KR102687035B1 - IL-2 muteins and their uses - Google Patents

Abstract

The present disclosure provides IL-2 muteins, compositions comprising them, and methods of using them.

Description

IL-2 muteins and their uses

Cross-reference to related applications

This application is related to U.S. Provisional Application No. 62/721,644 filed on August 23, 2018, U.S. Provisional Application No. 62/675,972 filed on May 24, 2018, and U.S. Provisional Application No. 62/595,357 filed on December 6, 2017. , claims priority of U.S. Provisional Application No. 16/109,875, filed on August 23, 2018, and U.S. Provisional Application No. 16/109,897, filed on August 23, 2018, each of which is incorporated by reference in its entirety.

Technology field

Embodiments provided herein relate to proteins referred to as IL-2 muteins, compositions containing them, and methods of using them.

IL-2 has three transmembrane receptor subunits: IL-2Rβ and IL-2Rγ, which together activate intracellular signaling events upon IL-2 binding, and are responsible for presenting IL-2 to the other two receptor subunits. Binds to CD25 (IL-2Rα). Signals transduced by IL-2Rβγ include signals from the PI3-kinase, Ras-MAP-kinase, and STAT5 pathways.

T cells require expression of CD25 to respond to the low concentrations of IL-2 normally present in tissues. T cells expressing CD25 include both CD4 ⁺ FOXP3 ⁺ regulatory T cells (T-reg cells) and FOXP3 ⁻ T cells, which are essential for suppressing autoimmune inflammation activated to express CD25. FOXP3 ^- CD4 ⁺ T effector cells (T-eff) can be either CD4 ⁺ or CD8 ⁺ cells, both of which can be inflammatory, causing autoimmune and other diseases in which the subject's immune system attacks organs or other tissues. You can contribute. IL-2-stimulated STAT5 signaling is important for normal T-reg cell growth and survival and high FOXP3 expression.

Because there is low affinity IL-2 for each of the three IL-2R chains, further reduction in affinity for IL-2Rβ and IL-2Rγ may be offset by increased affinity for CD25. Mutant variants of IL-2 have been generated. These IL-2 mutants may be referred to as IL-2 muteins and have been found to be useful in the treatment of various diseases. However, there is still a need for additional IL-2 muteins and compositions that can be used for a variety of applications. Embodiments of the present invention meet these needs as well as others.

summary

In some embodiments of the invention, a peptide comprising the amino acid sequence of SEQ ID NO: 1 is provided, including a mutation at positions 73, 76, 100, or 138.

In some embodiments of the invention, a peptide comprising the amino acid sequence of SEQ ID NO:2 is provided, including a mutation at positions 53, 56, 80, or 118.

In some embodiments of the invention, the peptide comprises the amino acid sequence of SEQ ID NO: 43, wherein at least one of X ₁ , X ₂ , and X ₃ and X ₄ is I and the others are L or I.

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

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

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

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

Figure 1 illustrates a non-limiting embodiment of the IL-2 muteins provided herein.

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

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

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

MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (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 prepared by mutating one or more IL-2 residues. Non-limiting examples of IL-2-muteins include WO2016/164937, US9580486, US7105653, US9616105, US 9428567, US2017/0051029, US2014/0286898A1, WO2014153111A2, 5, can be found in WO2016014428A2, WO2016025385A1, and US20060269515, Each of these is incorporated by reference in its entirety.

In some embodiments of the invention, 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 replaced by a cysteine at position 125 of the mature IL-2 sequence. Other combinations of mutations and substitutions in IL-2 mutein molecules are described in US20060269515, which is incorporated herein by reference in its entirety. In some embodiments, the cysteine at position 125 is also substituted with valine or alanine. In some embodiments, the IL-2 mutein molecule comprises the V91K substitution. In some embodiments, the IL-2 mutein molecule comprises the N88D substitution. In some embodiments, the IL-2 mutein molecule comprises the N88R substitution. In some embodiments, the IL-2 mutein molecule contains the substitution 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, T, D84A, D84E, D84G, D84I, D84M, D84Q D84R, D84S, D84T, S87R, N88A, N88D, N88E, N88I, N88F, N88G, N88M, N88R, N88S, N88V, N88W, V91D, V91E, V91S, , I92K , I92R, E95G, and Q126. In some embodiments, the amino acid sequence of the IL-2 mutein molecule has a C125A or C125S substitution and 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, 81T , D84A, D84E, D84G, D84I, D84M, D84Q, D84R, D84S, D84T, S87R, N88A, N88D, N88E, N88F, N88I, N88G, N88M, N88R, N88S, N88V, N88W, V91D, V91E, V91G, 91S , I92K, I92R, E95G, Q126I, Q126L, and Q126F. In some embodiments, the IL-2 mutein molecule has a C125A or C125S substitution and D20H, D20I, D20Y, D20E, D20G, D20W, D84A, D84S, H16D, H16G, H16K, H16R, H16T, H16V, I92K, I92R, L12K. , differs from the amino acid sequence presented in the mature IL-2 sequence with one substitution selected from 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 of the invention, the IL-2 mutein molecule comprises a mutation in the polypeptide sequence 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 mutation at position 30 is N30S. In some embodiments, the mutation at position 31 is Y31H. In some embodiments, the mutation at position 35 is K35R. In some embodiments, the mutation at position 69 is V69A. In some embodiments, the mutation at position 74 is Q74P. In some embodiments, the mutein does not contain a mutation at positions 30, 31, and/or 35.

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

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

Unless otherwise specified for IL-2 muteins, numbering referred to herein refers to the mature sequence. If the sequence or position represents SEQ ID NO: 1, it is an immature sequence. However, transposing a position from the immature sequence (SEQ ID NO: 1) to the mature sequence (SEQ ID NO: 2) simply involves subtracting 20 from the position mentioned in SEQ ID NO: 1 to obtain the corresponding position in SEQ ID NO: 2.

In addition to the substitutions or mutations described herein, in some embodiments, the IL-2 mutein has one or more of positions 73, 76, 100, or 138 corresponding to SEQ ID NO: 1 or positions 53, 56, 80 corresponding to SEQ ID NO: 2 or has a substitution/mutation at one or more positions among 118. In some embodiments, the IL-2 mutein has positions 73 and 76 corresponding to SEQ ID NO:1; 73 and 100; 73 and 138; 76 and 100; 76 and 138; 100 and 138; 73, 76 and 100; 73, 76 and 138; 73, 100 and 138; 76, 100 and 138; or mutations at 73, 76, 100 and 138, respectively. In some embodiments, the IL-2 mutein has positions 53 and 56 corresponding to SEQ ID NO:2; 53 and 80; 53 and 118; 56 and 80; 56 and 118; 80 and 118; 53, 56 and 80; 53, 56 and 118; 53, 80 and 118; 56, 80 and 118; or mutations at 53, 56, 80 and 118, respectively. Since IL-2 can be fused to or tethered to another protein, as used herein, the term may be used as a reference to SEQ ID NO: 6 or 15, such as the sequence may be used on the NCBI website. Corresponds to the default settings for the alignment software and refers to how it is aligned. In some embodiments, the mutation is leucine to isoleucine. Accordingly, the IL-2 mutein may comprise one or more isoleucines at positions 73, 76, 100, or 138, corresponding to SEQ ID NO: 1, or one or more positions 53, 56, 80, or 118, corresponding to SEQ ID NO: 2. . In some embodiments, the mutein comprises a mutation at L53 corresponding to SEQ ID NO:2. In some embodiments, the mutein comprises a mutation at L56 corresponding to SEQ ID NO:2. In some embodiments, the mutein comprises a mutation 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 leucine to isoleucine. In some embodiments, the muteins also include mutations at positions 69, 74, 88, 125, or any combination thereof in these muteins corresponding to SEQ ID NO:2. 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 a C125A or C125S mutation.

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

In some embodiments of the invention, the IL-2 mutein has one or more 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). These mutations are combined with other leucine mutations to isoleucine described herein or with mutations at positions 73, 76, 100, or 138 corresponding to SEQ ID NO: 1 or one or more positions 53, 56, 80, or 118 corresponding to SEQ ID NO: 2. It can be. 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 located at one or more positions 35, 36, 42, 104, 115, or 146 corresponding to SEQ ID NO: 1 or an equivalent position in SEQ ID NO: 2 (e.g., positions 15, 16, 22, 84, 95) or 126).

Mutations at these positions include, but are not limited to, substitutions 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, as described herein and above. can be combined with any of the other mutations described herein. 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 the C145A or C145S mutation corresponding to SEQ ID NO:1.

These substitutions can 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 has one or more 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) , 126 and 126).

In some embodiments of the invention, 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 can 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 has one or more 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).

