exclusion chromatography. served. Hence, muteins with L80I or L118I substitutions aggregated less. The difference in aggregation between the four molecules (IL-2 muteins including L80I, L118I, L53I, and L56I) was unexpected due to the type of mutations being made. Therefore, muteins with L80I or L118I mutations have a surprising advantage over other muteins in that they aggregate less than other muteins.

IL-2 Muteins Unexpectedly Increase Potency The muteins described in Example 12 were analyzed for potency in an in vitro assay. Briefly, PBMC were isolated from heparinized human whole blood and stimulated with fixed concentrations of various muteins for 30 minutes at 37°C. Stimulation was stopped by immobilization. After permeabilization, PBMCs were stained for intracellular FoxP3 and phospho-STAT5 levels and surface CD4 and CD25 expression and analyzed by flow cytometry. Regulatory T cells (Treg) and effector T cells (Teff) were gated as CD4+CD25hiFoxP3+ or CD4+CD25loFoxP3-, respectively. Percentage of cells staining positive for phospho-STAT5 is shown. This assay measured the ability of muteins to specifically stimulate Tregs without stimulating Teff. EC50 values were calculated using the best fit dose-response curve for each test substance.

Surprisingly, muteins with mutations L118I, L80I, L56I, or L53I had increased potency (stimulating Treg) compared to IL-2 muteins without either of these mutations. IL-2 muteins with the L118I, L80I, L56I, or L53I mutations, but without the V69A, Q74P, and N88D mutations, were approximately 51 pM. Each _EC50 of the muteins containing one of L118I, L80I, L56I, or L53I had _EC50s of approximately 30, 40, 41, and 45, respectively. The difference in EC50 for Treg stimulation (no change in Teff stimulation) was surprising and would not have been expected for a mutein with one of the mutations described in this example. Data could also be evaluated by comparing the ratio of parental IL-2 muteins (including V69A, Q74P, N88D, and C125S) to muteins containing also one of the L118I, L80I, L56I, or L53I mutations. This ratio was used to normalize different cell populations used in different experiments. Using this ratio, L118I had a mean increase in potency (mean 0.16 standard error) that was approximately 25% greater than the parental control, whereas the other mutations had a mean increase in potency of 0.16 using this ratio. decreased activity compared to

In vitro data were confirmed in vivo for muteins with one of the L118I, L80I, L56I, or L53I mutations. L118I was found in vivo to be more potent than muteins without the L118I mutation. Briefly, Nod-Scid-IL-2R gamma-deficient (NSG) mice reconstituted with human CD34+ hematopoietic stem cells were injected subcutaneously on days 0 and 7 with 1 microgram of the indicated test substance or vehicle. On day 11, mice were sacrificed and blood was collected by cardiac puncture into tubes containing heparin. Peripheral blood leukocytes (PBL) were isolated by erythrocyte lysis and stained with antibodies reactive to the human markers CD45, CD3, CD8, CD4, FoxP3, CD25 and CD56. 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 CD45+, total CD4+ and total CD8+ cells were unchanged. Similar results were observed in mouse spleen. The in vivo potency of IL-2 muteins with the L80I mutation, as measured in this assay, was slightly increased compared to muteins without the L80I mutation, and that of muteins with either the L56I or L53I mutation was slightly increased in this assay. The in vivo potency as measured was similar to the mutein without the mutation. The muteins were N-terminally linked to the Fc regions described herein by a 20 amino acid (GGGGS) ₄ (SEQ ID NO: 9) linker. Thus, the linker joined the C-terminus of the IL-2 mutein to the N-terminus of the Fc region.

An N-terminal Fc orientation with a 20 amino acid linker is most effective for Treg stimulation.
IL-2 muteins with V69A, Q74P and N88D were fused to the Fc region containing the L234A, L235A and G237A mutations using GGGGS (SEQ ID NO: 10) repeats of different lengths. IL2-mutein molecules fused to the n-terminus of human IgG1 Fc via the c-terminus of the mutein with a linker containing 3 and 4 GGGGS (SEQ ID NO: 10) repeats were tested and the linker length was determined whether it affects the potency of A mutein fused to the c-terminus of human IgG1 Fc via the n-terminus of the mutein by a single GGGGS (SEQ ID NO: 10) repeat was also tested. Briefly, Nod-Scid-IL-2R gamma-deficient (NSG) mice reconstituted with human CD34+ hematopoietic stem cells were injected subcutaneously on day 0 with 1 microgram of various IL-2 muteins with different linker lengths or vehicle. . On day 7, mice were sacrificed and blood was collected by cardiac puncture into tubes containing heparin. Peripheral blood leukocytes (PBL) were isolated by erythrocyte lysis and stained with antibodies reactive to the human markers CD45, CD3, CD8, CD4, FoxP3, CD25 and CD56. 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 CD45+, total CD4+ and total CD8+ cells were unchanged. Similar results were observed in mouse spleen.