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

In some embodiments of the invention, the IL-2 mutein comprises the following sequence:

MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATEIKHLQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 3)

In some embodiments of the invention, the IL-2 mutein comprises the following sequence:

MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHIQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 4)

In some embodiments of the invention, the IL-2 mutein comprises the following sequence:

MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHIRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 5)

In some embodiments of the invention, the IL-2 mutein comprises the following sequence:

MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFINRWITFSQSIISTLT (SEQ ID NO: 6)

In some embodiments of the invention, the IL-2 mutein sequences described herein do not include an IL-2 leader sequence. The IL-2 leader sequence can be represented by the sequence MYRMQLLSCIALSLALVTNS (SEQ ID NO: 7). Accordingly, in some embodiments, the sequences exemplified above may also include peptides without a leader sequence. SEQ ID NOs: 3-6 are exemplified only by mutations at one or more of positions 73, 76, 100, or 138, corresponding to SEQ ID NO: 1, or positions 53, 56, 80, or 118, corresponding to SEQ ID NO: 2, but the peptides It may contain 1, 2, 3 or 4 mutations at these positions. In some embodiments, the substitution at each position is isoleucine or another type of conservative amino acid substitution. In some embodiments, leucine at the recited position is independently replaced with isoleucine, valine, methionine or glycine, alanine, glutamine or glutamic acid.

In some embodiments of the invention, the IL-2 protein of SEQ ID NO: 2 comprises the following mutations: V69A, Q74P, N88D and C125S or C125A and one mutation selected from the group consisting of L53I, L56I, L80I and L118I. 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 include the L53I, L56I, L80I, or L118I mutation. In some embodiments, the IL-2 mutein comprises a T3A mutation.

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

In some embodiments of the invention, the IL-2 mutein molecule is fused to the Fc region or other linker region as described herein. Examples of such fusion proteins include US9580486, US7105653, US9616105, US 9428567, US2017/0051029, WO2016/164937, US2014/0286898A1, WO2014153111A2, WO2010/085495, WO20160 14428A2, WO2016025385A1, US2017/0037102, and US2006/0269515, Each of these is incorporated by reference in its entirety.

In some embodiments of the invention, the Fc region comprises known LALA mutations. In some embodiments, the Fc region includes L234A and L235A mutations (EU numbering). In some embodiments, the Fc region includes G237A (EU numbering). In some embodiments, the Fc region does not include a mutation at position G237 (EU numbering). Using Kabat numbering this would correspond to L247A, L248A and/or G250A. In some embodiments, the Fc region comprises an L234A mutation, an L235A mutation, and/or a G237A mutation using the EU numbering system. Regardless of the numbering system used, in some embodiments, the Fc portion may 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 will be based on the full-length sequence, but will be used in fragments based on the conventional alignment used by those skilled in the art for the Fc region (see, e.g., Kabat et al. (“Proteins for Immunological Purposes”) Sequence”, US Public Health Services, NIH Bethesda, MD, Publication No. 91, which is incorporated herein by reference). In some embodiments, the Fc region comprises the following sequence:

DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 8)

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

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 15)

In some embodiments of the invention, 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 only a non-limiting example and the linker may have various 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 of the invention, 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 illustrated in the table below:

Accordingly, the IL-2/Fc fusion can be expressed in the formula Z _IL-2M -L _gs -Z _Fc , where Z _IL-2M is an IL-2 mutein described herein and L _gs is an IL-2 mutein described herein. linker sequence (e.g., a glycine/serine linker) and Z _Fc is an Fc region described herein or known to those skilled in the art. In some embodiments, the formula may be reverse orientation Z _Fc -L _gs -Z _IL-2M .

In some embodiments of the invention, the IL-2/Fc fusion comprises the following sequence:

MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATEIKHLQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 11)

In some embodiments of the invention, the IL-2/Fc fusion comprises the following sequence:

MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHIQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 1 2)

In some embodiments of the invention, the IL-2/Fc fusion comprises the following sequence:

MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHIRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 1 3)

In some embodiments of the invention, the IL-2/Fc fusion comprises the following sequence:

MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFINRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 1 4).

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

Proteins described herein can also be fused to other proteins, such as antibodies or other types of therapeutic molecules.

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

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

In some embodiments of the invention, the sequences shown in the tables or throughout this specification may or may 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 specification may or may not include one or more mutations corresponding to positions L59I, L63I, I24L, L94I, L96I, or L132I or other substitutions at the same position. In some embodiments, the mutation is leucine to isoleucine. In some embodiments, the mutein does not contain further mutations 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: Comprises the sequence of SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, or SEQ ID NO: 43.

In some embodiments of the invention, 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 of the invention, a polypeptide comprising SEQ ID NO: 43 is provided, wherein one or more of X ₁ , X ₂ , X ₃ and X ₄ is I and the remainder is L or I. 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 of the invention, the IL-2 mutein may be in the format as illustrated in Figure 1. However, as described herein, in some embodiments, the IL-2 mutein can be used without an Fc domain or the Fc-domain is linked to the C-terminus of the IL-2 mutein, as opposed to the Fc domain linked to the C-terminus of the IL-2 mutein. Connected to the N-terminus of the mutein. Polypeptides described herein also include variants of the described peptides. In some embodiments, the IL-2 variant has an amino acid sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, Includes more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, and more than 99%. Variants include those described herein with various substitutions described herein and above. In some embodiments, the variant has 1, 2, 3, 4, or 5 additional substitutions. In some embodiments, the substitution is G to A, L to I, G to S, K to R, or other type of conservative substitution. In some embodiments, conservative substitutions are selected based on the following table:

The percent identity of two amino acids or two nucleic acid sequences can be determined by visual inspection and mathematical calculations, or, for example, the comparison is performed by comparing 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 basic parameters for the GAP program include: (1) Atlas of Polypeptide Sequence and Structure, Schwartz and Dayhoff, eds., National Biomedical Research Foundation, pp. the unary comparison matrix for nucleotides (with values 1 for identity and 0 for non-identity) described in 353-358 (1979) and the weighted amino acid comparison matrix of Gribskov and Burgess. matrix), the GCG implementation of ((1986) Nucleic Acids Res. 14: 6745), or another similar comparison matrix; (2) for amino acid sequences a penalty of 8 for each gap and an additional penalty of 2 for each symbol in each gap, or for nucleotide sequences a penalty of 50 for each gap and each An additional penalty of 3 for each symbol in the gap of; (3) No penalty for end gaps; and (4) no maximum penalty for long gaps. Other programs used by those skilled in the art of sequence comparison may also be used.

In some embodiments of the invention, the IL-2 muteins provided herein include proteins that alter signaling through specific pathways activated by wild-type IL-2 through the IL-2R and preferential proliferation of T-regs. Causes /survival/activation.

IL-2 muteins provided herein can be prepared using any suitable method known in the art, including those described in U.S. Pat. No. 6,955,807, which is incorporated herein by reference. It can be created. These methods involve constructing DNA sequences encoding IL-2 variants and expressing these sequences in an appropriately transformed host, such as a host cell. These methods will produce the recombinant proteins provided herein. Proteins can also be produced synthetically or by a combination of fragments produced synthetically and recombinantly in cells, and then the fragments can be combined to produce the entire protein of interest.

In some embodiments of the invention, nucleic acid molecules (e.g., DNA or RNA) are prepared by isolating or synthesizing nucleic acid molecules encoding the protein of interest. Alternatively, the wild-type sequence of IL-2 can be isolated and mutated using general techniques such as site-directed mutagenesis.

Another method of constructing DNA sequences encoding IL-2 variants is 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 include 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 can also be selected using codons that are preferred in the cell in which the recombinant variant will be generated. It is well known that the genetic code is degenerate, that is, an amino acid can be encoded by more than one codon. Accordingly, it will be understood that for a given DNA sequence encoding a particular IL-2 protein, there will be many DNA degenerate sequences that will encode variants of that IL-2. Accordingly, in some embodiments, nucleic acid molecules encoding proteins described herein are provided. Nucleic acid molecules can be DNA or RNA.

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

When a nucleic acid molecule or its complement contains codons that encode a protein, the nucleic acid molecule “encodes” a protein as used herein.

“Recombinant,” as applied to a polypeptide or protein, means that production of the protein relies on at least one step in which a nucleic acid, which may or may not encode the protein, is introduced into a cell where it is not found naturally.

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

Of course, it should be understood that not all vectors and expression control sequences function equally in expressing the DNA sequences described herein. Not all hosts operate identically with the same expression system. However, one skilled in the art can select among these vectors, expression control sequences and hosts without undue experimentation. For example, when choosing a vector, the host must be considered because the vector must be replicated. The vector copy number, its ability to regulate that copy number, and the expression of 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 vectors that allow DNA encoding an IL-2 variant to be propagated to copy number. Such proliferative vectors are well known in the art.

Vector and host cell

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

Host cells can be used as described herein.

pharmaceutical composition

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

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

As used herein, the phrases “parenteral administration” and “administered parenterally” refer to modes of administration other than enteral and topical, generally by injection, and intravenous. Intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injections. and injection.

Therapeutic compositions must generally be sterile and stable under the conditions of manufacture and storage. Compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or other array structures suitable for high therapeutic molecule concentrations. Sterile injectable solutions can be 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. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the necessary other ingredients from those enumerated above. For sterile powders for preparing sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze-drying, which yields the powder of the active ingredient and any additional desired ingredients from a previous sterile-filtered solution. Proper fluidity of the solution can be maintained, for example, by the use of coatings such as lecithin, maintenance of the required particle size during dispersion, and use of surfactants. Prolonged absorption of the injectable compositions can be achieved by including absorption delaying agents in the composition, such as monostearate salts and gelatin.

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

In certain embodiments of the invention, the therapeutic compounds may be administered orally, for example, with an inert diluent or assimilable edible carrier. The compound (and other ingredients, if desired) may also be encapsulated in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. You can. In order to administer the compounds of the present invention by methods other than parenteral administration, it may be necessary to coat the compounds with or co-administer them with a material to prevent their inactivation. Therapeutic compositions can also be administered in conjunction with medical products known in the art.

Dosage regimens are adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dosage may be proportionally reduced or increased depending on the exigencies of the treatment situation. It is particularly 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 suitable as a single dosage for the subject to be treated; Each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in relation to the desired pharmaceutical carrier. The specification for the dosage unit forms of the invention is influenced by (a) the unique properties of the active compounds and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the technique of mixing such active compounds for the treatment of individual susceptibilities. and is directly determined by it.

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 of therapeutic compounds and treatment regimens can be determined by those skilled in the art. In certain embodiments, the therapeutic compound is administered at about 1 to 40 mg/kg, such as 1 to 30 mg/kg, such as about 5 to 25 mg/kg, about 10 to 20 mg/kg, about 1 to 5 mg/kg, It is administered by injection (e.g., subcutaneously or intravenously) at doses of 1 to 10 mg/kg, 5 to 15 mg/kg, 10 to 20 mg/kg, 15 to 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 may be once a month, every 2 months, every 3 months, or once every 6 months. there is. In one embodiment, the therapeutic compound is administered at a dose of about 10 to 20 mg/kg every other week. The therapeutic compound is administered in doses greater than 20 mg/min, for example 20-40 mg/min, to reach a dose of about 35 to 440 mg/m2, typically about 70 to 310 mg/m2, and more typically, about 110 to 130 mg/m2. , and can be administered by intravenous infusion, generally at a rate of 40 mg/min or more. In an embodiment, an infusion rate of about 110 to 130 mg/m2 achieves a level of about 3 mg/kg. In other embodiments, the therapeutic compound is administered at a dose of less than 10 mg/min, e.g., to reach a dose of about 1 to 100 mg/m2, e.g., about 5 to 50 mg/m2, about 7 to 25 mg/m2, or about 10 mg/m2. For example, it may be administered by intravenous infusion at a rate of 5 mg/min or less. In some embodiments, the therapeutic compound is infused over a period of about 30 minutes. It should be noted that dosage values may vary depending on the type and severity of the condition to be alleviated. For any particular subject, the specific dosage regimen should be adjusted over time according to the personal needs and professional judgment of the person administering or supervising the administration of the composition, and the dosage ranges set forth herein are exemplary only. It should be further understood that there are no limitations on the scope or practice of the compositions of the present invention.

The pharmaceutical composition of the invention may comprise a “therapeutically effective amount” or a “prophylactically effective amount” of the therapeutic molecule of the invention. “Therapeutically effective amount” means an amount that is effective at the dosage and time required to achieve the desired therapeutic result. The therapeutically effective amount of a therapeutic molecule may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic compound to elicit the desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the therapeutic molecule (t) are outweighed by its therapeutically beneficial effects. A “therapeutically effective amount” refers to a parameter that is preferably measurable relative to an untreated subject, e.g., at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, even more preferably Suppresses immune attacks by approximately 80% or more. Measurable parameters, such as the ability of a compound to inhibit immune attack, can be assessed in animal model systems to predict efficacy in transplant rejection or autoimmune disorders. Alternatively, this property of the composition can be assessed by testing the ability of the compound to inhibit, such as inhibition in vitro, by assays known to those skilled in the art.

“Prophylactically effective amount” means an amount effective at the dosage and time required to achieve the desired prophylactic result. Typically, since prophylactic doses are used in subjects before the onset or early stages of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

Also within the scope of the present invention are kits comprising the therapeutic compounds described herein. The kit may include one or more other elements, including: instructions for use; Other reagents, such as labels, therapeutic agents, or agents useful for chelating or coupling a therapeutic molecule to a label or other therapeutic agent, or radioprotective compositions; Products or other substances for preparing therapeutic molecules for administration; Pharmaceutically acceptable carrier; and products or other substances for administration to a subject.

Combination

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

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

Treatment method

“Treatment” of any disease referred to herein means alleviating at least one symptom of the disease, reducing the severity of the disease, or in some cases delaying the progression of more severe symptoms that may accompany the disease or at least one other disease. Includes prevention. Treatment does not necessarily mean that the disease has been completely cured. Useful therapeutic agents reduce the severity of a disease, which may occur with any frequency depending on the condition treated, reduce the severity of symptom(s) associated with the disease or its treatment, or prevent more severe symptoms or the development of a more serious disease. All you have to do is delay it. For example, if the condition is inflammatory bowel disease, the therapeutic agent may be used to reduce the number of distinct areas of inflammation in the intestines, reduce the total size of the affected intestine, reduce pain and/or swelling, reduce symptoms such as diarrhea, constipation, or vomiting, and/ Or prevent intestinal perforation. The patient's condition can be assessed by standard techniques such as x-ray, endoscopy, colonoscopy and/or biopsy performed after a barium enema or enteroclysis. The appropriate procedure depends on the patient's condition and symptoms.

In some embodiments of the invention, the protein is used to treat inflammatory disorders. In some embodiments, the inflammatory disorder is inflammation, autoimmune disease, atopic disease, paraneoplastic autoimmune diseases, cartilage inflammation, arthritis, rheumatoid arthritis (e.g., active), juvenile arthritis, juvenile rheumatoid arthritis, Pauciarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathy arthritis, juvenile reactive arthritis, juvenile Reiter's Syndrome, SEA syndrome (Seronegativity, attachment Enthesopathy, Arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, ankylosing spondylitis. , Enteropathy arthritis, reactive arthritis, Reiter syndrome, SEA syndrome (Seronegativity, Enthesopathy, Arthropathy syndrome), dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, osteomyelitis, polyosteomyelitis, Dermatomyolitis, polyarteritis nodossa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, cirrhosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis , teardrop psoriasis, intertriginous psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, dermatitis herpetiformis, Behcet's disease, including but not limited to effects on the skin, alopecia, alopecia areata, frontalis Alopecia, arteriosclerosis, lupus, Still's disease, Systemic Lupus Erythematosus (SLE) (e.g. active), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, sinusitis, sinusitis with polyps, eosinophilic esophagitis, eosinophilic bronchitis, Guillain-Barré disease -Barre disease), type I diabetes mellitus, thyroiditis (e.g. Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, graft-versus-host disease, steroid refractoriness Sexual chronic graft-versus-host disease, transplant rejection (e.g., kidney, lung, heart, skin, etc.), kidney damage, hepatitis C vasculitis, spontaneous abortion, alopecia, vitiligo, focal segmental glomerulosclerosis (FSGS) ), minimal change disease, membranous nephropathy, ANCA-related glomerulonephropathy, membranoproliferative glomerulonephritis, IgA nephropathy, lupus nephritis, etc. In some embodiments, the protein is used to treat steroid-refractory chronic graft versus host disease. In some embodiments, the protein is used to treat active systemic lupus erythematosus. In some embodiments, the protein is used to treat active rheumatoid arthritis.

In some embodiments of the invention, the method includes administering to a subject a pharmaceutical composition comprising a protein described herein. In some embodiments, the subject is one in need thereof. Any of the above-described therapeutic proteins can be administered in the form of a composition (e.g., pharmaceutical composition) described herein. For example, the composition may comprise the IL-2 protein described herein and a buffer, antioxidants such as ascorbic acid, low molecular weight polypeptides (e.g. those with less than 10 amino acids), proteins, amino acids, carbohydrates such as glucose, water crosses or dextrins, chelating agents such as EDTA, glutathione and/or other stabilizers, excipients and/or preservatives. The composition may be formulated as a liquid or lyophilisate. Additional examples of ingredients that can be used in pharmaceutical preparations are given in Remington's Pharmaceutical Sciences, 16.sup.th Ed., Mack Publishing Company, Easton, Pa., (1980) and others described herein.

To treat a disease of interest, compositions comprising the therapeutic molecules described herein may be administered by any suitable method, including but not limited to parenteral, topical, oral, nasal, vaginal, rectal, or pulmonary (by inhalation) administration. It can be administered by. For injection, the composition(s) may be administered intraarticularly, intravenously, intraarterially, intramuscularly, intraperitoneally or subcutaneously by bolus injection or continuous infusion. Topical administration at the disease site is considered, such as transdermal delivery and sustained release from implants, skin patches, or suppositories. Delivery by inhalation includes, for example, nasal or oral inhalation, use of nebulizers, inhalation in aerosol form, etc. Administration via suppositories inserted into a body cavity can be accomplished, for example, by inserting and dissolving the composition in solid form within the selected body cavity. Other alternatives include oral preparations such as eye drops, pills, lozenges, syrups and chewing gum, and topical preparations such as lotions, gels, sprays and ointments. Therapeutic molecules, which are in most cases polypeptides, can be administered topically or by injection or inhalation.