Four GGGGS (SEQ ID NO: 10 ) muteins fused at the N-terminus of human IgG1 Fc by a linker containing repeats were found to be the most potent. Furthermore, a protein with four GGGGS (SEQ ID NO: 10) repeats was more effective in Treg expansion, whereas this configuration did not result in any difference in CD56+ NK cell expansion. It could not have been predicted that proteins containing N-terminal Fc fusion muteins with longer linkers would be the most potent and not produce any difference in CD56+ NK cell expansion.

Treatment of Patients with Active Rheumatoid Arthritis A pharmaceutical composition comprising an IL-2 mutein protein comprising the sequence of SEQ ID NO: 37, 38, 39, or 40 was administered to a patient with active rheumatoid arthritis. IL-2 muteins have been found to be effective in treating active rheumatoid arthritis in patients.

Treatment of Patients with Active Systemic Lupus Erythematosus A pharmaceutical composition comprising an IL-2 mutein protein comprising the sequence of SEQ ID NO: 37, 38, 39, or 40 was administered to a patient with active systemic lupus erythematosus. IL-2 muteins have been found to be effective in treating active systemic lupus erythematosus.

Treatment of Steroid-Resistant Chronic Graft-Versus-Host-Disease Patients A pharmaceutical composition comprising an IL-2 mutein protein comprising the sequence of SEQ ID NO: 37, 38, 39, or 40 is administered to a steroid-resistant chronic graft-versus-host disease patient. bottom. IL-2 muteins have been found effective in treating steroid-resistant chronic graft-versus-host disease.

IL-2 Muteins Induce pSTAT5 in Human Tregs Purified PBMC from heparinized whole blood from 6 healthy donors were tested using serial dilutions of IL-2 mutein proteins containing sequences of SEQ ID NOs: 39 or 40. for 30 minutes at 37°C. Cells were fixed, washed, permeabilized and washed. Cells were stained with antibodies that detect both surface and intracellular/nuclear markers (pSTAT5 and FOXP3). Data were collected on an Attune NxT cytometer. Tregs were gated as mononuclear, singlets, CD3pos, CD4pos, CD25hi, FoxP3pos. The percentage of gated Tregs expressing phosphorylated STAT5 was determined. Best fit curves were fitted to the pSTAT5 dose response and EC50 values were determined. Mean EC50 values for all six donors were compared to IL-2 of SEQ ID NO:39 (37.26±7.30; n=16) and IL-2 of SEQ ID NO:40 (23.11±5.35; n= 15) was determined. Data showed that IL-2 muteins can induce pSTAT5 in human Tregs. Although IL-2 containing the sequence of SEQ ID NO:40 was more potent than the IL-2 sequence containing SEQ ID NO:39, both were active across multiple populations of cells.

IL-2 muteins induce pSTAT5 in monkey PBMCs in vitro Purified PBMCs from heparinized whole blood from 3 healthy monkeys were serially diluted with IL-2 mutein proteins containing the sequences of SEQ ID NO: 39 or 40. for 30 minutes at 37°C. Fluorochrome-conjugated anti-CD25 and anti-CD4 were added for the last 30 minutes of IL-2 mutein treatment. Cells were fixed, washed, permeabilized and washed. Cells were stained with the remaining antibodies that detect both surface and intracellular/nuclear markers (pSTAT5 and FOXP3). Data were collected on an Attune NxT cytometer. Tregs were gated as mononuclear, singlet, CD4pos, CD25hi, FoxP3pos. The percentage of gated Tregs expressing phosphorylated STAT5 was determined. An IL-2 mutein was found to induce pSTAT5 in monkeys.