In carrying out a method of treatment, the therapeutic molecules described above may be administered as described herein and above. For example, the composition can be administered at any dosage, frequency, and duration effective to treat the condition being treated. Dosage depends on the molecular nature of the therapeutic molecule and the nature of the disorder being treated. Treatment may be continued for as long as necessary to achieve the desired results. The therapeutic molecules of the invention may be administered in a single dose or series of doses given periodically, including administration multiple times a day, daily, every other day, twice a week, three times a week, weekly, every other week, monthly, among other possible dosing regimens. It can be administered in dosage. Treatment cycles may or may not be constant during the treatment period. For example, treatments may initially occur at weekly intervals and then every other week. Treatments with a duration of days, weeks, months or years are encompassed by the invention. Treatment may be stopped and then restarted. Maintenance doses may or may not be administered after initial treatment.

Dosage may be measured in milligrams per kilogram of body weight (mg/kg) or 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 for promoting stimulation of STAT5 phosphorylation in T regulatory cells. In some embodiments, the method comprises administering a therapeutically effective amount of a peptide described herein or a pharmaceutical composition comprising the same to a subject in need thereof.

As used herein, the phrase “in need thereof” means that a subject (animal or mammal) has been identified as being in need of a particular method or treatment. In some embodiments, confirmation may be by any diagnostic means. In any of the methods and treatments described herein, an animal or mammal may be in need thereof. In some embodiments, the animal or mammal is in or will be moving into an environment where a particular disease, disorder or condition is prevalent.

Unless otherwise defined, all technical and scientific terms have the same meaning as commonly understood by a person skilled in the art to which the disclosed embodiments pertain.

As used herein, the term “a” or “an” means “at least one” or “one or more,” unless the context clearly indicates otherwise.

As used herein, the term “about” means that the values are approximate and that small changes would not significantly affect the practice of the described embodiments. Unless the context dictates otherwise, when numerical limits are used, “about” means that the numerical value can vary by ±10% and is within the range of the disclosed embodiments.

As used herein, the terms “individual” or “subject” or “patient” are used interchangeably and refer to mammals such as rats, mice, other rodents, rabbits, dogs, and cats. , means any animal, including primates such as pigs, cattle, sheep, horses or humans.

As used herein, the terms “comprising” (and any forms of comprising “comprise“, “comprises“, and “comprised“), “having” (and any forms of having “) “have” and “has”), “including” (and any forms of “includes” and “include”) or “containing” (and any forms of containing) and "contain") are inclusive or open-ended and do not exclude additional elements or method steps not mentioned. Any step or configuration using the phrases "comprise" or "comprising" is intended to be inclusive or open-ended. It can also be said to be described in the same way as the phrase “consisting of” or “consists.”

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

As used herein, the term "fused" or "linked", when used in reference to proteins having different domains or heterologous sequences, refers to chains of identical peptides in which the protein domains are linked together with peptide bonds or other covalent bonds. It means being part of it. Domains or sections may be directly connected or fused to each other, or another domain or peptide sequence may be between the two domains or sequences, and these sequences will still be considered to be fused or connected to each other. In some embodiments, the various domains or proteins provided herein are linked or fused directly to each other, or a linker sequence, such as the glycine/serine sequence described herein, joins the two domains together.

In some embodiments of the invention, embodiments provided herein also include, but are not limited to:

1. A peptide comprising the amino acid sequence of SEQ ID NO: 1, wherein the peptide contains a mutation at positions 73, 76, 100 or 138.

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

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

4. The method of embodiment 1, wherein it further comprises a mutation at one or more positions E35, H36, Q42, D104, E115 or Q146 or at 1, 2, 3, 4, 5 or each of E35, H36, Q42, D104, E115 or Q146 is the wild type peptide.

5. The peptide according to embodiment 4, wherein said mutation is one or more of E35Q, H36N, Q42E, D104N, E115Q or Q146E.

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

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

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

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

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

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

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

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

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

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

15.1. The peptide of any one of embodiments 1-15, wherein the peptide comprises V89A, Q94P, N108R or N108D, and one or more L73I, L76I, L100I, L118I mutations, and optionally C145A or C145S mutations.

16. As a peptide comprising the amino acid sequence of SEQ ID NO:2. Said peptide comprises a mutation at positions 53, 56, 80 or 118.

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

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

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

20. The peptide of embodiment 19, wherein said mutation is one or more of E15Q, H16N, Q22E, D84N, E95Q or Q126E.

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

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

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

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

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

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

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

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

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

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

31. The peptide of any one of embodiments 16-30, wherein the peptide comprises V69A, Q74P, N88R or N88D, and one or more L53I, L56I, L80I, L118I mutations, and optionally C125A or C125S mutations.

32. The peptide according to any one of the preceding embodiments, wherein the peptide does not contain mutations corresponding to positions 30, 31 and/or 35.

33. The peptide according to any one of the preceding embodiments, wherein the peptide further comprises an Fc peptide.

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

33B. The peptide of embodiment 33, wherein the Fc peptide comprises mutations such as L247A, L248A and G250A (Kabat numbering) or L234A L235A and G237A (EU numbering).

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

35. The peptide according to any one of the preceding embodiments, wherein the peptide further comprises a linker peptide connecting the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 and the Fc peptide.

36. The peptide of embodiment 35, wherein the linker comprises the sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 9) or GGGGSGGGGSGGGGS (SEQ ID NO: 16).

37. The peptide according to any one of the preceding embodiments, wherein the peptide comprises the sequence of SEQ ID NOs: 17-43.

38. A peptide comprising the amino acid sequence of SEQ ID NO: 27.

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

40. The peptide according to embodiment 1, wherein the peptide further comprises a linker peptide at the C-terminus.

41. The peptide of embodiment 40, wherein the linker peptide comprises the sequence of (GGGGS) _n (SEQ ID NO: 10), and n is 1, 2, 3 or 4.

42. The peptide according to embodiment 41, wherein n is 1.

43. The peptide according to embodiment 41, wherein n is 2.

44. The peptide according to embodiment 41, wherein n is 3.

45. The peptide according to embodiment 41, wherein n is 4.

46. The peptide according to any one of embodiments 48-45, wherein the peptide further comprises an Fc peptide.

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

48. The peptide according to embodiment 47, wherein the peptide further comprises a leader peptide having the sequence of SEQ ID NO: 7 at the N-terminus.

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

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

51. The peptide according to embodiment 1, wherein the peptide further comprises a linker peptide connecting the peptide of SEQ ID NO: 27 and the Fc peptide.

52. The peptide of embodiment 51, wherein the linker peptide is (GGGGS) _n (SEQ ID NO: 10), and n is 1, 2, 3 or 4.

53. The peptide according to embodiment 52, wherein n is 4.

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

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

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

58. A pharmaceutical composition comprising the peptide of any of the preceding embodiments.

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

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

61. The method of embodiment 60, wherein the inflammatory disorder is inflammation, autoimmune disease, atopic disease, paraneoplastic autoimmune disease, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular pediatric rheumatism. Arthritis, polyarticular juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathy arthritis, juvenile reactive arthritis, juvenile Reiter's Syndrome, SEA syndrome (Seronegativity, Enthesopathy) , Arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathy arthritis. , Reactive Arthritis, Reiter Syndrome, SEA Syndrome (Seronegativity, Enthesopathy, Arthropathy Syndrome), Dermatomyositis, Psoriatic Arthritis, Scleroderma, Vasculitis, Osteomyelitis, Multiple Osteomyelitis, Dermatomyolitis , polyarteritis nodossa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, droplet psoriasis, Interval psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, arteriosclerosis, lupus, Still's disease, Systemic Lupus Erythematosus (SLE), myasthenia gravis, inflammatory bowel disease (IBD) : inflammatory bowel disease, Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, sinusitis, sinusitis with polyps, eosinophilic esophagitis , eosinophilic bronchitis, Guillain-Barre disease, type I diabetes mellitus, thyroiditis (e.g. Graves' disease), Addison's disease, Raynaud's phenomenon, autologous Immune hepatitis, graft-versus-host disease, steroid-refractory chronic graft-versus-host disease, transplant rejection (e.g., kidney, lung, heart, skin, etc.), kidney damage, hepatitis C-vasculitis, spontaneous abortion, alopecia, vitiligo, Focal segmental glomerulosclerosis (FSGS), minimal change disease, membranous nephropathy, ANCA-related glomerulonephropathy, membranoproliferative glomerulonephritis, IgA nephropathy, lupus nephritis, etc.