IL-2 Muteins Expand Treg Cells and Induce Treg Expansion In Vivo ), and either SEQ ID NO: 39 (5 time points/mouse, 2 animals) or SEQ ID NO: 40 (5 time points/mouse, 3 animals) were collected after dosing in K2EDTA tubes. Samples were split in two and stained separately for two FACS panels. One was a "Treg panel" and one was a general immunophenotyping panel. RBCs were lysed and cells were stained for surface and intracellular markers after fixation and permeabilization. For FAC analysis, the number of total cells/ul was determined by ADVIA. The number of cells in a given subpopulation/ul was then calculated in total/ul and as a percentage of total cells. As the "fold change from pre-dose" was determined, the mean number of a given cell type/ul of the two pre-dose bleeds per monkey was averaged and used to normalize the post-dose bleeds. . Plasma from K2EDTA whole blood was frozen until the end of the study for analysis of serum cytokines and chemokines. Chemokine and cytokine levels were quantified by multiplex MSD assays using serial dilutions of reference controls. Mean and range of MCP-1 and IP-10 were determined at pre-dose bleeds. Both muteins were found to expand Tregs and induce Treg expansion in monkeys. These results demonstrate that the IL-2 mutein functions in an in vivo animal model similar to humans. It was also found that neither molecule significantly expanded Tconv cells, CD4 cells (Tnaive) or CD8 cells (cytotoxic T), NK cells in monkeys (non-human primates). It was also found that neither molecule significantly induced serum chemokines. This data indicates that IL-2 muteins can expand Treg cells and induce Treg cell proliferation without unwanted expansion or activation of other pathways. Thus, IL-2 muteins are surprisingly potent, efficacious and selective for Treg expansion and proliferation.

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

This specification contains numerous citations to patents, patent applications, and publications. each of which is incorporated herein by reference for all purposes.

Claims (91)