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

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

64. The method of embodiment 63, wherein the inflammatory disorder is inflammation, autoimmune disease, atopic disease, paraneoplastic autoimmune disease, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, oligoarthritis in children. Rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's Syndrome, SEA syndrome (Seronegativity, Enthesopathy) ), Arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, ankylosing spondylitis, enteropathy. Arthritis, reactive arthritis, Reiter syndrome, SEA syndrome (Seronegativity, Enthesopathy, Arthropathy syndrome), dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, osteomyelitis, multiple osteomyelitis, dermatomyolitis ), polyarteritis nodossa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, droplet psoriasis , intertriginous psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, arteriosclerosis, lupus, Still's disease, Systemic Lupus Erythematosus (SLE), myasthenia gravis, inflammatory bowel disease ( IBD: inflammatory bowel disease, Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, sinusitis, sinusitis with polyps, eosinophils Esophagitis, eosinophilic bronchitis, Guillain-Barre disease, type I diabetes mellitus, thyroiditis (e.g. Graves' disease), Addison's disease, Raynaud's phenomenon, Autoimmune hepatitis, graft-versus-host disease, steroid-refractory chronic graft-versus-host disease, transplant rejection (e.g., kidney, lung, heart, skin, etc.), kidney damage, hepatitis C vasculitis, spontaneous abortion, alopecia, vitiligo , focal segmental glomerulosclerosis (FSGS), minimal change disease, membranous nephropathy, ANCA-related glomerulonephropathy, membranous proliferative glomerulonephritis, IgA nephropathy, lupus nephritis, etc.

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

66. A vector containing 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. Cell containing the vector of embodiment 66.

71. A cell comprising or expressing the peptide of 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 variations known to those skilled in the art are within the scope of the following embodiments.

Example

Example 1:

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

Example 2:

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

Example 3: Generation of IL-muteins

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

Example 4: IL-2M Molecules Can Bind CD25

The immunosorbent plate was coated with CD25 at a concentration of 0.5 μg/mL in PBS pH 7.4, 75 μl/well, and incubated at 4°C overnight. Wells were washed three times with PBS pH 7.4 containing 0.05% Tween-20 (wash buffer), then washed with 200ul/well 1% BSA (block buffer) in PBS pH 7.4 for 2 hours at room temperature. ) was blocked. After three washes with wash buffer, IL-2M molecules were diluted to the highest concentration of 2 nM in 11- to 2-fold serial dilutions in PBS containing 1% BSA and 0.05% Tween-20 (assay buffer). The diluted material was added to CD25 coated plates at 75ul/well for 1 hour at room temperature. After three washes 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 μl/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 75ul/well for 15 minutes at room temperature. After three washes with wash buffer and one wash with wash buffer (no Tween-20), analysis was performed with TMB and stopped with 1N HCL. OD 450nm was measured. Experiments included appropriate controls for non-specific binding of IL-2M molecules to plates/blocks in the absence of CD25 and negative control molecules that cannot bind CD25.

The results show that at concentrations of 2nM-1.9pM, IL-2M molecules can bind CD25 with a subnanomolar EC50. Additionally, when CD25 was not present on the plate surface, there was no detection of any compound at any of the concentrations tested, indicating that none of the test compounds interacted non-specifically with the plate surface (data is not shown).

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

Fixed cells were sequentially permeabilized with BD Perm III followed by BioLegend FOXP3 permeabilization buffer. After blocking with human serum for 10 minutes, cells were stained with antibodies against phospho-STAT5 FITC, CD25 PE, FOXP3 AF647, and CD4 PerCP Cy5.5 for 30 minutes, followed by Attune NXT using a plate reader. got it from IL-2M of SEQ ID NO:23 strongly and selectively induces STAT5 phosphorylation in Tregs but not Teffs.

Example 6: Immunogenicity of IL-2 Muteins

IL-2 mutein mutant sequences were analyzed using NetMHCIIPan 3.2 software, which can be found at www.cbs.dtu.dk/services/NetMHCIIpan/. Peptide affinity for MHC class II alleles was measured using an artificial neural network. In this assay, a 9-residue peptide with potentially direct interactions with MHC class II molecules was recognized as the binding core. Residues adjacent to the binding core, which are likely to indirectly affect binding, were also identified as masking residues. Peptides containing both binding core and masking residues appeared to be strong binders for MHC class II molecules with their expected _K below 50 nM. Stronger binders are more likely to lead to T cell immunogenicity.

A total of nine MHCII alleles represented in North America and Europe were included in the in silico analysis. The panel of IL-2 mutein (IL-2M) molecules tested included IL-2 muteins with the L53I, L56I, L80I, or L118I mutations. Only MHCII alleles DRB1_1101, DRB1_1501, DRB1_0701 and DRB1_0101 generated peptide hits with any of the examined molecules. Peptide hits for DRB_1501 were identical between all constructs tested, including wild-type IL-2 with the C125S mutation. Addition of L80I removes 1 T cell epitope for DRB1-0101 [ALNLAPSKNFHLRPR (SEQ ID NO: 60)] and moderately modifies the affinity of two other T cell epitopes [EEALNLAPSKNFHLR (SEQ ID NO: 61) and EALNLAPSKNFHLRP (SEQ ID NO: 62)]. reduce. For MHCII allele DRB1-0701, L80I removes 1 T cell epitope [EEALNLAPSKNFHLR (SEQ ID NO:63)]. Therefore, the data demonstrate that IL-2 muteins containing the L80I mutation should be less immunogenic, which is a surprising and unexpected result from the in silico analysis.

Example 7: Generation of additional IL-2 muteins

Single IL-2M (IL-2 mutein) with human IL-2M or IL-2M fused to the N-terminus of a human IgG1 Fc domain SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 (and IL -2M control; SEQ ID NO: 34) pTT5 vector containing a single gene encoding polypeptide was transfected into HEK293 Expi cells. After 5-7 days, cell culture supernatants expressing SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40 (and IL-2M control; SEQ ID NO: 34) were obtained and filtered through centrifugation and 0.22um filtration. Purified by filtration. 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 obtained on proA dendrites. The resin was washed with PBS pH 7.4 and the obtained proteins were eluted using 0.25% acetic acid pH 3.5, neutralized using 10% volume of 1M Tris pH 8.0. Proteins were buffer exchanged into 30mM HEPES 150mM NaCl pH 7 and analyzed by size exclusion chromatography on a Superdex 200 3.2/300 column. 5ug of purified material was analyzed by reducing and non-reducing SDS-PAGE on Bis-Tris 4-12% gel.

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 expressed at >45 mg/L and were analyzed by size exclusion chromatography and reduced/non-reduced SDS- It was >95% monodisperse after purification as shown by PAGE.

Example 8: IL-2M can bind CD25

The immunosorbent plate was coated with CD25 at a concentration of 0.5 μg/mL in PBS pH 7.4, 75 μl/well, and incubated at 4°C overnight. Wells were washed three times with PBS pH 7.4 containing 0.05% Tween-20 (wash buffer) and then blocked with 200ul/well 1% BSA in PBS pH 7.4 (block buffer) for 2 hours at room temperature. After three washes with wash buffer, IL-2M SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40 were incubated at a maximum concentration of 2 nM in PBS (assay buffer) containing 1% BSA and 0.05% Tween-20. Dilutions were made in 11- to 2-fold serial dilutions. The diluted material was added to CD25 coated plates at 75ul/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 μl/well for 1 hour at room temperature. After washing three times with wash buffer, high-sensitivity streptavidin HRP diluted 1:5000 in assay buffer was added to the plate at 75ul/well for 15 minutes at room temperature. After three washes with wash buffer and one wash with wash buffer (without Tween-20), analysis was performed with TMB and stopped with 1N HCL. OD 450nm was measured. The experiment included appropriate controls for non-specific binding of molecules to the plate/block in the absence of CD25. The results show that the mutein of Example 7 at concentrations of 2nM-1.9pM can bind CD25 with a subnanomolar EC50. Additionally, when CD25 was not present on the plate surface, there was no detection of any compound at any of the concentrations tested, indicating that none of the test compounds interacted non-specifically with the plate surface. Therefore, the mutein of Example 7 can bind CD25.

Example 9: The IL-2 mutein of Example 7 is potent and selective

Peripheral blood mononuclear cells (PBMC) were prepared from freshly isolated heparinized human whole blood using FICOLL-PAQUE Premium and Sepmate tubes. PBMCs were cultured in 10% fetal bovine serum RPMI medium for 20 minutes in the presence of wild-type IL-2 or the mutein of Example 7, and then fixed with BD Cytofix for 10 minutes. Fixed cells were sequentially permeabilized with BD Perm III followed by 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. Acquired on Attune NXT using a plate reader. The IL-2 mutein of Example 7 was found to be potent and selective for Treg versus Teff. A mutein containing the L118I mutation was found to have increased activity and selectivity compared to other muteins.