A peptide that activates T regulatory cells comprising the amino acid sequence of SEQ ID NO:27. 2. The peptide of claim 1, further comprising an N-terminal leader peptide having the sequence of SEQ ID NO:7. 2. The peptide of claim 1, further comprising a linker peptide at its C-terminus. 4. The peptide of claim 3, wherein the linker peptide comprises the sequence (GGGGS)n, where n is 1, 2, 3, or 4. 5. The peptide of claim 4, wherein n is 1. 5. The peptide of claim 4, wherein n is two. 5. The peptide of claim 4, wherein n is 3. 5. The peptide of claim 4, wherein n is four. 2. The peptide of claim 1, further comprising an Fc peptide. 10. The peptide of claim 9, wherein the Fc peptide comprises the sequence of SEQ ID NO:8 or SEQ ID NO:15. 10. The peptide of claim 9, further comprising a leader peptide having the sequence of SEQ ID NO: 7 at its N-terminus. 10. The peptide of claim 9, wherein the Fc peptide is C-terminal. 10. The peptide of claim 9, wherein the Fc peptide is at the N-terminus. 2. The peptide of claim 1, further comprising a linker peptide connecting the peptide of SEQ ID NO:27 and the Fc peptide. 15. The peptide of claim 14, further comprising a leader peptide having the sequence of SEQ ID NO: 7 at its N-terminus. 15. The peptide of claim 14, wherein the linker peptide is (GGGGS)n, where n is 1, 2, 3, or 4. 17. The peptide of claim 16, wherein n is four. 15. The peptide of claim 14, wherein the Fc peptide comprises the sequence of SEQ ID NO:8 or SEQ ID NO:15. 15. The peptide of claim 14, wherein the Fc peptide is C-terminal. 15. The peptide of claim 14, wherein the Fc peptide is at the N-terminus. 15. The peptide of claim 14, comprising a peptide comprising the sequence of SEQ ID NO:40. A pharmaceutical composition comprising the peptide of claim 1. A pharmaceutical composition comprising the peptide of claim 14. A pharmaceutical composition comprising the peptide of claim 19. A pharmaceutical composition comprising the peptide of claim 21. A peptide that activates T regulatory cells comprising the amino acid sequence of SEQ ID NO:26. 17. The peptide of claim 16, further comprising an N-terminal leader peptide having the sequence of SEQ ID NO:7. 27. The peptide of claim 26, further comprising a linker peptide at its C-terminus. 29. The peptide of claim 28, wherein the linker peptide comprises the sequence (GGGGS)n, where n is 1, 2, 3, or 4. 30. The peptide of claim 29, wherein n is 1. 30. The peptide of claim 29, wherein n is two. 30. The peptide of claim 29, wherein n is 3. 30. The peptide of claim 29, wherein n is four. 27. The peptide of claim 26, further comprising an Fc peptide. 35. The peptide of claim 34, wherein the Fc peptide comprises the sequence of SEQ ID NO:8 or SEQ ID NO:15. 35. The peptide of claim 34, further comprising a leader peptide having the sequence of SEQ ID NO: 7 at its N-terminus. 35. The peptide of claim 34, wherein the Fc peptide is C-terminal. 35. The peptide of claim 34, wherein the Fc peptide is at the N-terminus. 27. The peptide of claim 26, further comprising a linker peptide connecting the peptide of SEQ ID NO:26 and the Fc peptide. 40. The peptide of claim 39, further comprising a leader peptide having the sequence of SEQ ID NO: 7 at its N-terminus. 40. The peptide of claim 39, wherein the linker peptide is (GGGGS)n, where n is 1, 2, 3, or 4. 42. The peptide of claim 41, wherein n is four. 40. The peptide of claim 39, wherein the Fc peptide comprises the sequence of SEQ ID NO:8 or SEQ ID NO:15. 40. The peptide of claim 39, wherein the Fc peptide is C-terminal. 40. The peptide of claim 39, wherein the Fc peptide is at the N-terminus. 40. The peptide of claim 39, comprising a peptide comprising the sequence of SEQ ID NO:39. A pharmaceutical composition comprising the peptide of claim 26. A pharmaceutical composition comprising the peptide of claim 39. 44. A pharmaceutical composition comprising the peptide of claim 43. 47. A pharmaceutical composition comprising the peptide of claim 46. An in vitro method of activating T regulatory cells, wherein the T regulatory cells are treated with a peptide according to any one of claims 1-21 and 26-46 or any one of claims 22-25 and 47-50. or the pharmaceutical composition of claim 1. 51. A pharmaceutical composition according to any one of claims 22-25 and 47-50 for use in a method of treating an inflammatory disorder in a subject, said method comprising administering to a subject in need thereof , administering a therapeutically effective amount of said pharmaceutical composition. Inflammatory disorders include alopecia areata, atopic dermatitis, dermatitis herpetiformis, psoriasis, lupus, graft-versus-host disease (GVHD), inflammation, autoimmune diseases, atopic diseases, paraneoplastic autoimmune diseases, cartilage Inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, small joint juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic Arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronegative, enthesopathy, arthritic syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, small arthritis, polyarticular rheumatoid arthritis, generalized rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome (seronegative, enthesopathy) , arthritic syndrome), dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myositis, polymyositis, dermatomyositis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatoid arthritis, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjögren's syndrome, psoriasis vulgaris, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythroderma psoriasis, dermatitis, atherosclerosis, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, sinusitis, Sinusitis with polyps, eosinophilic esophagitis, eosinophilic bronchitis, Guillain-Barré disease, type I diabetes, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, steroids resistant chronic graft-versus-host disease, transplant rejection, kidney injury, hepatitis C-induced vasculitis, spontaneous pregnancy loss, alopecia, vitiligo, focal segmental glomerulosclerosis (FSGS), minimal change group, 53. The pharmaceutical composition according to claim 52, which is membranous nephropathy, ANCA-associated glomerulonephropathy, membranous proliferative glomerulonephropathy, IgA nephropathy, lupus nephritis . 51. The pharmaceutical composition of any one of claims 22-25 and 47-50 for use in a method of promoting or stimulating STAT5 phosphorylation in T regulatory cells, said method comprising said pharmaceutical composition, comprising administering to a subject in need thereof a therapeutically effective amount of said pharmaceutical composition. A peptide according to any one of claims 1-21 and 26-46 or a medicament according to any one of claims 22-25 and 47-50 in the manufacture of a medicament for the treatment of inflammatory disorders Use of the composition. Inflammatory disorders include alopecia areata, atopic dermatitis, dermatitis herpetiformis, psoriasis, lupus, graft-versus-host disease (GVHD), inflammation, autoimmune diseases, atopic diseases, paraneoplastic autoimmune diseases, cartilage Inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, small joint juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic Arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronegative, enthesopathy, arthritic syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, small arthritis, polyarticular rheumatoid arthritis, generalized rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome (seronegative, enthesopathy) , arthritic syndrome), dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myositis, polymyositis, dermatomyositis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatoid arthritis, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjögren's syndrome, psoriasis vulgaris, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythroderma psoriasis, dermatitis, atherosclerosis, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, sinusitis, Sinusitis with polyps, eosinophilic esophagitis, eosinophilic bronchitis, Guillain-Barré disease, type I diabetes, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, steroids resistant chronic graft-versus-host disease, transplant rejection, kidney injury, hepatitis C-induced vasculitis, spontaneous pregnancy loss, alopecia, vitiligo, focal segmental glomerulosclerosis (FSGS), minimal change group, 56. Use according to claim 55, which is membranous nephropathy, ANCA-associated glomerulonephropathy, membranous proliferative glomerulonephropathy, IgA nephropathy, lupus nephritis . A nucleic acid molecule encoding the peptide of any one of claims 1-21 and claims 26-46 . 58. A vector comprising the nucleic acid molecule of claim 57. 58. A plasmid comprising the nucleic acid molecule of claim 57. 58. A cell comprising the nucleic acid molecule of claim 57. A cell comprising the plasmid of claim 59. A cell comprising the vector of claim 58. A cell comprising or expressing a peptide according to any one of claims 1-21 and claims 26-46 . 53. The pharmaceutical composition of claim 52, wherein the inflammatory disorder is alopecia areata. 53. The pharmaceutical composition according to claim 52, wherein the inflammatory disorder is atopic dermatitis. 53. The pharmaceutical composition of claim 52, wherein the inflammatory disorder is systemic lupus erythematosus (SLE). 53. The pharmaceutical composition of Claim 52, wherein the inflammatory disorder is ulcerative colitis. 53. The pharmaceutical composition of claim 52, wherein the inflammatory disorder is vitiligo. 53. The pharmaceutical composition of Claim 52, wherein the inflammatory disorder is psoriasis. 53. The pharmaceutical composition of claim 52, wherein the inflammatory disorder is lupus. 53. The pharmaceutical composition of Claim 52, wherein the inflammatory disorder is rheumatoid arthritis. 53. The pharmaceutical composition of claim 52, wherein the inflammatory disorder is juvenile systemic lupus erythematosus. 53. The pharmaceutical composition of claim 52, wherein the inflammatory disorder is ankylosing spondylitis. 53. The pharmaceutical composition of Claim 52, wherein the inflammatory disorder is psoriatic arthritis. 53. The pharmaceutical composition of claim 52, wherein the inflammatory disorder is Sjogren's Syndrome. 53. The pharmaceutical composition of Claim 52, wherein the inflammatory disorder is Crohn's disease. 53. The pharmaceutical composition of claim 52, wherein the inflammatory disorder is lupus nephritis. 56. Use according to claim 55, wherein the inflammatory disorder is alopecia areata. 56. Use according to claim 55, wherein the inflammatory disorder is atopic dermatitis. 56. Use according to claim 55, wherein the inflammatory disorder is systemic lupus erythematosus (SLE).
. 56. Use according to claim 55, wherein the inflammatory disorder is ulcerative colitis. 56. Use according to claim 55, wherein the inflammatory disorder is vitiligo. 56. Use according to claim 55, wherein the inflammatory disorder is psoriasis. 56. Use according to claim 55, wherein the inflammatory disorder is lupus. 56. Use according to claim 55, wherein the inflammatory disorder is rheumatoid arthritis. 56. Use according to claim 55, wherein the inflammatory disorder is juvenile systemic lupus erythematosus. 56. Use according to claim 55, wherein the inflammatory disorder is ankylosing spondylitis. 56. Use according to claim 55, wherein the inflammatory disorder is psoriatic arthritis. 56. Use according to claim 55, wherein the inflammatory disorder is Sjögren's syndrome. 56. Use according to claim 55, wherein the inflammatory disorder is Crohn's disease. 56. Use according to claim 55, wherein the inflammatory disorder is lupus nephritis.

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