Example 10: IL-2 Muteins Proliferate 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 1ug 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. On day 7, mice were euthanized and whole blood and spleen were collected. Whole blood was aliquoted into a 96-well deep well plate and fixed for 10 minutes using BD Fix Lyse. Splenocytes were isolated using a 70um filter (BD) and red blood cells were lysed using BioLegend's RBC lysis buffer. After washing with 2% fetal bovine serum PBS, splenocytes were labeled with near-infrared live-death staining (Invitrogen) for 20 minutes and then fixed using BioLegend fixation buffer for 20 minutes. Both whole blood cells and splenocytes were then permeabilized using BioLegend FOXP3 permeabilization buffer, blocked with human serum, and human CD8a FITC (BL), human CD25 PE (BD), human FOXP3 AF647 (BD) CD4 PerCP Cy5.5. (BD), plates were stained for 30 min with antibodies against human Siglec-8 PE Cy7 (BL), human CD3 BV421 (BL), human CD45 BV605 (L), human CD56 BV785 (BL), and rat CD45 (BV711). Acquired from Attune NXT using a plate loader.

Compared to vehicle controls, 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 spleens and whole blood of humanized mice. 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 demonstrate that IL-2 muteins increase the frequency of regulatory T cells.

Example 11: Durability of signaling induced by IL-2 muteins

Peripheral blood mononuclear cells (PBMC) were prepared from freshly isolated heparinized human whole blood using FICOLL-PAQUE Premium and Sepmate tubes. PBMCs were cultured in RPMI medium with 10% fetal bovine serum for 60 min 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 sequentially permeabilized with BD Perm III followed by BioLegend FOXP3 permeabilization buffer. After blocking with human serum for 10 minutes, cells were stained with antibodies against phospho-STAT5 FITC, CD25 PE, FOXP3 AF647, and CD4 PerCP Cy5.5 for 30 minutes, followed by Attune NXT using a plate reader. Obtained from . All four IL-2 muteins of Example 19 induced durable signaling in Tregs but not Teff compared to controls. SEQ ID NO: 40 is superior to SEQ ID NO: 39, SEQ ID NO: 38 or SEQ ID NO: 37. These results demonstrate that IL-2 can induce durable selective signaling in Tregs that requires greater Treg proliferation in vivo and requires less frequent administration to achieve Treg proliferation.

The examples provided herein demonstrate the surprising and unexpected result that IL-2 muteins can function to selectively and potently activate Tregs relative to Teff, which may lead to the molecule treating or ameliorating the diseases described herein. Prove that it can be used to IL-2 muteins provided herein can also be generated and used with or without fusion with an Fc domain or linker as provided herein.

Embodiments of the invention have been described with reference to specific examples. These examples are not intended to limit the embodiment in any way. For the purposes of this disclosure, it is understood that various changes and modifications may be made within the scope of this disclosure. Numerous other modifications may be made that will readily occur to those skilled in the art and are included within the spirit of the invention as disclosed herein and defined in the appended claims.

Example 12: Muteins show overall POI and less aggregation.

IL-2 muteins with one of the following mutations L53I, L56I, L80I or L118I linked to the Fc region comprising mutations of V69A, Q74P, N88D and C125S and the L234A, L235A and G237A mutations provided herein were transfected into HEK293 Expi cells. It was expressed in pTT5 vector by transfection. The IL-2 mutein was linked to the N-terminus of the Fc region with 4 GGGGS (SEQ ID NO: 10) repeats. After 5-7 days, cell culture supernatants expressing the different IL-2 muteins were harvested and clarified by centrifugation and filtration through a 0.22um filtration device. IL-2 mutein was captured on proA dendrites. The resin was washed with PBS pH 7.4 and the captured proteins were eluted using 0.25% acetic acid pH 3.5 while neutralizing using 10% volume of 1M Tris pH 8.0. The protein was buffer exchanged into 30mM HEPES 150mM NaCl pH 7 and analyzed by size exclusion chromatography on an AdvanceBio SEC column for percent peak of interest (POI). Results demonstrated that distinct muteins were expressed at >60 mg/L. However, surprisingly, size exclusion chromatography revealed that muteins with the L80I or L118I mutations were >90% monodisperse, whereas muteins with the L53I or L56I mutations did not. Accordingly, muteins with L80I or L118I substitutions showed less aggregation. The differences in aggregation between the four molecules (IL-2 muteins containing L80I, L118I, L53I and L56I) were surprising due to the types of mutations made. Therefore, muteins carrying the L80I or L118I mutation have a surprising advantage over other muteins in that they do not aggregate as much as other muteins.

Example 13: Unexpected increase in efficacy of IL-2 muteins

The muteins described in Example 12 were analyzed for efficacy in an in vitro assay. Briefly, PBMCs were isolated from heparinized human whole blood and stimulated with different muteins at different concentrations for 30 min at 37°C. Stimulation was interrupted by fixation. 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. The percentage of cells staining positive for phospho-STAT5 is shown. This assay measures the ability of muteins to specifically stimulate Tregs without stimulating Teff. The most appropriate dose-response curve at each test moment was used to calculate EC50 values.

Surprisingly, muteins with mutations of L118I, L80I, L56I or L53I had increased potency (stimulating Tregs) compared to IL-2 muteins without these mutations. IL-2 muteins without mutations of L118I, L80I, L56I or L53I but with mutations V69A, Q74P and N88D were approximately 51 pM. The EC ₅₀ for muteins containing either L118I, L80I, L56I or L53I each had an EC ₅₀ of approximately 30, 40, 41 and 45, respectively. This difference in EC50 for Treg stimulation (no change for Teff stimulation) is surprising and would not have been expected for a mutein with one of the mutations described in this example. Data can also be evaluated by comparing the ratio of the parent IL-2 muteins (including V69A, Q74P, N88D and C125S) to muteins containing one of the L118I, L80I, L56I or L53I mutations. Using this ratio, it is normalized for different cell populations used in different experiments. Using this ratio, L118I had a mean increase in potency of approximately 25% (standard error of the mean of 0.16) compared to the parental control, while the other mutations had reduced activity compared to the parental control using this ratio.

In vitro data were confirmed in vivo for muteins with either the L118I, L80I, L56I or L53I mutations. L118I was found to be more potent than muteins without the L118I mutation in vivo. Briefly, Nod-Scid-IL-2Rgamma-deficient (NSG) mice reconstituted with human CD34+ hematopoietic stem cells were injected subcutaneously with 1 microgram of the indicated test article or vehicle on days 0 and 7. On day 11, mice were killed and blood was collected by cardiac puncture into tubes containing heparin. Peripheral blood leukocytes (PBL) were isolated by lysis of red blood cells and stained with antibodies reactive to the human markers CD45, CD3, CD8, CD4, FoxP3, CD25 and CD56. Flow cytometric analysis of the percentages of human regulatory T cells (Tregs, CD45+CD3+CD4+CD25+FoxP3+), activated effector T cells (act Teff, CD45+CD3+CD4+CD25+FoxP3-), and NK cells (CD45+CD56+). Measured by analysis. The frequencies of total CD45+, total CD4+, and total CD8+ cells did not change. Similar results were observed in rat spleen. There was a slight increase in in vivo potency measured in this assay for IL-2 muteins with the L80I mutation compared to muteins without the L80I mutation, and the in vivo potency measured in this assay for muteins with the L56I or L53I mutation was significantly higher when the mutation was present. It was almost identical to the mutein without it. The mutein was N-terminally linked to the Fc region as described herein with a 20 amino acid (GGGGS) ₄ (SEQ ID NO: 9) linker. That is, the linker connected the C-terminus of the IL-2 mutein to the N-terminus of the Fc region.

Example 14: 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 mutations L234A, L235A and G237A mutations with GGGGS (SEQ ID NO: 10) repeats of different lengths. IL2-mutein molecules were tested fused to the n-terminus of human IgG1 Fc through the c-terminus of the mutein with linkers containing 3 and 4 GGGGS (SEQ ID NO: 10) repeats, such that the length of the linker was IL-2 It was measured whether it affects the efficacy of muteins. Muteins fused to the c-terminus of human IgG1 Fc with a single GGGGS (SEQ ID NO: 10) repeat through the n-terminus were also tested. Briefly, Nod-Scid-IL-2Rgamma-deficient (NSG) mice reconstituted with human CD34+ hematopoietic stem cells were injected subcutaneously on day 0 with 1 microgram of different 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 lysis of red blood cells and stained with antibodies reactive to the human markers CD45, CD3, CD8, CD4, FoxP3, CD25 and CD56. Flow cytometric analysis of the percentages of human regulatory T cells (Tregs, CD45+CD3+CD4+CD25+FoxP3+), activated effector T cells (act Teff, CD45+CD3+CD4+CD25+FoxP3-), and NK cells (CD45+CD56+). Measured by analysis. The frequencies of total CD45+, total CD4+, and total CD8+ cells did not change. Similar results were observed in rat spleen.

A mutein fused to the N-terminus of a human IgG1 Fc with a linker containing four GGGGS (SEQ ID NO: 10) repeats may be a mutein with a linker containing only three GGGGS (SEQ ID NO: 10) repeats or a single GGGGS (SEQ ID NO: It was found to be the most potent compared to muteins fused to the c-terminus of human IgG1 Fc with repeat number 10). Additionally, the protein with four GGGGS (SEQ ID NO: 10) repeats was more effective in proliferating Tregs, but this construct did not cause any differential proliferation of CD56+ NK cells. It was not predicted that proteins with N-terminal Fc fused muteins with longer linkers would be the most potent and would not cause differential proliferation of CD56+ NK cells.

Example 15: 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 is administered to a patient with active rheumatoid arthritis. IL-2 muteins have been shown to be effective in treating active rheumatoid arthritis in patients.

Example 16: 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 is administered to a patient with active systemic lupus erythematosus. IL-2 muteins have been shown to be effective in treating active systemic lupus erythematosus.

Example 17: Treatment of patients with steroid-refractory chronic graft-versus-host disease.

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 patient with steroid refractory chronic graft versus host disease. IL-2 muteins have been shown to be effective in the treatment of steroid-refractory chronic graft-versus-host disease.

Example 18: IL-2 muteins induce pSTAT5 in human Tregs. PBMCs purified from heparinized whole blood from six healthy donors were treated with serial dilutions of IL-2 mutein protein containing sequence SEQ ID NO: 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 markers and intracellular/nuclear markers (pSTAT5 and FOXP3). Data were collected on an Attune NxT cytometer. Tregs were gated as monocytes, singlets, CD3pos, CD4pos, CD25hi, and FoxP3pos. The percentage of gated Tregs expressing phosphorylated STAT5 was measured. The optimal curve was fitted to the dose-response of pSTAT5 and the EC50 values were determined. The average EC50 values of all six donors were determined for 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). Data demonstrate that IL-2 muteins can induce pSTAT5 in human Tregs. IL-2 comprising the sequence of SEQ ID NO: 40 is more potent than the IL-2 sequence comprising SEQ ID NO: 39, but both are active across multiple cell populations.

Example 19: IL-2 muteins induce pSTAT5 in monkey PBMC in vitro. PBMCs purified from heparinized whole blood from three healthy monkeys were treated with IL-2 mutein protein containing the sequence of SEQ ID NO: 39 or 40 in serial dilutions for 60 minutes at 37°C. Fluorochrome-conjugated anti-CD25 and anti-CD4 were added during the final 30 minutes of IL-2 mutein treatment. Cells were fixed, washed, permeabilized and washed. Cells were stained with residual antibodies that detect both surface markers and intracellular/nuclear markers (pSTAT5 and FOXP3). Data were collected on an Attune NxT cytometer. Tregs were gated as monocytes, singlets, CD4pos, CD25hi, and FoxP3pos. The percentage of gated Tregs expressing phosphorylated STAT5 was measured. IL-2 muteins were found to induce pSTAT5 in monkeys.

Example 20: IL-2 muteins induce proliferating Treg cells in vivo and induce Treg proliferation. Venous whole blood was administered prior to administration of IL-2 mutein at SEQ ID NO: 39 or 40 (2 timepoints/cyno, 5 cyno) and SEQ ID NO: 39 (5 time points/cyno, 2 cyno) or SEQ ID NO: 40 ( After administration, 5 time points/cyno, 3 cyno) were collected into K2EDTA tubes from monkeys (cynomolgus). Samples were divided into two and stained separately for two FACS panels. One was a “Treg panel” and the other was a general immunophenotypic panel. After fixation and permeabilization, RBCs were lysed and cells were stained for surface and intracellular markers. For FAC analysis, the total number of cells/ul was determined by ADVIA. The cell number/ul of a given subpopulation was then calculated as total number/ul and % total. For each monkey, the “fold-change from pre-dose” was determined by averaging and using the average number/ul of a given cell type from the two pre-dose bleeds to normalize the post-dose bleed. It has been done. To analyze serum cytokines and chemokines, plasma from K2EDTA whole blood was frozen until the end of the study. Chemokine and cytokine amounts were quantified by multiplex MSD assay using serial dilutions of standard controls. The mean and range of MCP-1 and IP-10 were determined in the pre-dose bleed. Both muteins were found to proliferate Tregs and induce Treg proliferation in monkeys. These results demonstrate that IL-2 muteins function in an in vivo animal model similar to humans. It was also found that neither molecule significantly increased Tconv cells, CD4 cells (Tnaive) or CD8 cells (Cytotoxic T), or NK cells in monkeys (non-human primates). It was also found that neither molecule significantly induced serum chemokines. These data demonstrate that IL-2 muteins can proliferate Treg cells and induce Treg cell proliferation without unwanted proliferation or activation of other pathways. Therefore, IL-2 muteins are surprisingly potent, effective, and selective for Treg proliferation and proliferation.

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

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

SEQUENCE LISTING <110> Pandion Therapeutics, Inc. Higginson-Scott, Nathan Viney, Joanne L Visweswaraiah, Jyothsna Sampson, Erik R Otipoby, Kevin L <120> IL-2 MUTEINS AND USES THEREOF <130> 145256.00402 <150> US 62/675,972 <151> 2018-05-24 < 150> US 62/721,644 <151> 2018-08-23 <150> US 16/109,875 <151> 2018-08-23 <150> US 16/109,897 <151> 2018-08-23 <150> US 62/ 595,357 <151> 2017-12-06 <160> 43 <170> PatentIn version 3.5 <210> 1 <211> 153 <212> PRT <213> homo sapiens <400> 1 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Cys Gln Ser Ile Ile Ser Thr Leu Thr 145 150 <210> 2 <211> 133 <212> PRT <213> Homo sapiens <400> 2 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 3 <211> 153 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 3 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Ser Asn His Lys Asn Pro Arg Leu Ala Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Glu Lys Ala Thr Glu Ile Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Leu Lys Pro Leu Glu Glu Ala Leu Arg Leu Ala Pro Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Ser Gln Ser Ile Ile Ser Thr Leu Thr 145 150 <210> 4 <211> 153 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 4 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Ser Asn His Lys Asn Pro Arg Leu Ala Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Glu Lys Ala Thr Glu Leu Lys His Ile Gln Cys Leu Glu 65 70 75 80 Glu Glu Leu Lys Pro Leu Glu Glu Ala Leu Arg Leu Ala Pro Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Ser Gln Ser Ile Ile Ser Thr Leu Thr 145 150 <210> 5 <211> 153 <212> PRT <213> Artificial Sequence <220 > <223> synthetic sequence <400> 5 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Ser Asn His Lys Asn Pro Arg Leu Ala Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Glu Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Leu Lys Pro Leu Glu Glu Ala Leu Arg Leu Ala Pro Ser Lys 85 90 95 Asn Phe His Ile Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Ser Gln Ser Ile Ile Ser Thr Leu Thr 145 150 <210> 6 <211 > 153 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 6 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Ser Asn His Lys Asn Pro Arg Leu Ala Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Glu Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Leu Lys Pro Leu Glu Glu Ala Leu Arg Leu Ala Pro Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Ile Asn Arg Trp Ile Thr Phe 130 135 140 Ser Gln Ser Ile Ile Ser Thr Leu Thr 145 150 <210> 7 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 7 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser 20 <210> 8 <211> 226 <212> PRT <213> artificial sequence <220> <223> synthetic sequence <400> 8 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly 225 <210> 9 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 9 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 10 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 10 Gly Gly Gly Gly Ser 1 5 <210> 11 <211> 399 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 11 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Ser Asn His Lys Asn Pro Arg Leu Ala Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Glu Lys Ala Thr Glu Ile Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Leu Lys Pro Leu Glu Glu Ala Leu Arg Leu Ala Pro Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly 145 150 155 160 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr 165 170 175 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser 180 185 190 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 195 200 205 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 210 215 220 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 225 230 235 240 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 245 250 255 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 260 265 270 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 275 280 285 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 290 295 300 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 305 310 315 320 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 325 330 335 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 340 345 350 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 355 360 365 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 370 375 380 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 385 390 395 <210> 12 <211> 399 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 12 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Ser Asn His Lys Asn Pro Arg Leu Ala Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Glu Lys Ala Thr Glu Leu Lys His Ile Gln Cys Leu Glu 65 70 75 80 Glu Glu Leu Lys Pro Leu Glu Glu Ala Leu Arg Leu Ala Pro Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly 145 150 155 160 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr 165 170 175 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser 180 185 190 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 195 200 205 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 210 215 220 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 225 230 235 240 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 245 250 255 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 260 265 270 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 275 280 285 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 290 295 300 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 305 310 315 320 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 325 330 335 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 340 345 350 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 355 360 365 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 370 375 380 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 385 390 395 <210> 13 <211> 399 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 13 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Ser Asn His Lys Asn Pro Arg Leu Ala Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Glu Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Leu Lys Pro Leu Glu Glu Ala Leu Arg Leu Ala Pro Ser Lys 85 90 95 Asn Phe His Ile Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe 130 135 140 Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly 145 150 155 160 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr 165 170 175 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser 180 185 190 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 195 200 205 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 210 215 220 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 225 230 235 240 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 245 250 255 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 260 265 270 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 275 280 285 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 290 295 300 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 305 310 315 320 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 325 330 335 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 340 345 350 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 355 360 365 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 370 375 380 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 385 390 395 <210> 14 <211> 399 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 14 Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu 1 5 10 15 Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu 20 25 30 Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile 35 40 45 Ser Asn His Lys Asn Pro Arg Leu Ala Arg Met Leu Thr Phe Lys Phe 50 55 60 Tyr Met Pro Glu Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu 65 70 75 80 Glu Glu Leu Lys Pro Leu Glu Glu Ala Leu Arg Leu Ala Pro Ser Lys 85 90 95 Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile 100 105 110 Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala 115 120 125 Asp Glu Thr Ala Thr Ile Val Glu Phe Ile Asn Arg Trp Ile Thr Phe 130 135 140 Ser Gln Ser Ile Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly 145 150 155 160 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr 165 170 175 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser 180 185 190 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 195 200 205 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 210 215 220 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 225 230 235 240 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 245 250 255 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 260 265 270 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 275 280 285 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 290 295 300 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 305 310 315 320 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 325 330 335 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Pro Val Leu Asp 340 345 350 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 355 360 365 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 370 375 380 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 385 390 395 <210> 15 <211> 226 <212> PRT <213> Artificial Sequence < 220> <223> synthetic sequence <400> 15 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly 225 <210> 16 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 16 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 17 <211> 133 <212> PRT <213> Artificial Sequence <220 > <223> synthetic sequence <400> 17 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 18 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 18 Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 19 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 19 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Arg Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210 > 20 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 20 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Ala Leu Asn Leu Ala Pro Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 21 <211> 133 <212> PRT < 213> Artificial Sequence <220> <223> synthetic sequence <400> 21 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Ala Leu Asn Leu Ala Pro Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 22 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 22 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Ala Leu Asn Leu Ala Pro Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Arg Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 23 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 23 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 24 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 24 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Ile Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Ala Leu Asn Leu Ala Pro Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 25 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 25 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Ile Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Ala Leu Asn Leu Ala Pro Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 26 <211> 133 <212> PRT <213> Artificial Sequence < 220> <223> synthetic sequence <400> 26 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Ala Leu Asn Leu Ala Pro Ser Lys Asn Phe His Ile 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 27 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 27 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Ala Leu Asn Leu Ala Pro Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Ile Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 28 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 28 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly 225 <210> 29 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 29 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gly Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly 225 <210> 30 <211> 374 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 30 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 145 150 155 160 Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 165 170 175 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 180 185 190 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 195 200 205 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 210 215 220 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 225 230 235 240 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 245 250 255 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 260 265 270 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 275 280 285 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 290 295 300 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 305 310 315 320 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 325 330 335 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 340 345 350 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 355 360 365 Leu Ser Leu Ser Pro Gly 370 <210> 31 <211> 374 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 31 Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 145 150 155 160 Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 165 170 175 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 180 185 190 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 195 200 205 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 210 215 220 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 225 230 235 240 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 245 250 255 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 260 265 270 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 275 280 285 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 290 295 300 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 305 310 315 320 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 325 330 335 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 340 345 350 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 355 360 365 Leu Ser Leu Ser Pro Gly 370 <210> 32 <211> 374 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 32 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Arg Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 145 150 155 160 Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 165 170 175 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 180 185 190 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 195 200 205 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 210 215 220 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 225 230 235 240 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 245 250 255 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 260 265 270 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 275 280 285 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 290 295 300 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 305 310 315 320 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 325 330 335 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 340 345 350 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 355 360 365 Leu Ser Leu Ser Pro Gly 370 <210> 33 <211> 374 <212> PRT <213> Artificial Sequence < 220> <223> synthetic sequence <400> 33 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Ala Leu Asn Leu Ala Pro Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 145 150 155 160 Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 165 170 175 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 180 185 190 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 195 200 205 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 210 215 220 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 225 230 235 240 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 245 250 255 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 260 265 270 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 275 280 285 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 290 295 300 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 305 310 315 320 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 325 330 335 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 340 345 350 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 355 360 365 Leu Ser Leu Ser Pro Gly 370 <210> 34 <211> 374 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 34 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Ala Leu Asn Leu Ala Pro Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 145 150 155 160 Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 165 170 175 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 180 185 190 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 195 200 205 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 210 215 220 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 225 230 235 240 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 245 250 255 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 260 265 270 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 275 280 285 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 290 295 300 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 305 310 315 320 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 325 330 335 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 340 345 350 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 355 360 365 Leu Ser Leu Ser Pro Gly 370 <210> 35 <211> 374 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 35 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Ala Leu Asn Leu Ala Pro Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Arg Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 145 150 155 160 Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 165 170 175 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 180 185 190 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 195 200 205 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 210 215 220 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 225 230 235 240 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 245 250 255 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 260 265 270 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 275 280 285 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 290 295 300 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 305 310 315 320 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 325 330 335 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 340 345 350 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 355 360 365 Leu Ser Leu Ser Pro Gly 370 <210> 36 <211> 374 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 36 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 145 150 155 160 Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 165 170 175 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 180 185 190 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 195 200 205 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 210 215 220 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 225 230 235 240 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 245 250 255 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 260 265 270 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 275 280 285 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 290 295 300 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 305 310 315 320 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 325 330 335 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 340 345 350 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 355 360 365 Leu Ser Leu Ser Pro Gly 370 <210> 37 < 211> 379 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 37 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Ile Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Ala Leu Asn Leu Ala Pro Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 370 375 <210> 38 <211> 379 <212> PRT <213> Artificial Sequence <220> < 223> synthetic sequence <400> 38 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Ile Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Ala Leu Asn Leu Ala Pro Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 370 375 <210> 39 <211> 379 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 39 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Ala Leu Asn Leu Ala Pro Ser Lys Asn Phe His Ile 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 370 375 <210> 40 <211> 379 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 40 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Ala Leu Asn Leu Ala Pro Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Ile Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 370 375 <210> 41 <211> 379 <212> PRT <213> Artificial Sequence <220 > <223> synthetic sequence <400> 41 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Ala Leu Asn Leu Ala Pro Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 370 375 <210> 42 <211> 364 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 42 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gly Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys 225 230 235 240 Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu 245 250 255 Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr 260 265 270 Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln 275 280 285 Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Ala Leu Asn Leu Ala 290 295 300 Pro Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asp Ile 305 310 315 320 Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys 325 330 335 Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp 340 345 350 Ile Thr Phe Ala Gln Ser Ile Ile Ser Thr Leu Thr 355 360 <210> 43 <211> 379 <212> PRT <213> Artificial Sequence <220 > <223> synthetic sequence <220> <221> MISC_FEATURE <222> (53)..(53) <223> L or I <220> <221> MISC_FEATURE <222> (56)..(56) <223 > L or I <220> <221> MISC_FEATURE <222> (80)..(80) <223> L or I <220> <221> MISC_FEATURE <222> (118)..(118) <223> L or I <400> 43 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asp Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 370 375

Claims (63)

A peptide comprising the amino acid sequence of SEQ ID NO: 27.
The peptide according to claim 1, wherein the peptide further comprises an N-terminal leader peptide having the sequence of SEQ ID NO:7.
The peptide according to claim 1, wherein the peptide further comprises a linker peptide at the C-terminus.
4. The peptide according to claim 3, wherein the linker peptide comprises a sequence of (GGGGS) _n , where n is 1, 2, 3 or 4.
The peptide according to claim 4, wherein n is 1.
The peptide according to claim 4, wherein n is 2.
The peptide according to claim 4, wherein n is 3.
The peptide according to claim 4, wherein n is 4.
The peptide according to claim 1, wherein the peptide further comprises an Fc peptide.
The peptide according to claim 9, wherein the Fc peptide comprises the sequence of SEQ ID NO: 8 or SEQ ID NO: 15.
10. The peptide according to claim 9, wherein the peptide further comprises a leader peptide having the sequence of SEQ ID NO: 7 at the N-terminus.
10. The peptide according to claim 9, wherein the Fc peptide is located at the C-terminus.
10. The peptide according to claim 9, wherein the Fc peptide is located at the N-terminus.
The peptide according to claim 1, wherein the peptide further comprises a linker peptide connecting the peptide of SEQ ID NO: 27 and the Fc peptide.
15. The peptide according to claim 14, wherein the peptide further comprises a leader peptide having the sequence of SEQ ID NO: 7 at the N-terminus.
15. The peptide according to claim 14, wherein the linker peptide is (GGGGS) _n , and n is 1, 2, 3, or 4.
17. The peptide according to claim 16, wherein n is 4.
15. The peptide according to claim 14, wherein the Fc peptide comprises the sequence of SEQ ID NO: 8 or SEQ ID NO: 15.
15. The peptide according to claim 14, wherein the Fc peptide is located at the C-terminus.
15. The peptide according to claim 14, wherein the Fc peptide is located at the N-terminus.
15. The peptide according to claim 14, wherein the peptide comprises a peptide comprising the sequence of SEQ ID NO: 40.
A pharmaceutical composition for the treatment of autoimmune diseases, comprising the peptide of claim 1.
A pharmaceutical composition for the treatment of autoimmune diseases, comprising the peptide of claim 14.
A pharmaceutical composition for the treatment of autoimmune diseases, comprising the peptide of claim 19.
A pharmaceutical composition for the treatment of autoimmune diseases, comprising the peptide of claim 24. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete