Interleukin-2 mutant proteins for expansion of regulatory T cells

Abstract

Provided herein are IL-2 mutant proteins and IL-2 mutant protein Fc fusion molecules that preferentially expand and activate cells and are suitable for large-scale production. Also provided herein are methods for preparing and using the compositions of the invention.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. patent application Ser. No. 62 / 886,283, filed Aug. 13, 2019, which is incorporated herein by reference in its entirety. Background Art

[0003] IL-2 binds to three transmembrane receptor subunits: IL-2Rβ and IL-2Rγ, which together activate intracellular signaling events when IL-2 binds, and CD25 (IL-2Rα), which serves to stabilize the interaction between IL-2 and IL-2Rβγ. Signals delivered by IL-2Rβγ include those in the PI3 kinase, Ras-MAP kinase, and STAT5 pathways.

[0004] T cells need to express CD25 in response to the low concentration of IL-2 typically present in tissues. The T cells expressing CD25 include FOXP3+ regulatory T cells (Treg cells) that are necessary for suppressing autoimmune inflammation, and both FOXP3-T cells activated to express CD25. FOXP3-CD25+ effector T cells (Teff) can be CD4+ or CD8+ cells, both of which may contribute to inflammation, autoimmune diseases, organ transplant rejection or graft-versus-host disease. The STAT5 signaling stimulated by IL-2 is critical for normal T-reg cell growth and survival, and for high FOXP3 expression.

[0005] In co-owned WO 2010 / 085495, we describe the use of IL-2 mutant proteins to preferentially expand or stimulate Treg cells. When administered to a subject, the effect on Treg cells can be used to treat inflammatory diseases and autoimmune diseases. Although the IL-2 mutant proteins described herein can be used to expand Tregs over Teff cells in vivo, it is desirable to produce IL-2 mutant proteins with optimal properties for human therapeutics. Summary of the invention

[0006] IL-2 mutant proteins suitable for high-yield manufacturability and pharmacological activity are described herein. In particular, the IL-2 mutant proteins of the present invention have an improved Treg:Teff selectivity window. Efforts have been made to produce such molecules for use as human therapeutic agents, and many unexpected and unpredictable observations have occurred. Compositions and methods described herein have been produced from such efforts.

[0007] IL-2 mutant proteins described herein have a lower probability of immune response for IL-2 mutant proteins and / or endogenous IL-2, and provide Treg preferential expansion and activation. In addition, in certain embodiments, IL-2 mutant proteins are fused to molecules, such as antibody Fc, and when applied to a subject, this increases serum half-life. IL-2 mutant proteins have short serum half-life (for subcutaneous injection, 3 to 5hr). Exemplary IL-2 mutant proteins described herein have a half-life of at least 1 day, at least 3 days, at least 5 days, at least 10 days, at least 15 days, at least 20 days or at least 25 days in people. This effect on the pharmacokinetics of IL-2 mutant proteins allows the reduction of IL-2 mutant protein therapeutic agents or less frequent administration.

[0008] Furthermore, when producing drug macromolecules, the ability to produce large quantities of the macromolecule while minimizing aggregation and maximizing the stability of the molecule must be considered. IL-2 mutein Fc fusion molecules display such attributes.

[0009] In addition, in certain embodiments, the IL-2 mutant protein Fc fusion protein contains an IgG1 Fc region. When it is desired to eliminate the effector function (e.g., ADCC activity) of IgG1, it is found that the asparagine mutation at position 297 is glycine (N297G; EU numbering scheme) provides greatly improved purification efficiency and biophysical properties, exceeding other mutations that lead to deglycosylated IgG1 Fc. In a preferred embodiment, cysteine ​​is engineered into Fc to allow disulfide bonds that increase the stability of molecules containing deglycosylated Fc. The utility of deglycosylated Fc exceeds the IL-2 mutant protein Fc fusion background. Therefore, provided herein are Fc-containing molecules, Fc fusions, and antibodies that include N297G substitutions and, optionally, substitutions to one or more additional residues of cysteine.

[0010] In one aspect, the present invention provides a human interleukin-2 (IL-2) mutant protein comprising an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the IL-2 mutant protein has at least one mutation selected from the group consisting of: V91K, D20L; D84R, E61Q; V91K, D20A, E61Q, M104T; N88K, M104L; V91H, M104L; V91K, H16E, M104V; V91K, H16R, M104V; V91K, H16R, M104T; V91K, D20A, M104T; V91K, H16E, M104T; V91K, H16E, E61Q, M104T; V91K, H16R, E61Q, M104T; V91K, H16E; V91H, D20A, M104T; H16E, V91H, M104V; V9 1H, D20A, E61Q, M104T; V91H, H16R, E16Q; V91K, D20A, M104V; H16E, V91H; V 91H, D20A, M104V; H16E, V91H, M104T; H16E, V91H, E61Q, M104T; V91K, E61Q , H16E; V91K, H16R, M104L; H16E, V91H, E16Q; V91K, E61Q, H16R; D20W, V91K , E61Q; V91H, H16R; V91K, H16R; D20W, V91K, E61Q, M104T; V91K, D20A; V91 H, D20A, E16Q; V91K, D20A, M104L; V91H, D20A; V91K, E61Q, D20A; V91H, M10 4T; V91H, M104V; V91K, E61Q; V91K, N88K, E61Q, M104T; V91K, N88K, E61Q; V91H, E61Q; V91K, N88K; D20A, H16E, M104T; D20A, M104T; H16E, N88K; D20A , M104V; D20A, M104L; H16E, M104T; H16E, M104V; N88K, M104V; N88K, E61Q ;D20A, E61Q; H16R, D20A; D20W, E61Q; H16E, E61Q; H16E, M104L; N88K, M104 T; D20A, H16E; D20A, H16E, E16Q; D20A, H16R, E16Q; V91K, D20W; V91A, H16A ;V91A, H16D; V91A, H16E; V91A, H16S; V91E, H16A; V91E, H16D; V91E, H16E;V91E, H16S; V91K, H16A; V91K, H16D; V91K, H16S; and V91S, H16E; and preferentially stimulates regulatory T cells relative to other T cells or NK cells in both in vitro assays and in humanized mice (NSG mice reconstituted with CD34+ hematopoietic stem cells). In one embodiment, the mutant protein has at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 1. In another embodiment, the mutant protein has at least 97% identity to the amino acid sequence set forth in SEQ ID NO: 1. In another embodiment, the amino acid sequence of the mutant protein has at least 98% identity to the amino acid sequence set forth in SEQ ID NO: 1. The amino acid sequence listed in ID NO: 1 differs only at C125A and at one position selected from the group consisting of: V91K, D20L; D84R, E61Q; V91K, D20A, E61Q, M104T; N88K, M104L; V91H, M104L; V91K, H16E, M104V; V91K, H16R, M104V; V91K, H16R, M104T; V91K, D20A, M104T; V91K, H16E, M104T; V91K, H16E , E61Q, M104T; V91K, H16R, E61Q, M104T; V91K, H16E; V91H, D20A, M104T; H16E, V91H, M104V; V91H, D20A, E61Q, M104T ;V91H, H16R, E16Q; V91K, D20A, M104V; H16E, V91H; V91H, D20A, M104V; H16E, V91H, M104T; H16E, V91H, E61Q, M104T; V 91K, E61Q, H16E; V91K, H16R, M104L; H16E, V91H, E16Q; V91K, E61Q, H16R; D20W, V91K, E61Q; V91H, H16R; V91K, H16R; D20W, V91K, E61Q, M104T; V91K, D20A; V91H, D20A, E16Q; V91K, D20A, M104L; V91H, D20A; V91K, E61Q, D20A; V91H, M104 T; V91H, M104V; V91K, E61Q; V91K, N88K, E61Q, M104T; V91K, N88K, E61Q; V91H, E61Q; V91K, N88K; D20A, H16E, M104T; D20A, M104T; H16E, N88K; D20A, M104V; D20A, M104L; H16E, M104T; H16E, M104V; N88K, M104V; N88K, E61Q; D20A, E61Q;H16R, D20A; D20W, E61Q; H16E, E61Q; H16E, M104L; N88K, M104T; D20A, H16E; D20A, H16E, E16Q; D20A, H16R, E16Q; V91K, D20W; V91A, H16A; V91A, H16D; V91A, H16E; V91A, H16S; V91E, H16A; V91E, H16D; V91E, H16E; V91E, H16S; V91K, H16A; V91K, H16D; V91K, H16S; and V91S, H16E. ;

[0011] In another aspect, the present invention provides an Fc fusion protein comprising an Fc and a human IL-2 mutant protein as described above. In one embodiment, the Fc is a human IgG1 Fc. In another embodiment, the human IgG1 Fc comprises one or more mutations that change the effector function of the Fc. In another embodiment, the human IgG1 comprises a substitution at N297. In another embodiment, the substitution at N297 is N297G. In another embodiment, the Fc fusion protein comprises a substitution or deletion of the C-terminal lysine of the human IgG Fc. In another embodiment, the C-terminal lysine of the human IgG Fc is deleted. In another embodiment, a linker connects the Fc and human IL-2 mutant protein portions of the protein. In another embodiment, the linker is GGGGS (SEQ ID NO: 5), GGNGT or (SEQ ID NO: 6), and YGNGT (SEQ ID NO: 7). In another embodiment, the linker is GGGGS (SEQ ID NO: 5). In another embodiment, when expressed in a mammalian cell, the IL-2 mutant protein further comprises an amino acid addition, substitution, or deletion that changes the glycosylation of the Fc fusion protein. In another embodiment, the IL-2 mutant protein comprises a T3 substitution. In another embodiment, the IL-2 mutant protein comprises a T3N or T3A substitution. In another embodiment, the IL-2 mutant protein comprises a T3N substitution. In another embodiment, the IL-2 mutant protein further comprises an S5 mutation. In another embodiment, the IL-2 mutant protein further comprises an S5T mutation. In another embodiment, the Fc fusion protein comprises an Fc dimer. In another embodiment, the Fc fusion protein comprises two IL-2 mutant proteins. In another embodiment, the Fc fusion protein comprises a single IL-2 mutant protein.

[0012] In another aspect, the present invention provides an isolated nucleic acid encoding a human IL-2 mutant protein as described above.

[0013] In another aspect, the present invention provides an isolated nucleic acid encoding an Fc portion of an antibody and a human IL-2 mutant protein as described above. In one embodiment, the Fc portion of an antibody and the human IL-2 mutant protein are encoded in a single open reading frame. In another embodiment, the Fc is a human IgG1 Fc. In another embodiment, the human IgG1 Fc comprises one or more mutations that change the effector function of the Fc. In another embodiment, the human IgG1 comprises a substitution at N297. In another embodiment, the substitution at N297 is N297G. In another embodiment, the nucleic acid encodes a substitution or deletion of the C-terminal lysine of the human IgG Fc. In another embodiment, the C-terminal lysine of the human IgG Fc is deleted. In another embodiment, the nucleic acid further encodes a linker connecting the Fc portion of an antibody and a human IL-2 mutant protein. In another embodiment, the linker is GGGGS (SEQ ID NO: 5), GGNGT or (SEQ ID NO: 6), and YGNGT (SEQ ID NO: 7). In another embodiment, the linker is GGGGS (SEQ ID NO: 5). In another embodiment, when expressed in a mammalian cell, the IL-2 mutant protein further comprises an amino acid addition, substitution, or deletion that changes the glycosylation of the protein, and the protein comprises the IL-2 mutant protein. In another embodiment, the IL-2 mutant protein comprises a T3 substitution. In another embodiment, the IL-2 mutant protein comprises a T3N or T3A substitution. In another embodiment, the IL-2 mutant protein comprises a T3N substitution. In another embodiment, the IL-2 mutant protein further comprises an S5 mutation. In another embodiment, the IL-2 mutant protein further comprises an S5T mutation.

[0014] In another aspect, the present invention provides an expression vector comprising the isolated nucleic acid described above operably linked to a promoter.

[0015] In another aspect, the invention provides a host cell comprising the isolated nucleic acid described above. In one embodiment, the isolated nucleic acid is operably linked to a promoter. In another embodiment, the host cell is a prokaryotic cell. In another embodiment, the host cell is an Escherichia coli. In another embodiment, the host cell is a eukaryotic cell. In another embodiment, the host cell is a mammalian cell. In another embodiment, the host cell is a Chinese hamster ovary (CHO) cell line.

[0016] In another aspect, the present invention provides a method for preparing human IL-2 mutant protein, the method comprising culturing the host cell as described above under conditions where the promoter is expressed, and harvesting the human IL-2 mutant protein from the culture.

[0017] In another aspect, the present invention provides a method for preparing an Fc fusion protein, the method comprising culturing the host cell as described above under conditions wherein the promoter is expressed, and harvesting the Fc fusion protein from the culture.

[0018] In another aspect, the invention provides a method for increasing the ratio of regulatory T cells (Treg) to non-regulatory T cells in a population of T cells, the method comprising contacting a population of T cells with an effective amount of human IL-2 mutant proteins as described above. In one embodiment, the ratio of CD3+FoxP3+ cells to CD3+FoxP3- increases. In another embodiment, the ratio of CD3+FoxP3+ cells to CD3+FoxP3- increases by at least 50%.

[0019] In another aspect, the invention provides a method for increasing the ratio of regulatory T cells (Treg) to non-regulatory T cells in a population of T cells, the method comprising contacting a population of T cells with an effective amount of an Fc fusion protein as described above. In one embodiment, the ratio of CD3+FoxP3+ cells to CD3+FoxP3- increases. In another embodiment, the ratio of CD3+FoxP3+ cells to CD3+FoxP3- increases by at least 50%.

[0020] In another aspect, the present invention provides a method for increasing the ratio of regulatory T cells (Treg) to non-regulatory T cells in the peripheral blood of a subject, the method comprising administering an effective amount of human IL-2 mutant proteins as described above. In one embodiment, the ratio of CD3+FoxP3+ cells to CD3+FoxP3- increases. In another embodiment, the ratio of CD3+FoxP3+ cells to CD3+FoxP3- increases by at least 50%.

[0021] In another aspect, the present invention provides a method for increasing the ratio of regulatory T cells (Treg) to non-regulatory T cells in the peripheral blood of a subject, the method comprising administering an effective amount of an Fc fusion protein as described above. In one embodiment, the ratio of CD3+FoxP3+ cells to CD3+FoxP3- increases. In another embodiment, the ratio of CD3+FoxP3+ cells to CD3+FoxP3- increases by at least 50%.

[0022] In another aspect, the present invention provides a method for increasing the ratio of regulatory T cells (Treg) to natural killer (NK) cells in the peripheral blood of a subject, the method comprising administering an effective amount of a human IL-2 mutant protein as described above. In one embodiment, the ratio of CD3+FoxP3+ cells to CD3-CD19- lymphocytes expressing CD56 and / or CD16 is increased. In another embodiment, the ratio of CD3+FoxP3+ cells to CD3-CD19- lymphocytes expressing CD56 and / or CD16 is increased by at least 50%.

[0023] In another aspect, the present invention provides a method for increasing the ratio of regulatory T cells (Treg) to natural killer (NK) cells in the peripheral blood of a subject, the method comprising administering an effective amount of an Fc fusion protein as described above. In one embodiment, the ratio of CD3+FoxP3+ cells to CD3-CD19- lymphocytes expressing CD56 and / or CD16 increases. In another embodiment, the ratio of CD3+FoxP3+ cells to CD3-CD19- lymphocytes expressing CD56 and / or CD16 increases by at least 50%.

[0024] In another aspect, the invention provides a method for treating a subject suffering from an inflammatory disease or an autoimmune disease, the method comprising administering to the subject a therapeutically effective amount of an IL-2 mutant protein as described above or a therapeutically effective amount of an Fc fusion protein as described above. In one embodiment, administration causes the alleviation of at least one symptom of the disease. In another embodiment, after administration, the ratio of regulatory T cells (Treg) to non-regulatory T cells in the peripheral blood of the subject increases. In another embodiment, after administration, the ratio of regulatory T cells (Treg) to non-regulatory T cells in the peripheral blood of the subject remains substantially the same. In another embodiment, the inflammatory disease or autoimmune disease is lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type I diabetes, type II diabetes, multiple sclerosis, rheumatoid arthritis, alopecia areata, atherosclerosis, psoriasis, organ transplant rejection, Sjögren's syndrome ( Syndrome, Behcet's disease, spontaneous pregnancy loss, atopic disease, asthma, or inflammatory bowel disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Shown are the results of DSC Tm measurements of IL-2 muteins with stability at 40°C for 10 days measured by SEC chromatography.

[0026] Figure 2Shown are dose titration curves of three attenuated mutant proteins, H16R, V91K D20A M104V, D20W, and control, wild-type human IL-2.Fc, recombinant human IL-2, and recombinant mouse IL-2 in an in vitro pSTAT5 assay.

[0027] Figure 3 Evaluation of human IL-2 muteins V91K D20A M104V, H16R, D20W, and control wild-type IL-2-Fc for in vivo activity in mice is shown. DETAILED DESCRIPTION

[0028] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.All references cited in the text of this specification are expressly incorporated by reference in their entirety.

[0029] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, tissue culture and transformation, protein purification, etc. Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as routinely implemented in the art or as described herein. The following procedures and techniques can be generally performed according to conventional methods well known in the art and as described in different general and more specific references cited and discussed in this specification throughout. See, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manuel [Molecular Cloning: Laboratory Manual], 3rd edition, Cold Spring Harbor Laboratory Press [Cold Spring Harbor Laboratory Press], New York, which is incorporated herein by reference for any purpose. Unless a specific definition is provided, the nomenclature and laboratory processes and techniques used in connection with analytical chemistry, organic chemistry, and medicine and pharmaceutical chemistry described herein are those well known in the art and commonly used. Standard techniques can be used for chemical synthesis, chemical analysis, drug preparation, preparation and delivery, and treatment of patients.

[0030] IL-2

[0031] The IL-2 mutant proteins described herein are variants of wild-type human IL-2. As used herein, "wild-type human IL-2," "wild-type IL-2," or "WT IL-2" will refer to a polypeptide having the following amino acid sequence:

[0032] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFXQSIISTLT

[0033] wherein X is C, S, V, or A (SEQ ID NO: 2).

[0034] Variants may contain one or more substitutions, deletions, or insertions within the wild-type IL-2 amino acid sequence. Residues are designated herein as the one-letter amino acid code preceding the IL-2 amino acid position, e.g., K35 is the lysine residue at position 35 of SEQ ID NO: 2. Substitutions are designated herein as the one-letter amino acid code preceding the IL-2 amino acid position preceding the one-letter amino acid code for substitution, e.g., K35A is a substitution of a lysine residue with an alanine residue at position 35 of SEQ ID NO: 2.

[0035] IL-2 mutant protein

[0036] Provided herein is a human IL-2 mutant protein and an anti-IL-2 antibody that preferentially stimulates T regulatory (Treg) cells. As used herein, "preferentially stimulating regulatory T cells" means that mutant proteins or antibodies promote the proliferation, survival, activation and / or function of CD3+FoxP3+ T cells beyond CD3+FoxP3-T cells. The method for measuring the ability of preferentially stimulating Treg can be measured by flow cytometry of peripheral blood leukocytes, wherein there is an increase in the percentage of FOXP3+CD4+ T cells in total CD4+ T cells, an increase in the percentage of FOXP3+CD8+ T cells in total CD8+ T cells, an increase relative to the percentage of FOXP3+ T cells of NK cells, and / or an increase in the expression level of CD25 on the surface of FOXP3+ T cells relative to the increase of CD25 expression on other T cells. As detected by sequencing of polymerase chain reaction (PCR) products from bisulfite-treated genomic DNA, the preferential growth of Treg cells can also be detected as an increase in the demethylated FOXP3 promoter DNA (i.e., Treg-specific demethylation region, or TSDR) relative to the demethylated CD3 gene in DNA extracted from whole blood (J. Sehouli et al., 2011, Epigenetics [Epigenetics] 6:2, 236-246). In particular, the IL-2 mutant proteins of the present invention have an enhanced Treg:Teff window, i.e., those that retain high levels of activity in Treg cells while showing significant attenuation in Teff cells. Since activated Teff cells express increased levels of CD25, and patients with autoimmune and inflammatory diseases have elevated numbers of these cells, we apply CD25+ gating to Teff cells, thereby simulating a more realistic differentiation in terms of CD25 expression in patients.

[0037] The IL-2 mutant protein or its Fc fusion protein that preferentially stimulates Treg cells increases the ratio of CD3+FoxP3+ T cells relative to CD3+FoxP3- T cells in a subject or in a peripheral blood sample by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%.

[0038] In some embodiments, using the protocol of Example 1, the IL-2 mutant protein or its Fc fusion protein has less than 50% pSTAT activation of effector T cells when treated with 1 nM IL-2. In some embodiments, using the protocol of Example 1, the IL-2 mutant protein or its Fc fusion protein has less than 40% pSTAT activation of effector T cells when treated with 1 nM IL-2. In some embodiments, using the protocol of Example 1, the IL-2 mutant protein or its Fc fusion protein has less than 30% pSTAT activation of effector T cells when treated with 1 nM IL-2. In some embodiments, using the protocol of Example 1, the IL-2 mutant protein or its Fc fusion protein has less than 20% pSTAT activation of effector T cells when treated with 1 nM IL-2. In some embodiments, using the protocol of Example 1, the IL-2 mutant protein or its Fc fusion protein has less than 10% pSTAT activation of effector T cells when treated with 1 nM IL-2. In some embodiments, using the regimen of Example 2, the IL-2 mutant protein or its Fc fusion protein also has a 10-day stability of greater than 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0039] In some embodiments, using the protocol of Example 1, the IL-2 mutant protein or its Fc fusion protein has less than 50% pSTAT activation of effector T cells when treated with 200nM IL-2. In some embodiments, using the protocol of Example 1, the IL-2 mutant protein or its Fc fusion protein has less than 40% pSTAT activation of effector T cells when treated with 200nM IL-2. In some embodiments, using the protocol of Example 1, the IL-2 mutant protein or its Fc fusion protein has less than 30% pSTAT activation of effector T cells when treated with 200nM IL-2. In some embodiments, using the protocol of Example 1, the IL-2 mutant protein or its Fc fusion protein has less than 20% pSTAT activation of effector T cells when treated with 200nM IL-2. In some embodiments, using the protocol of Example 1, the IL-2 mutant protein or its Fc fusion protein has less than 10% pSTAT activation of effector T cells when treated with 200nM IL-2. In some embodiments, using the regimen of Example 2, the IL-2 mutant protein or its Fc fusion protein also has a 10-day stability of greater than 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0040] In some embodiments, using the protocol of Example 1, the IL-2 mutant protein or its Fc fusion protein has greater than 30% pSTAT activation of regulatory T cells when treated with 1 nM IL-2. In some embodiments, using the protocol of Example 1, the IL-2 mutant protein or its Fc fusion protein has greater than 40% pSTAT activation of regulatory T cells when treated with 1 nM IL-2. In some embodiments, using the protocol of Example 1, the IL-2 mutant protein or its Fc fusion protein has greater than 50% pSTAT activation of regulatory T cells when treated with 1 nM IL-2. In some embodiments, using the protocol of Example 1, the IL-2 mutant protein or its Fc fusion protein has greater than 60% pSTAT activation of regulatory T cells when treated with 1 nM IL-2. In some embodiments, using the protocol of Example 1, the IL-2 mutant protein or its Fc fusion protein has greater than 70% pSTAT activation of regulatory T cells when treated with 1 nM IL-2. In some embodiments, using the regimen of Example 2, the IL-2 mutant protein or its Fc fusion protein also has a 10-day stability of greater than 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0041] In some embodiments, using the protocol of Example 1, the IL-2 mutant protein or its Fc fusion protein has greater than 30% pSTAT activation of regulatory T cells when treated with 200nM IL-2. In some embodiments, using the protocol of Example 1, the IL-2 mutant protein or its Fc fusion protein has greater than 40% pSTAT activation of regulatory T cells when treated with 200nM IL-2. In some embodiments, using the protocol of Example 1, the IL-2 mutant protein or its Fc fusion protein has greater than 50% pSTAT activation of regulatory T cells when treated with 200nM IL-2. In some embodiments, using the protocol of Example 1, the IL-2 mutant protein or its Fc fusion protein has greater than 60% pSTAT activation of regulatory T cells when treated with 200nM IL-2. In some embodiments, using the protocol of Example 1, the IL-2 mutant protein or its Fc fusion protein has greater than 70% pSTAT activation of regulatory T cells when treated with 200nM IL-2. In some embodiments, using the regimen of Example 2, the IL-2 mutant protein or its Fc fusion protein also has a 10-day stability of greater than 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0042] Examples of IL-2 mutant proteins include, but are not limited to, IL-2 mutant proteins comprising the following in the amino acid sequence set forth in SEQ ID NO:2: one or more of V91K, D20L; D84R, E61Q; V91K, D20A, E61Q, M104T; N88K, M104L; V91H, M104L; V91K, H16E, M104V; V91K, H16R, M104V; V91K, H16R, M104T; V91K, D20A, M104T; V91K, H16E, M104T; T; H16E, V91H, M104V; V91H, D20A, E61Q, M104T; V91H, H16R, E16Q; V91K, D 20A, M104V; H16E, V91H; V91H, D20A, M104V; H16E, V91H, M104T; H16E, V91H , E61Q, M104T; V91K, E61Q, H16E; V91K, H16R, M104L; H16E, V91H, E16Q; V91 K, E61Q, H16R; D20W, V91K, E61Q; V91H, H16R; V91K, H16R; D20W, V91K, E61Q , M104T; V91K, D20A; V91H, D20A, E16Q; V91K, D20A, M104L; V91H, D20A; V9 1K, E61Q, D20A; V91H, M104T; V91H, M104V; V91K, E61Q; V91K, N88K, E61Q, M 104T; V91K, N88K, E61Q; V91H, E61Q; V91K, N88K; D20A, H16E, M104T; D20A , M104T; H16E, N88K; D20A, M104V; D20A, M104L; H16E, M104T; H16E, M104V; N88K, M104V; N88K, E61Q; D20A, E61Q; H16R, D20A; D20W, E61Q; H16E, E61Q ;H16E, M104L; N88K, M104T; D20A, H16E; D20A, H16E, E16Q; D20A, H16R, E16 Q; V91K, D20W; V91A, H16A; V91A, H16D; V91A, H16E; V91A, H16S; V91E, H16A ;V91E, H16D; V91E, H16E; V91E, H16S; V91K, H16A; V91K, H16D; V91K, H16S;and / or V91S, H16E substitutions. The IL-2 mutant proteins of the present invention optionally comprise a C125A substitution. Although it may be advantageous to reduce the number of additional mutations of the wild-type IL-2 sequence, the present invention includes IL-2 mutant proteins that also include truncations and / or additional insertions, deletions, and / or substitutions in addition to the following: V91K, D20L; D84R, E61Q; V91K, D20A, E61Q, M104T; N88K, M104L; V91H, M104L; V91K, H16E, M104V; V91K, H16R, M104V; V91K, H16R, M104T; V91K, D20A, M104T; V91K, H16E, M104T; V91K, H16E, E61Q, M104T; V91K, H16R, E61Q, M104T; V91K, H16E; V91H, D20A, M104T; H16E, V9 1H, M104V; V91H, D20A, E61Q, M104T; V91H, H16R, E16Q; V91K, D20A, M104V ;H16E, V91H; V91H, D20A, M104V; H16E, V91H, M104T; H16E, V91H, E61Q, M1 04T; V91K, E61Q, H16E; V91K, H16R, M104L; H16E, V91H, E16Q; V91K, E61Q, H16R; D20W, V91K, E61Q; V91H, H16R; V91K, H16R; D20W, V91K, E61Q, M104 T; V91K, D20A; V91H, D20A, E16Q; V91K, D20A, M104L; V91H, D20A; V91K, E6 1Q, D20A; V91H, M104T; V91H, M104V; V91K, E61Q; V91K, N88K, E61Q, M104T ;V91K, N88K, E61Q; V91H, E61Q; V91K, N88K; D20A, H16E, M104T; D20A, M10 4T; H16E, N88K; D20A, M104V; D20A, M104L; H16E, M104T; H16E, M104V; N88 K, M104V; N88K, E61Q; D20A, E61Q; H16R, D20A; D20W, E61Q; H16E, E61Q; H1 6E, M104L; N88K, M104T; D20A, H16E; D20A, H16E, E16Q; D20A, H16R, E16Q; V91K, D20W; V91A, H16A; V91A, H16D; V91A, H16E; V91A, H16S; V91E, H16A;V91E, H16D; V91E, H16E; V91E, H16S; V91K, H16A; V91K, H16D; V91K, H16S; and / or V91S, H16E substitutions, provided that the mutant protein maintains activity that preferentially stimulates Tregs. Thus, embodiments include IL-2 mutant proteins that preferentially stimulate Treg cells and comprise an amino acid sequence having: V91K, D20L; D84R, E61Q; V91K, D20A, E61Q, M104T; N88K, M104L; V91H, M104L; V91K, H16E, M104V; V91K, H16R, M104V; V91K, H16R, M104T; V91K, D20A, M104T; V91K, H16E, M104T; V91K, H16E, E61Q, M104T; V91K, H16R, E 61Q, M104T; V91K, H16E; V91H, D20A, M104T; H16E, V91H, M104V; V91H, D20A, E61Q, M104T; V91H, H16R, E16Q; V91K, D20A, M104V; H16E, V91 H; V91H, D20A, M104V; H16E, V91H, M104T; H16E, V91H, E61Q, M104T; V91K, E61Q, H16E; V91K, H16R, M104L; H16E, V91H, E16Q; V91K, E61Q, H 16R; D20W, V91K, E61Q; V91H, H16R; V91K, H16R; D20W, V91K, E61Q, M104T; V91K, D20A; V91H, D20A, E16Q; V91K, D20A, M104L; V91H, D20A; V 91K, E61Q, D20A; V91H, M104T; V91H, M104V; V91K, E61Q; V91K, N88K, E61Q, M104T; V91K, N88K, E61Q; V91H, E61Q; V91K, N88K; D20A, H16E, M104T; D20A, M104T; H16E, N88K; D20A, M104V; D20A, M104L; H16E, M104T; H16E, M104V; N88K, M104V; N88K, E61Q; D20A, E61Q; H16R, D20A; D20W, E61Q; H16E, E61Q; H16E, M104L; N88K, M104T; D20A, H16E; D20A, H16E, E16Q; D20A, H16R, E16Q; V91K, D20W; V91A, H16A; V91A, H16D;V91A, H16E; V91A, H16S; V91E, H16A; V91E, H16D; V91E, H16E; V91E, H16S; V91K, H16A; V91K, H16D; V91K, H16S; and / or V91S, H16E substitutions, and having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence listed in SEQ ID NO:2. In particularly preferred embodiments, such IL-2 mutant proteins comprise an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 2. ;

[0043] For amino acid sequences, sequence identity and / or similarity is determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search by similarity method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. USA 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Avenue, Madison, Wisconsin), and the like. Drive, Madison, Wis.), Devereux et al., 1984, Nucl. Acid Res. [Nucleic Acids Research] 12: 387-395 described best match sequence program, preferably using default settings, or by inspection. Preferably, the identity percentage is calculated by FastDB based on the following parameters: mismatch penalty is 1; gap penalty is 1; gap size penalty is 0.33; and connection penalty is 30, "Current Methods in Sequence Comparison and Analysis [Current Methods in Sequence Comparison and Analysis]", Macromolecule Sequencing and Synthesis [Macromolecule Sequencing and Synthesis], Selected Methods and Applications [Selected Methods and Applications], pp. 127-149 (1988), Alan R. Liss Company.

[0044] An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a set of related sequences using progressive pairwise alignments. It can also draw dendrites showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle, 1987, J. Mol. Evol. [Journal of Molecular Evolution] 35:351-360; the method is similar to that described by Higgins and Sharp, 1989, CABIOS 5:151-153. Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

[0045] Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402; and Karin et al., 1993, Proc. Natl. Acad. Sci. USA 90:5873-5787. A particularly useful BLAST program is the WU-BLAST-2 program, which is available from Altschul et al., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses a number of search parameters, most of which are set to default values. The adjustable parameters are set to the following values: overlap interval = 1, overlap fraction = 0.125, word threshold (T) = II. The HSP S and HSP S2 parameters are dynamic values ​​and are established by the program itself based on the composition of the particular sequence and the composition of the particular database against which the sequence of interest is being searched; however, these values ​​can be adjusted to increase sensitivity.

[0046] Another useful algorithm is Gap BLAST reported by Altschul et al., 1993, Nucl. Acids Res. [Nucleic Acids Research] 25:3389-3402. Gap BLAST uses BLOSUM-62 instead of scoring; the threshold T parameter is set to 9; the double-click method triggers non-gap extension, and a cost of 10+k is charged for a gap length of k; X u Set to 16, and X g Set to 40 (for the database search phase) and 67 (for the output phase of the algorithm). Gapped alignments are triggered by a score corresponding to approximately position 22.

[0047] Although the site or region for introducing amino acid sequence variation can be predetermined, the mutation itself does not need to be predetermined. For example, in order to optimize the performance of the mutation at a given site, random mutagenesis can be carried out at the target codon or region, and the IL-2 mutant protein expressed can be screened for the best combination of desired activity. The technology of carrying out substitution mutation at a predetermined site in a DNA with a known sequence is well known, such as M13 primer mutagenesis and PCR mutagenesis. The screening of mutants can be carried out using the assay described herein, for example.

[0048] Amino acid substitutions are typically single residue substitutions; insertions will generally be on the order of from about one (1) to about twenty (20) amino acid residues, although significantly larger insertions can be tolerated. Deletions range from about one (1) to about twenty (20) amino acid residues, although in some cases, deletions can be much larger.

[0049] Replacement, deletion, insertion or any combination thereof can be used to realize final derivative or variant.Usually these changes are carried out on a minority of amino acids, thereby the change of molecule, particularly the immunogenicity and specificity of antigen-binding proteins are minimized.However, in some cases, larger changes can be tolerated.Usually conservative substitutions are carried out according to the following chart as described in Table 1.

[0050] Table 1

[0051]

[0052] Substantial changes in function or immunological properties are caused by selecting substitutions that are less conservative than those shown in Table 1. For example, substitutions that have a more significant effect on the structure of the polypeptide backbone (e.g., alpha helical or beta-sheet structure) in the altered region; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain. Substitutions that are generally expected to produce the greatest changes in polypeptide properties are those in which (a) a hydrophilic residue, such as seryl or threonyl, is substituted with (or is substituted with) a hydrophobic residue, such as leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) cysteine ​​or proline is substituted with (or is substituted with) any other residue; (c) a residue with a positively charged side chain (e.g., lysyl, arginyl, or histidyl) is substituted with (or is substituted with) a negatively charged residue, such as glutamyl or aspartyl; or (d) a residue with a bulky side chain (e.g., phenylalanine) is substituted with (or is substituted with) a residue without a side chain, such as glycine.

[0053] Compared to naturally occurring analogs, these variants typically exhibit the same qualitative biological activity and will elicit the same immune response, although variants are also selected to modify the characteristics of the IL-2 mutant protein as desired. Alternatively, variants can be designed so that the biological activity of the IL-2 mutant protein is altered. For example, as discussed herein, glycosylation sites can be changed or removed.

[0054] IL-2 mutant proteins with extended serum half-life

[0055] Because the IL-2 muteins provided herein preferentially expand Tregs relative to, for example, Teff or NK cells, it is expected that the safety profile will be different from that of wild-type IL-2 or (Aldesleukin; Novartis, Basel, Switzerland). Related side effects include flu-like symptoms, feeling cold / chills, joint pain, fever, rash, itching, injection site reactions, hypotension, diarrhea, nausea, anxiety, mental confusion, and depression. The IL-2 mutant proteins provided herein can also be altered to include or fused to molecules that extend the serum half-life of the mutant protein without increasing the risk that such half-life extension will increase the likelihood or intensity of side effects in patients. Subcutaneous administration of such a mutant protein with an extended serum half-life can allow for lower systemic maximum exposure (C 最大 ) in the absence of a mutated protein. The extended serum half-life may allow for a lower or less frequent dosing regimen of the mutant protein.

[0056] The serum half-life of the IL-2 mutant proteins provided herein can be extended by any method known in the art substantially. Such methods include changing the sequence of the IL-2 mutant proteins to include binding to neonatal Fcγ receptors or binding to a peptide of a protein (e.g., IgG or human serum albumin) with a prolonged serum half-life. In other embodiments, the IL-2 mutant proteins are fused to a polypeptide that grants a half-life that is prolonged by the fusion molecule. Such polypeptides include IgG Fc or other polypeptides that bind neonatal Fcγ receptors, human serum albumin, or a polypeptide that binds a protein with a prolonged serum half-life. In a preferred embodiment, the IL-2 mutant proteins are fused to an IgG Fc molecule.

[0057] The IL-2 mutein can be fused to the N-terminus or C-terminus of the IgG Fc region. As shown in the Examples, fusion to the C-terminus of the IgG Fc region maintains IL-2 mutein activity to a greater extent than when fused to the N-terminus of IgG Fc.

[0058] One embodiment of the present invention is directed to a dimer comprising two Fc fusion polypeptides produced by fusing an IL-2 mutant protein to the Fc region of an antibody. For example, the dimer can be prepared by inserting a gene fusion encoding the fusion protein into an appropriate expression vector, expressing the gene fusion in a host cell transformed with the recombinant expression vector, and allowing the expressed fusion protein to assemble much like an antibody molecule, thereby forming an interchain bond between the Fc portions to produce a dimer.

[0059] As used herein, the term "Fc polypeptide" or "Fc region" includes native and mutant forms of polypeptides derived from the Fc region of an antibody, and may be part of an IL-2 mutant protein fusion protein or an anti-IL-2 antibody of the present invention. Such polypeptides in truncated forms containing hinge regions that promote dimerization are also included. In certain embodiments, the Fc region comprises antibody CH2 and CH3 domains. Along with an extended serum half-life, fusion proteins comprising the Fc portion (and oligomers formed therefrom) provide the advantage of simple purification by affinity chromatography on a protein A or protein G column. A preferred Fc region is derived from human IgG, which includes IgG1, IgG2, IgG3, and IgG4. Herein, specific residues within Fc are identified by position. All Fc positions are based on the EU numbering scheme.

[0060] One of the functions of the Fc portion of an antibody is to communicate with the immune system when the antibody binds to its target. This is considered an "effector function". Communication leads to antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). ADCC and ADCP are mediated by Fc binding to Fc receptors on the surface of immune system cells. CDC is mediated by binding of Fc to proteins of the complement system, such as C1q.

[0061] IgG subclasses differ in their ability to mediate effector functions. For example, IgG1 is far superior to IgG2 and IgG4 in mediating ADCC and CDC. Therefore, in embodiments where effector functions are not desired, IgG2 Fc would be preferred. However, it is known that IgG2 Fc-containing molecules are more difficult to manufacture and have less attractive properties, such as a shorter half-life, than IgG1 Fc-containing molecules.

[0062] By introducing one or more mutations into the Fc, the effector function of the antibody can be increased or decreased. Embodiments of the present invention include IL-2 mutant protein Fc fusion proteins with Fc engineered to increase effector function (US 7,317,091 and Strohl, Curr. Opin. Biotech. [Biotech Review], 20:685-691, 2009; both of which are incorporated herein by reference in their entirety). Exemplary IgG1 with increased effector function Fc molecules include those with the following substitutions: S239D / I332E; S239D / A330S / I332E; S239D / A330L / I332E; S298A / D333A / K334A; P247I / A339D; P247I / A339Q; D280H / K290S; D280H / K290S / S298D; D280H / K290S / S298V; F243L / R292P / Y300L; F243L / R292P / Y300L / P396L; F243L / R292P / Y300L / V305I / P396L; G236A / S239D / I332E; K326A / E333A; K326W / E333S; K290E / S298G / T299A; K290N / S298G / T299A; K290E / S298G / T299A / K326E; and / or K290N / S298G / T299A / K326E.

[0063] Another method of increasing the effector function of IgG Fc protein is by reducing the fucosylation of Fc. Removing core fucose from the biantennary complex oligosaccharide attached to Fc increases ADCC effector function without changing antigen binding or CDC effector function. Several methods are known to reduce or eliminate the fucosylation of Fc molecules (such as antibodies). These include recombinant expression in certain mammalian cell lines, including FUT8 knockout cell lines, variant CHO cell lines Lec13, rat hybridoma cell lines YB2 / 0, cell lines containing small interfering RNA specific for FUT8 genes, and cell lines co-expressing β-1,4-N-acetylglucosamine transferase III and Golgi α-mannosidase II. Alternatively, Fc molecules can be expressed in non-mammalian cells (such as plant cells, yeast, or prokaryotic cells, such as Escherichia coli).

[0064] In certain embodiments, the IL-2 mutein Fc fusion protein or anti-IL-2 antibody of the invention comprises an Fc engineered to reduce effector function. Exemplary Fc molecules with reduced effector function include those with the following substitutions: N297A or N297Q (IgG1); L234A / L235A (IgG1); V234A / G237A (IgG2); L235A / G237A / E318A (IgG4); H268Q / V309L / A330S / A331S (IgG2); C220S / C226S / C229S / P238S (IgG1); C226S / C229S / E233P / L234V / L235A (IgG1); L234F / L235E / P331S (IgG1); and / or S267E / L328F (IgG1).

[0065] It is known that human IgG1 has a glycosylation site at N297 (EU numbering system), and glycosylation contributes to the effector function of IgG1 antibodies. An exemplary IgG1 sequence is provided in SEQ ID NO: 3:

[0066]

[0067] In an effort to make deglycosylated antibodies, several groups have mutated N297. The mutations have focused on replacing N297 with an amino acid with physiochemical properties similar to asparagine, such as glutamine (N297Q) or alanine (N297A), which mimic asparagine without a polar group.

[0068] As used herein, "deglycosylated antibody" or "deglycosylated Fc" refers to the glycosylation state of the residue at position 297 of Fc. An antibody or other molecule may contain glycosylation at one or more other positions but may still be considered a deglycosylated antibody or a deglycosylated Fc fusion protein.

[0069] In an effort to prepare an IgG1 Fc without effector function, it was found that mutating the amino acid N297 of human IgG1 to glycine, i.e., N297G, provided far superior purification efficacy and biophysical properties than other amino acid substitutions at this residue. See Example 8. Therefore, in a preferred embodiment, the IL-2 mutant protein Fc fusion protein comprises a human IgG1 Fc having an N297G substitution. The Fc comprising the N297G substitution is useful in any context in which the molecule comprises a human IgG1 Fc, and is not limited to use in the context of an IL-2 mutant protein Fc fusion. In certain embodiments, the antibody comprises an Fc having an N297G substitution.

[0070] The Fc comprising a human IgG1 Fc with a N297G mutation may also comprise additional insertions, deletions, and substitutions. In certain embodiments, the human IgG1 Fc comprises an N297G substitution and has at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the amino acid sequence listed in SEQ ID NO:3. In a particularly preferred embodiment, the C-terminal lysine residue is substituted or deleted. The amino acid sequence of a human IgG1 comprising an N297G substitution and a C-terminal lysine deletion is listed in SEQ ID NO:4.

[0071] It is shown that compared with glycosylated IgG1 Fc molecules, glycosylated IgG1 Fc molecules are more unstable. The Fc region can be further engineered to increase the stability of deglycosylated molecules. In certain embodiments, one or more amino acid residues are substituted with cysteine ​​to form disulfide bonds in a dimer state. Cysteine ​​can be used to replace the residues V259, A287, R292, V302, L306, V323, or I332 of the amino acid sequence listed in SEQ ID NO:3. In a preferred embodiment, the specific residue pair is the following substitution, which makes it preferentially form disulfide bonds with each other, thereby limiting or preventing disulfide bond confusion. Preferred pairs include, but are not limited to, A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C.

[0072] Provided herein are Fc-containing molecules wherein one or more of residues V259, A287, R292, V302, L306, V323, or I332 are substituted with cysteine, examples of which include those comprising A287C and L306C, V259C and L306C, R292C and V302C, or V323C and I332C substitutions.

[0073] Additional mutations that can be applied to IgG1 Fc include those that promote heterodimer formation in Fc-containing polypeptides. In some embodiments, the Fc region is engineered to produce a "knob" and a "hole" that, when co-expressed in a cell, promote heterodimer formation of two different Fc-containing polypeptide chains. US7,695,963. In other embodiments, the Fc region is altered to promote heterodimer formation of two different Fc-containing polypeptides when co-expressed in a cell using electrostatic manipulation while preventing homodimer formation. WO 09 / 089,004, which is incorporated herein by reference in its entirety. Preferred heterodimeric Fcs include those in which one chain of the Fc comprises D399K and E356K substitutions and the other chain of the Fc comprises K409D and K392D substitutions. In other embodiments, one chain of the Fc comprises D399K, E356K, and E357K substitutions and the other chain of the Fc comprises K409D, K392D, and K370D substitutions.

[0074] In certain embodiments, it may be advantageous that the IL-2 mutant protein Fc fusion protein is monomeric, i.e., contains only a single IL-2 mutant protein molecule. Similarly, it may be desirable to specifically bind to one or more other targets of the double, three or four specific antibodies. In such embodiments, the Fc region of the fusion protein or antibody may contain one or more mutations that promote heterodimer formation. The fusion protein or antibody is co-expressed with the Fc region, and the Fc region has the reciprocal mutations (reciprocal mutation) of those mutations in the IL-2 mutant protein Fc fusion polypeptide, but lacks IL-2 mutant protein or anti-IL-2 heavy chain variable domains. When two heterodimers containing Fc polypeptides are formed, the resulting protein only contains a single IL-2 mutant protein or anti-IL-2 binding domain.

[0075] Another method of producing monomeric IL-2 mutant protein Fc fusion protein is to fuse IL-2 mutant protein to monomeric Fc, i.e., Fc region that does not dimerize. Suitable monomeric Fc includes mutations that prevent dimerization and stabilize the molecule in monomeric form. Preferred monomeric Fc is disclosed in WO2011 / 063348, which is incorporated herein by reference in its entirety. In certain embodiments, IL-2 mutant protein Fc fusion protein includes Fc, and the Fc includes negatively charged amino acids at positions 392 and 409, together with threonine substitutions at Y349, L351, L368, V397, L398, F405, or Y407.

[0076] In certain embodiments, the IL-2 mutant protein Fc fusion protein is included in a linker between Fc and IL-2 mutant protein. Many different linker polypeptides are known in the art, and these linker polypeptides can be used in the context of IL-2 mutant protein Fc fusion protein. In a preferred embodiment, the IL-2 mutant protein Fc fusion protein is included in one or more copies of a peptide consisting of the following between Fc and IL-2 mutant protein: GGGGS (SEQ ID NO: 5), GGNGT (SEQ ID NO: 6), or YGNGT (SEQ ID NO: 7). In some embodiments, the polypeptide region between the Fc region and the IL-2 mutant protein includes a single copy of the following: GGGGS (SEQ ID NO: 5), GGNGT (SEQ ID NO: 6), or YGNGT (SEQ ID NO: 7). As shown herein, when expressed in appropriate cells, the linker GGNGT (SEQ ID NO: 6) or YGNGT (SEQ ID NO: 7) is glycosylated, and such glycosylation can help stabilize the protein in solution and / or when administered in vivo. Thus, in certain embodiments, the IL-2 mutein fusion protein comprises a glycosylation linker between the Fc region and the IL-2 mutein region.

[0077] It is expected that when placed in the context of a polypeptide, a glycosylation linker will be useful. Provided herein are polypeptides comprising GGNGT (SEQ ID NO: 6) or YGNGT (SEQ ID NO: 7), wherein the GGNGT (SEQ ID NO: 6) or YGNGT (SEQ ID NO: 7) is inserted into the amino acid sequence of the polypeptide or replaces one or more amino acids within the amino acid sequence of the polypeptide. In a preferred embodiment, GGNGT (SEQ ID NO: 6) or YGNGT (SEQ ID NO: 7) is inserted into a loop of the tertiary structure of the polypeptide. In other embodiments, one or more amino acids of the loop are replaced with GGNGT (SEQ ID NO: 6) or YGNGT (SEQ ID NO: 7).

[0078] The C-terminal portion of Fc and / or the amino-terminal portion of IL-2 mutant protein may contain one or more mutations that change the glycosylation profile of IL-2 mutant protein Fc fusion protein when expressed in mammalian cells. In certain embodiments, IL-2 mutant protein further comprises T3 substitution, such as T3N or T3A. IL-2 mutant protein may further comprise S5 substitution, such as S5T.

[0079] Covalent modifications of IL-2 mutant proteins and IL-2 mutant protein Fc fusion proteins and anti-IL-2 antibodies are included within the scope of the present invention and are usually, but not always, performed post-translationally. For example, several types of covalent modifications are introduced into the molecule by reacting certain amino acid residues of the molecule with an organic derivatizing agent that is capable of reacting with selected side chains or N or C terminal residues.

[0080] Cysteinyl residues are most commonly reacted with α-haloacetates (and corresponding amines) such as chloroacetic acid or chloroacetamide to give carboxymethyl or carboxamidomethyl derivatives. Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidazolyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuryl benzoate, 2-chloromercuryl-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0081] Histidyl residues are derivatized by reaction with diethyl pyrocarbonate at pH 5.5-7.0, as this reagent is relatively specific for histidyl side chains. p-Bromophenacyl bromide is also useful; the reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0.

[0082] Lysyl and amino terminal residues react with succinic acid or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysyl residues. Other suitable agents for derivatizing residues containing α-amino groups include imidoesters such as methyl pyridinium imide; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactions with glyoxylate.

[0083] Arginyl residues are modified by reaction with one or more conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. a High, therefore, derivatization of arginine residues requires the reaction to be carried out under alkaline conditions. In addition, these reagents can react with lysine groups as well as with the arginine ε-amino group.

[0084] Specific modifications of tyrosyl residues can be performed, with particular interest in the introduction of spectral labels into tyrosyl residues by reaction with aromatic diazo compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to form o-acetyl tyrosyl species and 3-nitro derivatives, respectively. 125 I or 131 I iodinate tyrosyl residues to prepare labeled proteins for radioimmunoassay; the chloramine T method described above is suitable.

[0085] The carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimide (R'-N=C=N--R'), wherein R and R' are optionally different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. In addition, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0086] Derivatization with bifunctional agents can be used to crosslink antigen binding proteins to water-insoluble carrier matrices or surfaces for use in a variety of methods. Commonly used crosslinking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters (e.g., esters with 4-azidosalicylic acid), homobifunctional imidoesters (including disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl propionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane). Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propionimide ester produce photoactivatable intermediates that are able to form crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide activated carbohydrates and reactive substrates described in US Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.

[0087] Glutaminyl and asparaginyl residues are usually deamidated to corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mild acidic conditions. Any form of these residues falls within the scope of the present invention.

[0088] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, WH Freeman Company, San Francisco, 1983, pp. 79-86), acetylation of the N-terminal amine and amidation of any C-terminal carboxyl group.

[0089] Another type of covalent modification of IL-2 mutant proteins, IL-2 mutant protein Fc fusions, or anti-IL-2 antibodies included within the scope of the present invention includes changing the glycosylation pattern of the protein. As is known in the art, the glycosylation pattern may depend on both the sequence of the protein (e.g., the presence or absence of specific glycosylated amino acid residues discussed below) or the host cell or organism in which the protein is produced. Specific expression systems are discussed below.

[0090] Glycosylation of polypeptides is usually N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0091] Addition of glycosylation sites to an IL-2 mutant protein, IL-2 mutant protein Fc fusion, or anti-IL-2 antibody can be conveniently accomplished by altering the amino acid sequence to contain one or more of the above tripeptide sequences (for N-linked glycosylation sites). The alteration can also be made by adding one or more serine or threonine residues to the starting sequence or replacing them with one or more serine or threonine residues (for O-linked glycosylation sites). For convenience, the IL-2 mutant protein, IL-2 mutant protein Fc fusion, or anti-IL-2 antibody amino acid sequence is preferably altered by changes at the DNA level, particularly by mutating the DNA encoding the target polypeptide at preselected bases so that codons are generated that will translate into the desired amino acids.

[0092] Another means of increasing the number of carbohydrate moieties on an IL-2 mutant protein, IL-2 mutant protein Fc fusion, or anti-IL-2 antibody is by chemically or enzymatically coupling glycosides to the protein. These methods are advantageous because they do not require the production of proteins in host cells with glycosylation capabilities for N- and O-linked glycosylation. Depending on the coupling method used, one or more sugars may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as those of cysteine, (d) free hydroxyl groups, such as those of serine, threonine or hydroxyproline, (e) aromatic residues, such as those of phenylalanine, tyrosine or tryptophan, or (f) amide groups of glutamine. These methods are described in WO 87 / 05330 published on September 11, 1987, and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem. [CRC Critical Review of Biochemistry], pages 259-306.

[0093] Removal of carbohydrate moieties present on the starting IL-2 mutant protein, IL-2 mutant protein Fc fusion, or anti-IL-2 antibody can be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid or an equivalent compound. The treatment results in the cleavage of most or all sugars except the linked sugar (N-acetylglucosamine or N-acetylgalactosamine) while leaving the polypeptide intact. Chemical deglycosylation is described by Hakimuddin et al., 1987, Arch. Biochem. Biophys. [Biochemistry and Biophysics] 259: 52 and Edge et al., 1981, Anal. Biochem. [Analytical Biochemistry] 118: 131. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by using a variety of endoglycosidases and exoglycosidases, such as described by Thotakura et al., 1987, Meth. Enzymol. [Enzymology Methods] 138: 350. Glycosylation at potential glycosylation sites can be prevented by using the compound tunicamycin, as described by Duskin et al., 1982, J. Biol. Chem. 257: 3105. Tunicamycin blocks the formation of protein-N-glycosidic linkages.

[0094] For example, another type of covalent modification of IL-2 mutant protein, IL-2 mutant protein Fc fusion, or anti-IL-2 antibody includes connecting the protein to various non-protein polymers in a manner described in U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337, including but not limited to various polyols, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene. In addition, amino acid substitutions can be made at different positions within IL-2 mutant protein, IL-2 mutant protein Fc fusion, or anti-IL-2 antibody to facilitate the addition of polymers (e.g., PEG). Therefore, embodiments of the present invention include PEGylated IL-2 mutant protein, IL-2 mutant protein Fc fusion, or anti-IL-2 antibody. Such PEGylated proteins can have increased half-life and / or reduced immunogenicity compared to their non-PEGylated forms.

[0095] Polynucleotides encoding IL-2 mutant protein and IL-2 mutant protein Fc fusion protein

[0096] The invention encompasses nucleic acids encoding IL-2 muteins, IL-2 mutein Fc fusions, or anti-IL-2 antibodies.Aspects of the invention include polynucleotide variants (eg, due to degeneracy) encoding the amino acid sequences described herein.

[0097] The nucleotide sequence corresponding to the amino acid sequence described herein can be obtained by "back translation" from the amino acid sequence, which is used as a probe or primer for isolating nucleic acid, or as a query sequence for database retrieval. The DNA sequence encoding IL-2 mutant protein and IL-2 mutant protein Fc fusion protein can be separated and amplified using a well-known polymerase chain reaction (PCR) program. Oligonucleotides defining the desired ends of the combination of DNA fragments are used as 5' and 3' primers. The oligonucleotides may additionally contain a recognition site for a restriction endonuclease to facilitate the insertion of the amplified combination of DNA fragments into an expression vector. PCR technology is described in Saiki et al., Science, 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds., Academic Press, Inc., San Diego (1989), pp. 189-196, and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press, Inc. (1990).

[0098] Nucleic acid molecules of the present invention include DNA and RNA in single-stranded and double-stranded forms, and corresponding complementary sequences. "Isolated nucleic acid" refers to nucleic acid separated from adjacent gene sequences present in the genome of an organism from which nucleic acid is isolated, in the case of nucleic acids isolated from naturally occurring sources. In the case of nucleic acids synthesized by template enzymatic method or chemically synthesized, such as PCR products, cDNA molecules or oligonucleotides, it should be understood that the nucleic acid obtained by such methods is an isolated nucleic acid. Isolation nucleic acid molecules refer to nucleic acid molecules in the form of independent fragments or as a component of a larger nucleic acid construct. In a preferred embodiment, nucleic acid is substantially free of contaminating endogenous substances. The nucleic acid molecules are preferably derived from substantially pure forms and can utilize standard biochemical methods (such as Sambrook et al., Molecular Cloning: A Laboratory Manual [Molecular Cloning: Laboratory Manual], 2nd edition, Cold Spring Harbor Laboratory [Cold Spring Harbor Laboratory], Cold Spring Harbor [Cold Spring Harbor], NY [New York] (1989) outlined in the method) to identify, operate and reclaim the amount or concentration of its component nucleotide sequences to separate at least once DNA or RNA. Such sequences are preferably provided and / or constructed in the form of an open reading frame that is free of internal non-translated sequences or introns that are typically present in eukaryotic genes. Non-translated DNA sequences may be present 5' or 3' to the open reading frame, where these sequences do not interfere with the manipulation or expression of the coding region.

[0099] The IL-2 mutant protein according to the present invention is generally prepared by: using cassette or PCR mutagenesis, or other techniques well known in the art, site-specific mutagenesis of nucleotides in the DNA encoding the IL-2 mutant protein or IL-2 mutant protein Fc fusion protein to produce DNA encoding the variant, and then expressing the recombinant DNA in cell culture as outlined herein. However, IL-2 mutant proteins and IL-2 mutant protein Fc fusions can be prepared by in vitro synthesis using established techniques. These variants typically exhibit the same biological activity as naturally occurring analogs, such as Treg expansion, although variants with improved features as will be described more fully below can also be selected.

[0100] As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, each IL-2 mutant protein, IL-2 mutant protein Fc fusion, and anti-IL-2 antibody of the present invention is encoded by a very large number of nucleic acids, each of which is within the scope of the present invention and can be prepared using standard techniques. Therefore, by simply modifying one or more codon sequences to identify a specific amino acid sequence in a manner that does not change the amino acid sequence of the encoded protein, a person of ordinary skill in the art can prepare a variety of different nucleic acids.

[0101] The present invention also provides expression systems and constructs in the form of plasmids, expression vectors, transcription or expression cassettes, which comprise at least one polynucleotide as above. In addition, the present invention provides host cells comprising such expression systems or constructs.

[0102] Typically, expression vectors used in any host cell will contain sequences for plastid maintenance and for cloning and expressing exogenous nucleotide sequences. In certain embodiments, collectively referred to as "flanking sequences", typically will include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a selectable marker component. These sequences are discussed separately below.

[0103] Optionally, the vector may contain a "tag" coding sequence, i.e., an oligonucleotide molecule located at the 5' or 3' end of the IL-2 mutein, IL-2 mutein Fc fusion, or anti-IL-2 antibody coding sequence; the oligonucleotide sequence encodes poly-His (e.g., hexameric His (SEQ ID NO: 8)), or another "tag" for which there is a commercially available antibody, such as FLAG, HA (hemagglutinin influenza virus), or myc. This tag is typically fused to the antibody when the polypeptide is expressed, and can be used as a means of affinity purification or detection from host cells. Affinity purification can be achieved, for example, by column chromatography, using an antibody to the tag as an affinity matrix. Optionally, the tag can then be removed by various means, such as cleavage using certain peptidases.

[0104] The flanking sequences may be homologous (i.e., from the same species and / or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), synthetic, or natural. Thus, the source of the flanking sequences may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, as long as the flanking sequences function in and can be activated by the host cell machinery.

[0105] The flanking sequences that can be used in the vectors of the present invention can be obtained by any of several methods well known in the art. Typically, the flanking sequences that can be used herein will be identified in advance by positioning and / or by restriction endonuclease digestion and therefore can be separated from appropriate tissue sources using appropriate restriction endonucleases. In some cases, the complete nucleotide sequence of the flanking sequence may be known. At this, the flanking sequences can be synthesized using the method described herein for nucleic acid synthesis or cloning.

[0106] Whether all or only a portion of the flanking sequence is known, it can be obtained using the polymerase chain reaction (PCR) and / or by screening a genomic library with suitable probes, such as oligonucleotides and / or flanking sequence fragments from the same or another species. If the flanking sequence is unknown, the DNA fragment containing the flanking sequence can be isolated from a larger DNA fragment that may contain, for example, the coding sequence or even another gene or genes. Isolation can be achieved by restriction endonuclease digestion to produce the appropriate DNA fragments, followed by agarose gel purification, Separation is performed by column chromatography (Chatsworth, CA) or other methods known to the skilled artisan. The selection of suitable enzymes to achieve this purpose will be readily apparent to one of ordinary skill in the art.

[0107] The replication origin is typically a part of a commercially available prokaryotic expression vector, and the starting point helps to amplify the vector in the host cell. If the selected vector does not contain a replication origin, it can be chemically synthesized based on a known sequence, and connected to the vector. For example, the replication origin from plasmid pBR322 (New England Biolabs, Beverly, Massachusetts (MA)) is applicable to most gram-negative bacteria, and various viral starting points (such as SV40, polyoma virus, adenovirus, vesicular stomatitis virus (VSV), or papillomavirus (such as HPV or BPV)) can be used for cloning vectors in mammalian cells. Generally speaking, mammalian expression vectors do not need a replication origin component (for example, usually only using the SV40 starting point, because it also contains a viral early promoter).

[0108] The transcription termination sequence is typically located at the 3' end of the polypeptide coding region and is used to terminate transcription. Usually, the transcription termination sequence in prokaryotes is a GC-rich fragment followed by a poly-T sequence. Although the sequence can be easily cloned from a library or even purchased from the market as part of a vector, it can also be easily synthesized using nucleic acid synthesis methods such as those described herein.

[0109] Selectable marker genes encode proteins required for survival and growth of host cells grown in selective media. Typical selectable marker genes encode proteins that (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells); (b) supplement nutritional deficiencies of cells; or (c) provide important nutrients that are not available from complex media or defined media. Specific selectable markers are kanamycin resistance genes, ampicillin resistance genes, and tetracycline resistance genes. Advantageously, the neomycin resistance gene can also be used to select in both prokaryotic and eukaryotic host cells.

[0110] Other selectable genes can be used to amplify the genes to be expressed. Amplification is a process in which genes required for the production of proteins that are important for growth or cell survival are repeated in tandem within the chromosomes of successive generations of recombinant cells. Examples of suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and promoterless thymidine kinase genes. Mammalian cell transformants are placed under selection pressure, wherein only the transformants are uniquely suitable for survival due to the presence of selectable genes in the vector. The selection pressure is applied by culturing the transformed cells under conditions of continuously increasing the concentration of the selector in the culture medium, thereby amplifying both the selectable gene and the gene encoding the desired polypeptide (e.g., IL-2 mutant protein, IL-2 mutant protein Fc fusion, or heavy and / or light chain of an anti-IL-2 antibody). Therefore, a larger amount of polypeptide is synthesized from the amplified DNA.

[0111] Ribosome binding sites are usually necessary for the initiation of mRNA translation and are characterized by Shine-Dalgarno sequences (prokaryotes) or Kozak sequences (eukaryotes). This component is typically located at the 3' end of the promoter and at the 5' end of the polypeptide coding sequence to be expressed. In certain embodiments, one or more coding regions can be operably linked to an internal ribosome binding site (IRES), thereby allowing translation of two open reading frames from a single RNA transcript.

[0112] In some cases, such as when glycosylation is needed in a eukaryotic host cell expression system, various pre-sequences or pro-sequences can be operated to improve glycosylation or yield. For example, the peptidase cleavage site of a specific signal peptide can be changed, or a pro-sequence can be added, which can also affect glycosylation. The final protein product can have one or more other amino acids that are easy to express at position-1 (relative to the first amino acid of the mature protein), which may not be completely removed. For example, the final protein product may have one or two amino acid residues found in the peptidase cleavage site attached to the amino terminal. Alternatively, when the enzyme is cut in such regions in the mature polypeptide, some enzyme cleavage sites may be used to produce a slightly truncated form of the desired polypeptide.

[0113] The expression and cloning vectors of the present invention typically contain a promoter that is recognized by the host organism and operably connected to a molecule encoding the heavy chain and / or light chain of an IL-2 mutant protein, an IL-2 mutant protein Fc fusion, or an anti-IL-2 antibody. A promoter is a non-transcribed sequence located upstream (i.e., 5') of the start codon of a structural gene that controls transcription of the structural gene (generally within about 100 to 1000 bp). Promoters are generally grouped into one of two categories: an inducible promoter and a constitutive promoter. An inducible promoter initiates transcription of the DNA under its control at an increased level in response to a certain change in culture conditions (such as the presence or absence of nutrients, or temperature changes). On the other hand, a constitutive promoter consistently transcribes the gene to which it is operably connected, i.e., has minimal or no control over gene expression. Many promoters recognized by a variety of potential host cells are well known.

[0114] Suitable promoters for yeast hosts are also well known in the art. Yeast enhancers are preferably used with yeast promoters. Suitable promoters for mammalian host cells are well known and include, but are not limited to, those obtained from viral genomes such as polyoma virus, transmissible epithelioma virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and most preferably simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and actin promoters.

[0115] Other promoters that may be of interest include, but are not limited to, the SV40 early promoter (Benoist and Chambon, 1981, Nature 290:304-310); the CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad. USA 81:659-663); the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797); the herpes virus thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sc i. USA [Proceedings of the National Academy of Sciences of the United States of America] 78: 1444-1445); promoter and regulatory sequences from metallothionein genes (Prinster et al., 1982, Nature 296: 39-42); and prokaryotic promoters, such as the β-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 75: 3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 80: 21-25). Also of interest are the following animal transcriptional control regions that show tissue specificity and have been used in transgenic animals: the elastase I gene control region active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Laboratory Press, 1996). Symp. Quant. Biol. [Cold Spring Harbor Symposium on Quantitative Biology] 50:399-409; MacDonald, 1987, Hepatology [Hepatology] 7:425-515); the insulin gene control region active in pancreatic β cells (Hanahan, 1985, Nature [Nature] 315:115-122); the immunoglobulin gene control region active in lymphoid cells (Grosschedl et al., 1984, Cell [Cell] 38:647-658; Adames et al., 1985, Nature [Nature] 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.[Molecular Cell Biol.] 7:1436-1444); the control region of the mouse mammary tumor virus, which is active in testicular cells, mammary cells, lymphoid cells, and mast cells (Leder et al., 1986, Cell 45:485-495); the control region of the albumin gene, which is active in the liver (Pinkert et al., 1987, Genes and Development 1:268-276); the control region of the α-fetoprotein gene, which is active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 253:53-58); the control region of the α1-antitrypsin gene, which is active in the liver (Kelsey et al., 1987, Genes and Development 1:268-276). Devel. [Genes and Development] 1: 161-171); the beta-globin gene control region active in myeloid cells (Mogram et al., 1985, Nature 315: 338-340; Kollias et al., 1986, Cell 46: 89-94); the myelin basic protein gene control region active in oligodendrocytes of the brain (Readhead et al., 1987, Cell 48: 703-712); the myosin light chain-2 gene control region active in skeletal muscle (Sani, 1985, Nature 314: 283-286); and the gonadotropin-releasing hormone gene control region active in the hypothalamus (Mason et al., 1986, Science 234: 1372-1378). .

[0116] Enhancer sequences can be inserted into the vector to increase transcription of higher eukaryotic organisms. Enhancers are cis-acting elements of DNA, and lengths are generally about 10-300bp, acting on promoters to increase transcription. Enhancers are relatively independent in direction and position, and have been found in 5' and 3' positions of transcription units. Some enhancer sequences (e.g., globulin, elastase, albumin, alpha-fetoprotein and insulin) known to be available from mammalian genes. However, enhancers from viruses are typically used. SV40 enhancers, cytomegalovirus early promoter enhancers, polyoma enhancers and adenovirus enhancers known in the art are exemplary reinforcing elements for activating eukaryotic promoters. Although enhancers can be positioned at 5' or 3' of the coding sequence in the vector, they are typically located at the site of 5' of the promoter. Sequences encoding appropriate native or heterologous signal sequences (leader sequences or signal peptides) can be incorporated into expression vectors to promote extracellular secretion of the heavy and / or light chains of IL-2 mutant proteins, IL-2 mutant protein Fc fusions, or anti-IL-2 antibodies. The choice of signal peptide or leader sequence depends on the type of host cell in which the protein is to be produced, and the heterologous signal sequence can replace the native signal sequence. Examples of signal peptides that are functional in mammalian host cells include the following: the signal sequence of interleukin-7 (IL-7) described in U.S. Patent No. 4,965,195; the interleukin-2 receptor signal sequence described in Cosman et al., 1984, Nature [Nature] 312: 768; the interleukin-4 receptor signal peptide described in European Patent No. 0367566; the type I interleukin-1 receptor signal peptide described in U.S. Patent No. 4,968,607; the type II interleukin-1 receptor signal peptide described in EP Patent No. 0460846. In one embodiment, the IL-2 mutein Fc fusion of the present invention comprises a leader sequence as shown in FIG. 24 .

[0117] The vector may contain one or more elements that promote expression when the vector is integrated into the host cell genome. Examples include EASE elements (Aldrich et al., 2003, Biotechnol Prog. [Progress in Biotechnology], 19: 1433-38) and matrix attachment regions (MARs). MAR mediates the structural organization of chromatin and can insulate the integrated vector from "position" effects. Therefore, MARs are particularly useful when the vector is used to produce stable transfectants. A plurality of natural or synthetic MAR-containing nucleic acids are known in the art, such as U.S. Patent Nos. 6,239,328; 7,326,567; 6,177,612; 6,388,066; 6,245,974; 7,259,010; 6,037,525; 7,422,874; 7,129,062.

[0118] The expression vectors of the present invention can be constructed from a starting vector (e.g., a commercially available vector). These vectors may or may not contain all desired flanking sequences. In the case where one or more flanking sequences described herein are not present in the vector, they can be independently obtained and connected to the vector. Methods for obtaining each flanking sequence are well known to those of ordinary skill in the art.

[0119] After the vector has been constructed and the nucleic acid molecules encoding the heavy chain and / or light chain of the IL-2 mutant protein, IL-2 mutant protein Fc fusion, or anti-IL-2 antibody have been inserted into the appropriate site of the vector, the complete vector can be inserted into a suitable host cell for amplification and / or polypeptide expression. The expression vector can be transformed into the selected host cell by the following well-known methods, including: transfection, infection, calcium phosphate coprecipitation, electroporation, microinjection, lipofection, DEAE-dextran mediated transfection or other known techniques. The selected method will vary in part with the type of host cell to be used. These methods and other suitable methods are well known to technicians and are described in, for example, Sambrook et al., 2001, supra.

[0120] The host cells, when cultured under appropriate conditions, synthesize the heavy and / or light chains of the IL-2 mutant proteins, IL-2 mutant protein Fc fusions, or anti-IL-2 antibodies, which can then be collected from the culture medium (if the host cells secrete them into the culture medium) or directly from the host cells that produced them (if they are not secreted). The selection of appropriate host cells will depend on various factors, such as the desired expression level, the desired or required polypeptide modification (e.g., glycosylation or phosphorylation) for activity, and the ease of folding into a biologically active molecule. The host cells can be eukaryotic or prokaryotic.

[0121] Mammalian cell lines that can be used as expression hosts are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), and any cell line known in the art that can be used in the expression system for preparing the recombinant polypeptide of the present invention. Host cells are usually transformed with a recombinant expression vector comprising a DNA encoding the desired IL-2 mutant protein, IL-2 mutant protein Fc fusion, or anti-IL-2 antibody. The host cells that can be used are prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes include gram-negative or gram-positive organisms, such as Escherichia coli or bacillus. Higher eukaryotic cells include established cell lines of insect cells and mammalian origin. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC CCL163), Chinese hamster ovary (CHO) cells, or derivatives thereof (e.g., Veggie CHO cells) and related cell lines grown in serum-free medium (Rasmussen et al., 1998, Cytotechnology 28:31), HeLa cells, BHK (ATCC CRL10) cell lines, and the CVI / EBNA cell line derived from the African green monkey kidney cell line CVI (ATCC CCL70) (as described by McMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cells (e.g., 293, 293EBNA or MSR 293), human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, in vitro cultured cell lines derived from primary tissues, primary explants, HL-60, U937, HaK or Jurkat cells. Optionally, for example, when it is desired to use the polypeptide in different signal transduction or receptor assays, mammalian cell lines such as HepG2 / 3B, KB, NIH 3T3 or S49 can be used to express the polypeptide.

[0122] Alternatively, the polypeptide may be produced in a lower eukaryote, such as a yeast, or in a prokaryote, such as a bacterium. Suitable yeasts include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing a heterologous polypeptide. Suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing a heterologous polypeptide. If the polypeptide is prepared in yeast or bacteria, it may be desirable to modify the polypeptide produced herein, such as by phosphorylation or glycosylation of appropriate sites, to obtain a functional polypeptide. Such covalent attachment may be accomplished using known chemical or enzymatic methods.

[0123] The polypeptides can be produced by operably linking the isolated nucleic acids of the invention to suitable control sequences in one or more insect expression vectors and employing an insect expression system. Materials and methods for baculovirus / insect cell expression systems are available in kit form from, for example, Invitrogen, San Diego, CA, USA ( Kits) are commercially available, and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin, 1555 (1987), and Luckow and Summers, Bio / Technology 6:47 (1988). Cell-free translation systems can be employed to produce polypeptides using RNA derived from the nucleic acid constructs disclosed herein. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985). A host cell containing an isolated nucleic acid of the invention, preferably operably linked to at least one expression control sequence, is a "recombinant host cell".

[0124] In certain aspects, the invention comprises an isolated nucleic acid encoding a human IL-2 mutant protein that preferentially stimulates regulatory T cells and comprises: V91K, D20L; D84R, E61Q; V91K, D20A, E61Q, M104T; N88K, M104L; V91H, M104L; V91K, H16E, M104V; V91K, H16R, M104V; V91K, H16R, M104T; V91K, D20A, M104T; V91K, H16E, M104T; E; V91H, D20A, M104T; H16E, V91H, M104V; V91H, D20A, E61Q, M104T; V91H, H1 6R, E16Q; V91K, D20A, M104V; H16E, V91H; V91H, D20A, M104V; H16E, V91H, M1 04T; H16E, V91H, E61Q, M104T; V91K, E61Q, H16E; V91K, H16R, M104L; H16E, V 91H, E16Q; V91K, E61Q, H16R; D20W, V91K, E61Q; V91H, H16R; V91K, H16R; D20 W, V91K, E61Q, M104T; V91K, D20A; V91H, D20A, E16Q; V91K, D20A, M104L; V91 H, D20A; V91K, E61Q, D20A; V91H, M104T; V91H, M104V; V91K, E61Q; V91K, N88 K, E61Q, M104T; V91K, N88K, E61Q; V91H, E61Q; V91K, N88K; D20A, H16E, M104 T; D20A, M104T; H16E, N88K; D20A, M104V; D20A, M104L; H16E, M104T; H16E, M 104V; N88K, M104V; N88K, E61Q; D20A, E61Q; H16R, D20A; D20W, E61Q; H16E, E 61Q; H16E, M104L; N88K, M104T; D20A, H16E; D20A, H16E, E16Q; D20A, H16R, E 16Q; V91K, D20W; V91A, H16A; V91A, H16D; V91A, H16E; V91A, H16S; V91E, H16 A; V91E, H16D; V91E, H16E; V91E, H16S; V91K, H16A; V91K, H16D; V91K, H16S;and / or V91S, H16E substitutions and an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1. ;

[0125] Also included are isolated nucleic acids encoding any of the exemplary IL-2 mutein Fc fusion proteins described herein. In preferred embodiments, the Fc portion of an antibody and the human IL-2 mutein are encoded within a single open reading frame, optionally with a linker encoded between the Fc region and the IL-2 mutein.

[0126] In another aspect, provided herein is an expression vector comprising the above IL-2 mutein or IL-2 mutein Fc fusion protein encoding nucleic acid operably linked to a promoter.

[0127] In another aspect, provided herein is a host cell comprising an isolated nucleic acid encoding the above IL-2 mutant protein, IL-2 mutant protein Fc fusion protein, or anti-IL-2 antibody. The host cell can be a prokaryotic cell, such as Escherichia coli, or can be a eukaryotic cell, such as a mammalian cell. In certain embodiments, the host cell is a Chinese hamster ovary (CHO) cell line.

[0128] In another aspect, the present invention provides a method for preparing human IL-2 mutant proteins. The method includes culturing host cells under conditions where a promoter operably linked to human IL-2 mutant proteins is expressed. Subsequently, human IL-2 mutant proteins are harvested from the culture. IL-2 mutant proteins can be harvested from culture medium and / or host cell lysates.

[0129] In another aspect, the present invention provides a method for preparing a human IL-2 mutant protein Fc fusion protein. The method includes culturing a host cell under conditions in which a promoter operably linked to a human IL-2 mutant protein Fc fusion protein is expressed. Subsequently, the human IL-2 mutant protein Fc fusion protein is harvested from the culture. The human IL-2 mutant protein Fc fusion protein can be harvested from a culture medium and / or a host cell lysate.

[0130] In another aspect, the present invention provides a method for preparing an anti-IL-2 antibody. The method comprises culturing a host cell under conditions in which a promoter operably linked to a heavy chain and a light chain of an anti-IL-2 antibody is expressed. Subsequently, the anti-IL-2 antibody is harvested from the culture. The anti-IL-2 antibody can be harvested from a culture medium and / or a host cell lysate.

[0131] Pharmaceutical composition

[0132] In some embodiments, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an IL-2 mutant protein or an anti-IL-2 antibody together with a pharmaceutically effective diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant. In certain embodiments, the IL-2 mutant protein is in the context of an IL-2 mutant protein Fc fusion protein. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.

[0133] Preferably, acceptable formulation materials are non-toxic to recipients at the dosages and concentrations employed. In certain embodiments, pharmaceutical compositions comprising a therapeutically effective amount of an IL-2 mutein containing a therapeutic molecule, such as an IL-2 mutein Fc fusion, are provided.

[0134] In certain embodiments, pharmaceutical compositions may contain formulation materials to adjust, maintain or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, proline, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers; the pharmaceutical composition may be a pharmaceutical composition comprising: ... See, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th ed. (AR Genrmo, ed.), 1990, Mack Publishing Company.

[0135] In certain embodiments, the optimal drug composition will be determined by those skilled in the art according to the expected route of administration, delivery form and required dosage, for example.See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, the same. In certain embodiments, such compositions can affect the physical state, stability, in vivo release rate and in vivo clearance rate of the antigen-binding proteins of the present invention. In certain embodiments, the main vehicle or carrier in the pharmaceutical composition can be aqueous or non-aqueous. For example, a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, which may be supplemented with other substances common in parenteral administration compositions. Neutral buffered saline or saline mixed with serum albumin are other exemplary vehicles. In a particular embodiment, the pharmaceutical composition comprises a Tris buffer of about pH 7.0-8.5 or an acetate buffer of about pH 4.0-5.5, and may further include sorbitol or a suitable substitute thereof. In certain embodiments of the invention, IL-2 mutant protein or anti-IL-2 antibody compositions can be prepared for storage by mixing the selected components having the desired purity with optional formulating agents (REMINGTON'S PHARMACEUTICAL SCIENCES, supra) in the form of a lyophilized cake or aqueous solution. In addition, in certain embodiments, the IL-2 mutant protein or anti-IL-2 antibody product can be formulated as a lyophilizate using an appropriate excipient (e.g., sucrose).

[0136] The pharmaceutical composition of the present invention can be selected for parenteral delivery. Alternatively, the composition can be selected for suction or for delivery via the digestive tract (e.g., oral). The preparation of such pharmaceutically acceptable compositions is within the technical scope of those skilled in the art. The formulation components are preferably present in concentrations acceptable to the site of administration. In certain embodiments, a buffer is used so that the composition is maintained at a physiological pH or slightly lower pH, typically in the pH range of about 5 to about 8.

[0137] When considering parenteral administration, the therapeutic composition for the present invention can be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution, the aqueous solution being included in the desired IL-2 mutant protein or anti-IL-2 antibody composition in a pharmaceutically acceptable vehicle. The particularly suitable vehicle for parenteral injection is sterile distilled water, wherein the mutant protein or anti-IL-2 antibody composition is formulated into a sterile isotonic solution appropriately preserved. In certain embodiments, the preparation can include preparing the desired molecule with a reagent, such as an injectable microsphere, a bioerodible particle, a polymeric compound (such as polylactic acid or polyglycolic acid), a bead or a liposome, which can provide a controlled release or sustained release of a product delivered via an accumulation injection. In certain embodiments, hyaluronic acid can also be used, which has the effect of promoting the duration in circulation. In certain embodiments, an implantable drug delivery device can be used to introduce an IL-2 mutant protein or an anti-IL-2 antibody composition.

[0138] Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations involving IL-2 mutant proteins or anti-IL-2 antibody compositions in the form of sustained or controlled delivery formulations. Techniques for preparing various other sustained or controlled delivery modes (such as liposome carriers, bioerodible microparticles or porous beads, and depot injections) are also known to those skilled in the art. See, for example, International Patent Application No. PCT / US93 / 00829, which is incorporated herein by reference and describes the controlled release of porous polymer microparticles for delivering pharmaceutical compositions. Sustained release formulations may include semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides (as disclosed in U.S. Pat. No. 3,773,919 and European Patent Application Publication No. EP 058481, each of which is incorporated herein by reference), copolymers of L-glutamic acid and γ-L-glutamic acid ethyl ester (Sidman et al., 1983, Biopolymers [Biopolymers] 2: 547-556), poly (2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. [Journal of Biomedical Materials Research] 15: 167-277 and Langer, 1982, Chem. Tech. [Chemical Technology] 12: 98-105), ethylene vinyl acetate (Langer et al., 1981, supra) or poly-D (-) -3-hydroxybutyric acid (European Patent Application Publication No. EP 133,988). Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art. See, for example, Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-3692; European Patent Application Publication Nos. EP 036,676; EP 088,046 and EP 143,949, which are incorporated by reference.

[0139] Pharmaceutical compositions for in vivo administration are typically provided in the form of sterile preparations. Sterilization can be accomplished by filtration through a sterile filter membrane. When the composition is freeze-dried, sterilization using the method can be performed before or after freeze-drying and reconstitution. Compositions for parenteral administration can be stored in freeze-dried form or in solution. Parenteral compositions are typically placed in a container with a sterile access port, such as an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle.

[0140] Aspects of the invention include self-buffered IL-2 mutein or anti-IL-2 antibody formulations that can be used as pharmaceutical compositions as described in International Patent Application WO 06138181 A2 (PCT / US2006 / 022599), which is incorporated herein by reference in its entirety.

[0141] As discussed above, certain embodiments provide IL-2 mutant or anti-IL-2 antibody compositions, particularly pharmaceutical IL-2 mutant protein Fc fusion proteins, which contain, in addition to the IL-2 mutant or anti-IL-2 antibody composition, one or more excipients, such as those illustratively described in this section and elsewhere herein. Excipients in this regard can be used for a variety of purposes in the present invention, such as adjusting the physical, chemical or biological properties of the formulation (such as adjusting viscosity), and or for improving the efficacy and or stabilizing the process of such formulations and against degradation and spoilage due to stresses that occur during, for example, manufacturing, transportation, storage, preparation prior to use, administration and subsequent processes.

[0142] There are various discussions of protein stabilization and formulation materials and methods useful in this regard, for example, Arakawad et al., "Solvent interactions in pharmaceutical formulations," Pharm Res. 8(3):285-91 (1991); Kendrick et al., "Physical stabilization of proteins in aqueous solution," in RATIONAL DESIGN OF STABLE PROTEIN FORMULATIONS: THEORY AND PRACTICE, Carpenter and Manning, eds. Pharmaceutical Biotechnology. 13:61-84 (2002), and Randolph et al., "Surfactant-protein interactions," Pharm Biotechnol. 13:159-75 (2002), each of which is incorporated herein by reference in its entirety, particularly with respect to the portions relating to excipients for self-buffering protein formulations according to the present invention and methods for their preparation, especially protein pharmaceutical products and methods for veterinary and / or human medical use.

[0143] According to certain embodiments of the invention, salts may be used, for example, to adjust the ionic strength and / or isotonicity of a formulation and / or to improve the solubility and / or physical stability of a protein or other component of a composition according to the invention.

[0144] It is well known that ions can stabilize the native state of proteins by binding to charged residues on the surface of proteins and by shielding charged and polar groups in proteins and reducing the strength of their electrostatic interactions, attractive and repulsive interactions. Ions can also stabilize the denatured state of proteins by binding, in particular, to denatured peptide bonds (--CONH) of proteins. In addition, the interaction of ions with charged and polar groups in proteins can also reduce intermolecular electrostatic interactions, thereby preventing or reducing protein aggregation and insolubility.

[0145] The effects of ionic species on proteins vary widely. Many classified rankings of ions and their effects on proteins have been developed, and the proteins can be used to formulate pharmaceutical compositions according to the present invention. An example is the Hofmeister series, which ranks ions and polar non-ionic solutes by their effects on the conformational stability of proteins in solution. Stable solutes are called "lyophilic". Unstable solutes are called "chaotropic". High concentrations of lyophilic agents (e.g., >1 molar ammonium sulfate) are often used to precipitate proteins from solution ("salting out"). Chaotropic agents are often used to denature and / or dissolve proteins ("salting out"). The relative effectiveness of ions for "salting out" and "salting out" defines their position in the Hofmeister series.

[0146] According to various embodiments of the present invention, free amino acids can be used in IL-2 mutant proteins or anti-IL-2 antibody formulations as bulking agents, stabilizers and antioxidants and other standard uses. Lysine, proline, serine and alanine can be used to stabilize the protein in the formulation. Glycine can be used for freeze drying to ensure the correct cake structure and characteristics. In both liquid and freeze-dried formulations, arginine can be used to inhibit protein aggregation. Methionine can be used as an antioxidant.

[0147] Polyols include sugars (e.g., mannitol, sucrose, and sorbitol) and polyhydroxy alcohols (e.g., glycerol and propylene glycol) and, for the purposes of this discussion, polyethylene glycol (PEG) and related substances). Polyols are lyophilic. They are useful stabilizers in both liquid and lyophilized formulations to protect proteins from physical and chemical degradation processes. Polyols can also be used to adjust the tonicity of the formulation.

[0148] The polyol that can be used for the selected embodiment of the present invention is mannitol, which is generally used to ensure the structural stability of cakes in freeze-dried formulations. It ensures the structural stability for cakes. Mannitol is usually used together with freeze-dried protective agents, such as sucrose. Sorbitol and sucrose are preferred agents for adjusting tension and are used as stabilizers for preventing freeze-thaw stress during the preparation of powders during transportation or during the manufacturing process. Reducing sugars (which contain free aldehydes or ketone groups) such as glucose and lactose can saccharify surface lysine and arginine residues. Therefore, they are not generally preferred polyols used according to the present invention. In addition, the sugars such as sucrose that form this reactive species are hydrolyzed to fructose and glucose under acidic conditions and therefore produce saccharification, and this sugar is not a preferred polyol of the present invention in this regard. PEG can be used for stabilizing proteins and as a cryoprotectant, and can be used for the present invention in this regard.

[0149] Embodiments of IL-2 mutant proteins and / or anti-IL-2 antibody formulations further comprise surfactants. Protein molecules may be susceptible to adsorption on surfaces and are susceptible to denaturation and subsequent aggregation at gas-liquid, solid-liquid, and liquid-liquid interfaces. These effects are generally inversely proportional to protein concentration. These deleterious interactions are generally inversely proportional to protein concentration and are generally aggravated by physical agitation (e.g., physical agitation generated during transportation and handling of the product).

[0150] Surfactants are generally used to prevent, minimize or reduce surface adsorption. In this regard, surfactants useful in the present invention include polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan polyethoxylates, and poloxamer 188.

[0151] Surfactants are also commonly used to control protein conformational stability. In this regard, the use of surfactants is protein-specific, as any given surfactant will typically stabilize some proteins while destabilizing others.

[0152] Polysorbates are susceptible to oxidative degradation and generally contain sufficient peroxide as provided to cause oxidation of protein residue side chains, particularly methionine. Therefore, polysorbates should be used with caution and, when used, the lowest effective concentration should be used. In this respect, polysorbates exemplify the general rule that excipients should be used at their lowest effective concentration.

[0153] Embodiments of IL-2 mutant proteins or anti-IL-2 antibody formulations further comprise one or more antioxidants. To a certain extent, by maintaining an appropriate level of ambient oxygen and temperature and avoiding illumination, harmful oxidation of proteins in pharmaceutical formulations can be prevented. Antioxidant excipients can also be used to prevent oxidative degradation of proteins. In this regard, useful antioxidants are reducing agents, oxygen / free radical scavengers and chelating agents. Antioxidants for therapeutic protein formulations according to the present invention are preferably water-soluble and maintain their activity throughout the shelf life of the product. In this regard, EDTA is a preferred antioxidant according to the present invention.

[0154] Antioxidants can damage proteins. For example, reducing agents such as glutathione can particularly damage intramolecular disulfide bonds. Therefore, the antioxidants used in the present invention are selected, among other aspects, to eliminate or substantially reduce the possibility that they themselves will damage the proteins in the formulation.

[0155] The formulations according to the invention may contain metal ions which are protein cofactors and are necessary for the formation of protein coordination complexes, such as zinc which is necessary for the formation of certain insulin suspensions. Metal ions may also inhibit some processes that degrade proteins. However, metal ions also catalyze physical and chemical processes that degrade proteins.

[0156] Magnesium ions (10-120 mM) can be used to inhibit the isomerization of aspartate to isoaspartate. +2 ions (up to 100 mM) can increase the stability of human DNAse. +2 , Mn +2 and Zn +2 Can destabilize rhDNase. Similarly, Ca +2 and Sr +2 Can stabilize factor VIII, which can be absorbed by Mg +2 , Mn +2 and Zn +2 , Cu +2 and Fe +2 destabilization, and the aggregation of this stabilizing factor can be achieved by Al +3 Ion strengthening.

[0157] Embodiments of IL-2 mutant proteins or anti-IL-2 antibody formulations further include one or more preservatives. When developing a multi-dose parenteral formulation that involves more than one removal from the same container, a preservative is necessary. Their main function is to inhibit microbial growth and ensure product sterility throughout the shelf life or service life of the drug product. Commonly used preservatives include benzyl alcohol, phenol, and meta-cresol. Although preservatives have been used with small molecule parenteral drugs for a long time, the development of protein formulations containing preservatives may be challenging. Preservatives almost always have an unstable effect (aggregation) on proteins, and this has become a major factor limiting their use in multi-dose protein formulations. To date, most protein drugs are only formulated for single use. However, when multi-dose formulations become possible, they have the additional advantage of making it convenient for patients and increasing marketability. A good example is the preservative of human growth hormone (hGH), where the development of the preservation formulation has led to the commercialization of a more convenient, multi-purpose injection pen. At least four such pen devices containing preserved hGH formulations are currently available on the market. Norditropin (liquid, Novo Nordisk), Nutropin AQ (liquid, Genentech) and Genotropin (lyophilized - dual chamber cartridge, Pharmacia & Upjohn) contain phenol, whereas Somatrope (Eli Lilly) is formulated with m-cresol.

[0158] In one embodiment, an IL-2 mutein or Fc fusion of an IL-2 mutein, such as any of the IL-2 muteins or Fc fusions of an IL-2 mutein described herein, is formulated in 10 mM KPi, 161 mM L-arginine at pH 7.6.

[0159] Several aspects need to be considered during formulation and development of preserved dosage forms. Effective preservative concentrations in the drug product must be optimized. This requires testing a range of concentrations of a given preservative in the dosage form that imparts antimicrobial effectiveness without compromising protein stability.

[0160] In another aspect, the invention provides IL-2 mutant proteins or Fc fusions of IL-2 mutant proteins in freeze-dried formulations. Freeze-dried products can be freeze-dried in the absence of preservatives and reconstituted with a diluent containing a preservative when used. This shortens the time that the preservative is in contact with the protein, thereby significantly reducing the relevant stability risks. In the case of liquid formulations, the effectiveness and stability of the preservative should be maintained throughout the product shelf life (about 18 to 24 months). It should be noted that the effectiveness of the preservative should be confirmed in the final formulation containing the active drug and all excipient components.

[0161] IL-2 mutein formulations will typically be designed for a specific route and method of administration, a specific dosage and frequency of administration, a specific treatment for a specific disease, with a range of bioavailability and persistence, etc. Thus, formulations can be designed in accordance with the present invention for delivery by any suitable route, including but not limited to oral, intraaural, ophthalmic, rectal, and vaginal, as well as by parenteral routes, including intravenous and intraarterial injections, intramuscular injections, and subcutaneous injections.

[0162] Once the pharmaceutical composition is prepared, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, crystal or as a dehydrated or lyophilized powder. Such preparations can be stored in a ready-to-use form or in a form (e.g., lyophilized) reconstructed before administration. The present invention also provides a kit for producing a single-dose administration unit. The kit of the present invention can each contain a first container with dry protein and a second container with an aqueous preparation. In certain embodiments of the present invention, a kit containing a single-chamber and multi-chamber prefilled syringe (e.g., a liquid syringe and a lyophilized syringe) is provided.

[0163] The therapeutically effective amount of the IL-2 mutant protein pharmaceutical composition to be adopted will depend on, for example, the treatment context and purpose. It will be appreciated by those skilled in the art that the appropriate dosage level for treatment will depend in part on the delivered molecule, the indication for the use of the IL-2 mutant protein or anti-IL-2 antibody, the route of administration, and the size (weight, body surface area or organ size) and / or condition (age and general health) of the patient. In certain embodiments, the clinician may titrate the dose and modify the route of administration to obtain the best therapeutic effect. Depending on the above factors, a typical dosage range may be from about 0.1 μg / kg to up to about 1 mg / kg or higher. In a particular embodiment, the dosage range may be from 0.5 μg / kg to up to about 100 μg / kg, optionally from 2.5 μg / kg to up to about 50 μg / kg.

[0164] A therapeutically effective amount of an IL-2 mutein or anti-IL-2 antibody preferably results in a reduction in the severity of disease symptoms, an increase in the frequency or duration of disease symptom-free periods, or prevention of impairment or disability due to disease affliction.

[0165] The pharmaceutical composition may be administered using a medical device. Examples of medical devices for administering pharmaceutical compositions are described in U.S. Pat. Nos. 4,475,196; 4,439,196; 4,447,224; 4,447,233; 4,486,194; 4,487,603; 4,596,556; 4,790,824; 4,941,880; 5,064,413; 5,312,335; 5,312,335; 5,383,851; and 5,399,163, all of which are incorporated herein by reference.

[0166] In one embodiment, a pharmaceutical composition is provided, comprising:

[0167] Methods of treating autoimmune or inflammatory disorders

[0168] In certain embodiments, the IL-2 mutant protein or anti-IL-2 antibody of the present invention is used to treat autoimmune disorders or inflammatory disorders. In a preferred embodiment, IL-2 mutant protein Fc fusion protein is used.

[0169] Disorders particularly suitable for treatment with the IL-2 muteins or anti-IL-2 antibodies disclosed herein include, but are not limited to, inflammation, autoimmune diseases, atopic diseases, paraneoplastic autoimmune diseases, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular juvenile 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 (seronegative, enthesopathy, arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome (Reiter's Syndrome), SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myositis, polymyositis, dermatomyositis, polyarteritis nodosa, Wegener's granulomatosis granulomatosis), arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjögren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, sinusitis, sinusitis with polyps, eosinophilic esophagitis, eosinophilic bronchitis, Guillain-Barre disease, type I diabetes, thyroiditis (e.g., Graves' disease), Addison's disease disease), Raynaud's phenomenon, autoimmune hepatitis, GVHD, transplant rejection, renal injury, hepatitis C-induced vasculitis, spontaneous pregnancy loss, etc.

[0170] In preferred embodiments, the autoimmune or inflammatory disorder is lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type I diabetes, multiple sclerosis, spontaneous pregnancy loss, atopic disease, and inflammatory bowel disease.

[0171] In another embodiment, a patient with an autoimmune disorder or inflammatory disorder or a risk of developing these disorders is treated with an IL-2 mutant protein or an anti-IL-2 antibody (e.g., an IL-2 mutant protein disclosed herein, e.g., an IL-2 mutant protein Fc fusion as disclosed herein, or another IL-2 mutant protein known in the art, or wild-type IL-2, optionally as part of an Fc fusion molecule of the type disclosed herein), and the patient's response to the treatment is monitored. The monitored patient's response can be any detectable or measurable response of the patient to the treatment, or any combination of such responses. For example, the response can be a change in the patient's physiological state, such as body temperature or fever, cravings, sweating, headache, nausea, fatigue, hunger, thirst, mental acuity, etc. Alternatively, the response can be a change in the amount of a cell type or gene product (e.g., a protein, peptide, or nucleic acid) in a sample taken from the patient's peripheral blood. In one embodiment, if the patient has a detectable or measurable response to the treatment, or if such a response exceeds a specific threshold, the patient's treatment regimen is changed. The change can be a decrease or increase in the frequency of dosing, or a decrease or increase in the amount of IL-2 mutant protein or anti-IL-2 antibody administered per dosing, or a "holiday" in dosing (i.e., a temporary stop in treatment for a specified period of time, or until the treating physician decides that treatment should continue, or until the patient's monitored response indicates that treatment should be resumed or can be resumed), or termination of treatment. In one embodiment, the response is a change in the patient's temperature or CRP level. For example, the response can be an increase in the patient's body temperature, or an increase in the CRP level in a sample of peripheral blood, or both. In a specific embodiment, if during the course of treatment, the patient's body temperature increases by at least 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.7°, 1°, 1.5°, 2°, or 2.5°C, the patient's treatment is reduced, suspended, or terminated. In another specific embodiment, if during the course of treatment, the concentration of CRP in a sample of the patient's peripheral blood increases by at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1, 1.5, or 2 mg / mL, the patient's treatment is reduced, suspended, or terminated. Other patient responses that can be monitored and used in deciding whether to modify, reduce, suspend, or terminate treatment include the development or worsening of capillary leak syndrome (hypotension and cardiovascular instability), impaired neutrophil function (e.g., development or worsening of infection caused or detected), thrombocytopenia, thrombotic vasculopathy, injection site reaction, vasculitis (e.g., hepatitis C virus vasculitis), or inflammatory symptoms or diseases. Additional patient responses that can be monitored and used in deciding whether to modify, reduce, increase, suspend, or terminate treatment include increases in the number of NK cells, Treg cells, FOXP3 - CD4 T cells, FOXP3 +CD4 T cells, FOXP3-CD8 T cells, or eosinophils. An increase in these cell types can be detected, for example, as an increase in the number of such cells per unit of peripheral blood (e.g., expressed as an increase in cells per milliliter of blood), or as an increase in the percentage of such cell types compared to one or more cells of another type in a blood sample. Another patient response that can be monitored is the expression of CD25 in a sample of the patient's peripheral blood. + An increase in the amount of cell surface-bound IL-2 mutein or anti-IL-2 antibody on a cell.

[0172] Methods for expanding Treg cells

[0173] IL-2 mutant proteins, anti-IL-2 antibodies, or IL-2 mutant protein Fc fusion proteins can be used to expand Treg cells in a subject or sample. A method for increasing the ratio of Treg to non-regulatory T cells is provided herein. The method includes contacting a population of T cells with an effective amount of human IL-2 mutant proteins, anti-IL-2 antibodies, or IL-2 mutant protein Fc fusions. The ratio can be measured by determining the ratio of CD3+FOXP3+ cells to CD3+FOXP3- cells in a population of T cells. In human blood, a typical Treg frequency is 5%-10% of total CD4+CD3+ T cells, however, in the diseases listed above, this percentage may be lower or higher. In a preferred embodiment, the percentage of Treg increases by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%. For a particular disease, the maximum multiple increase of Treg can vary; however, the maximum Treg frequency that may be obtained by IL-2 mutant protein treatment is 50% or 60% of total CD4+CD3+ T cells. In certain embodiments, IL-2 mutant proteins, anti-IL-2 antibodies, or IL-2 mutant protein Fc fusion proteins are administered to a subject, and the ratio of regulatory T cells (Treg) to non-regulatory T cells in the subject's peripheral blood increases.

[0174] Because IL-2 mutant proteins, anti-IL-2 antibodies, and IL-2 mutant protein Fc fusion proteins preferentially expand Tregs over other cell types, they can also be used to increase the ratio of regulatory T cells (Tregs) to natural killer (NK) cells in the peripheral blood of a subject. The ratio can be measured by determining the ratio of CD3+FOXP3+ cells to CD16+ and / or CD56+ lymphocytes (which are CD19- and CD3-).

[0175] It is expected that IL-2 mutant proteins, anti-IL-2 antibodies, or IL-2 mutant protein Fc fusion proteins may have a therapeutic effect on a disease or disorder in a patient without significantly expanding the ratio of Treg to non-regulatory T cells or NK cells in the patient's peripheral blood. The therapeutic effect can be attributed to the local activity of IL-2 mutant proteins, anti-IL-2 antibodies, or IL-2 mutant protein Fc fusion proteins at sites of inflammation or autoimmunity.

[0176] Examples

[0177] The following examples (both actual and predicted) are provided to illustrate specific embodiments or features of the invention and are not intended to limit the scope thereof.

[0178] Example 1 - pSTAT5 signaling of IL-2 mutant proteins

[0179] IL-2 mutant protein is studied for relative pSTAT5 activation. Design activity screening to identify mutant proteins with enhanced Treg:Teff window, that is, high-level activity retained in Treg cells simultaneously shows those significantly weakened in Teff cells. Because activated Teff cells express increased levels of CD25, and patients with autoimmune diseases and inflammatory diseases have these cells with elevated numbers, we apply CD25+ gating to Teff cells, thereby simulating the more realistic differentiation of CD25 expression in patients. By responding to phosphor-STAT5 measured by FACS-based assay in cells, the activity of IL-2 mutant protein is assessed. In short, previously frozen human PBMCs are thawed, and rested in complete medium for 0.5-2hr. Press 5-10 million cells / ml suspended cells, and divide into 96-well deep well plates (0.5-1 million cells / well) by 100ul / well. By the final volume of 10 μ l, with the 10x dosage titration ranging from 1nM to 200nM, cells are stimulated with IL-2 mutant proteins for 30min. The level of STAT5 phosphorylation is measured using the BD phospho-flow buffer kit. In short, 1ml of BD lysis / fixed phospho-flow buffer is added to stop stimulation. Cells are fixed at 37°C for 10-15min, and permeabilized with 1x BD phospho-flow permeabilization buffer on ice, and then stained for CD3, CD4, CD25, FOXP3, CD8 and pSTAT5. The results of two donors of PBMC are shown in Table 2 below.

[0180] Table 2. pSTAT5 activation by IL-2 mutant proteins

[0181]

[0182]

[0183]

[0184] Example 2 - Stability of IL-2 mutant proteins

[0185] The molecule evaluation assay of DSC Tm measurement was tested together with the 10-day stability at 40°C measured by SEC chromatography to select stable molecules with good manufacturability potential. Figure 1 The results for %MP after 10 days are shown in FIG.

[0186] Example 3 - Attenuation of human IL-2 mutant protein

[0187] Attenuated human IL-2 mutant proteins were evaluated for activity in mouse splenocytes in an in vitro pSTAT5 assay. We confirmed the activity of selected mutant proteins on mouse immune cells in a mouse splenocyte pSTAT5 assay ( Figure 2 ). Shown are three weakened mutant proteins H16R, V91K D20A M104V, D20W, and control, wild-type human IL-2.Fc, recombinant human IL-2, and recombinant mouse IL-2 dose titration curves. Mouse splenocytes were stimulated for 30 min in a culture medium containing titrated concentrations of mutant proteins and analyzed by FACS. In mouse Treg cells, mutant proteins showed similar activity rankings to their performance in human Treg cells (i.e. WT>H16R>V91KD20A M104V>D20W). The ranking is also similar for effector T cells.

[0188] Example 4 - In vivo activity of human IL-2 mutant proteins in mice

[0189] At the 0th day, C57Bl6 mice were given a single dose of PBS (vehicle control), wild-type IL-2-Fc, V91K D20AM104V, H16R or D20W, and spleen cells were harvested at the 4th day, and the effects on Treg cells, CD8 T cells and NK cells were analyzed. Three doses (1ug, 5ug or 25ug / mouse) were evaluated, except that D20W was only given at 25ug. The percentage of Treg cells (as defined by CD4+CD25+FoxP3+ cells) in total live gated cells is shown in (A), and the calculated total number of Treg cells (B), CD8 T cells (C) and NK cells (D) is shown. As Figure 3As shown in , in vivo, wild-type IL-2, H16R, and V91KD20A M104V induce significant expansion of Treg cells in a dose-dependent manner., Two mutant proteins H16R and V91K D20A M104V showed surprisingly robust activity against Treg cells, with a degree similar to or even greater than that of wild-type IL-2.Fc. In contrast, compared with wild-type IL-2.Fc, neither H16R nor V91K D20A M104V showed significant activity against CD8 T or NK cells. These results demonstrate that, although activity is significantly reduced as measured by in vitro pSTAT5 readout, the reduced mutant protein retains the ability to induce robust Treg responses in vivo while inducing minimal CD8 T and NK cell responses. Therefore, reduction disproportionately affects Treg: non-Treg selectivity in vivo.

[0190] Example 5 - pSTAT5 Signaling of IL-2 Mutant Proteins

[0191] IL-2 mutant protein is studied for relative pSTAT5 activation. Design activity screening to identify mutant proteins with enhanced Treg:Teff window, that is, high-level activity retained in Treg cells simultaneously shows those significantly weakened in Teff cells. Because activated Teff cells express increased levels of CD25, and patients with autoimmune diseases and inflammatory diseases have these cells with elevated numbers, we apply CD25+ gating to Teff cells, thereby simulating the more realistic differentiation of CD25 expression in patients. By responding to phosphor-STAT5 measured by FACS-based assay in cells, the activity of IL-2 mutant protein is assessed. In short, previously frozen human PBMCs are thawed, and rested in complete medium for 0.5-2hr. Press 5-10 million cells / ml suspended cells, and divide into 96-well deep well plates (0.5-1 million cells / well) by 100ul / well. By the final volume of 10 μ l, with the 10x dosage titration ranging from 0.4nM to 25nM, cells are stimulated with IL-2 mutant proteins for 30min. The level of STAT5 phosphorylation is measured using the BD phosphorus flow buffer kit. In short, 1ml of BD lysis / fixed phosphorus flow buffer is added to stop stimulation. Cells are fixed at 37°C for 10-15min, and permeabilized with 1x BD phosphorus flow permeabilization buffer on ice, and then stained for CD3, CD4, CD25, FOXP3, CD8 and pSTAT5. The results of two donors of PBMC are shown in Table 2 below.

[0192] Table 3. pSTAT5 activation by IL-2 mutant proteins

[0193]

[0194]

Claims

1. A human interleukin-2 (IL-2) mutant protein, wherein compared with the amino acid sequence listed in SEQ ID NO: 1, one of the following mutation pairs is present: V91K, H16E; H16E, V91H; V91H, H16R; V91A, H16D; V91A, H16E; V91A, H16S; V91E, H16A; V91E, H16D; V91E, H16E; V91E, H16S; V91K, H16A; V91K, H16D; V91K, H16S; or V91S, H16E, and wherein the IL-2 mutant protein preferentially stimulates regulatory T cells relative to other T cells.

2. The human IL-2 mutant protein of claim 1, wherein the IL-2 mutant protein further preferentially stimulates regulatory T cells relative to NK cells.

3. A human interleukin-2 (IL-2) mutant protein, wherein compared with the amino acid sequence listed in SEQ ID NO:1, one of the following mutation pairs is present: V91K, H16E; H16E, V91H; V91H, H16R; V91A, H16D; V91A, H16E; V91A, H16S; V91E, H16A; V91E, H16D; V91E, H16E; V91E, H16S; V91K, H16A; V91K, H16D; V91K, H16S; or V91S, H16E; wherein a C125A mutation is also present, and wherein the IL-2 mutant protein preferentially stimulates regulatory T cells relative to other T cells.

4. A human interleukin-2 (IL-2) mutant protein, wherein compared with the amino acid sequence listed in SEQ ID NO:1, one of the following mutation pairs is present: V91K, H16E; H16E, V91H; V91H, H16R; V91A, H16D; V91A, H16E; V91A, H16S; V91E, H16A; V91E, H16D; V91E, H16E; V91E, H16S; V91K, H16A; V91K, H16D; V91K, H16S; or V91S, H16E; wherein T3N or T3A mutations are also present, and wherein the IL-2 mutant protein preferentially stimulates regulatory T cells relative to other T cells.

5. A human interleukin-2 (IL-2) mutant protein, wherein compared with the amino acid sequence listed in SEQ ID NO:1, one of the following mutation pairs is present: V91K, H16E; H16E, V91H; V91H, H16R; V91A, H16D; V91A, H16E; V91A, H16S; V91E, H16A; V91E, H16D; V91E, H16E; V91E, H16S; V91K, H16A; V91K, H16D; V91K, H16S; or V91S, H16E; wherein an S5T mutation is also present, and wherein the IL-2 mutant protein preferentially stimulates regulatory T cells relative to other T cells.

6. An Fc fusion protein comprising Fc and the human IL-2 mutant protein according to any one of claims 1 to 5. The Fc fusion protein of claim 6 , wherein the Fc is human IgG1 Fc.

8. The Fc fusion protein of claim 7, wherein the human IgG1 Fc comprises one or more mutations that alter the effector function of the Fc.

9. The Fc fusion protein of claim 8, wherein the human IgG1 Fc comprises a substitution at N297.

10. The Fc fusion protein of claim 9, wherein the substitution at N297 is N297G. The Fc fusion protein of claim 7 , comprising a substitution or deletion of the C-terminal lysine of the human IgG Fc.

12. The Fc fusion protein of claim 11, wherein the C-terminal lysine of the human IgG Fc is deleted.

13. The Fc fusion protein of claim 7, wherein a linker connects the Fc and human IL-2 mutant protein portions of the protein.

14. The Fc fusion protein of claim 13, wherein the linker is GGGGS (SEQ ID NO: 5), GGNGT (SEQ ID NO: 6) or YGNGT (SEQ ID NO: 7).

15. The Fc fusion protein of claim 14, wherein the linker is GGGGS (SEQ ID NO: 5).

16. The Fc fusion protein of claim 7, wherein the Fc portion of the fusion comprises amino acid additions, substitutions, or deletions that alter the glycosylation of the Fc fusion protein when expressed in mammalian cells.

17. The Fc fusion protein of claim 7, wherein the Fc fusion protein comprises an Fc dimer.

18. The Fc fusion protein of claim 17, wherein the Fc fusion protein comprises two IL-2 mutant proteins.

19. The Fc fusion protein of claim 17, wherein the Fc fusion protein comprises a single IL-2 mutein.

20. An isolated nucleic acid encoding the human IL-2 mutant protein according to any one of claims 1 to 5.

21. An isolated nucleic acid encoding the Fc portion of an antibody and the human IL-2 mutant protein according to any one of claims 1 to 5.

22. The isolated nucleic acid of claim 21, wherein the Fc portion of the antibody and the human IL-2 mutein are encoded in a single open reading frame.

23. The isolated nucleic acid of claim 21, wherein the Fc is human IgG1 Fc.

24. The isolated nucleic acid of claim 23, wherein the human IgGl Fc comprises one or more mutations that alter the effector function of said Fc.

25. The isolated nucleic acid of claim 24, wherein the human IgG1 Fc comprises a substitution at N297.

26. The isolated nucleic acid of claim 25, wherein the substitution at N297 is N297G.

27. The isolated nucleic acid of claim 23, encoding a substitution or deletion of the C-terminal lysine of the human IgG Fc.

28. The isolated nucleic acid of claim 27, wherein the C-terminal lysine of the human IgG Fc is deleted.

29. The isolated nucleic acid of claim 21, further encoding a linker connecting the Fc portion of an antibody and the human IL-2 mutant protein.

30. The isolated nucleic acid of claim 29, wherein the linker is GGGGS (SEQ ID NO: 5), GGNGT (SEQ ID NO: 6), or YGNGT (SEQ ID NO: 7).

31. The isolated nucleic acid of claim 30, wherein the linker is GGGGS (SEQ ID NO: 5).

32. The isolated nucleic acid of claim 21, wherein the Fc portion of the fusion comprises amino acid additions, substitutions, or deletions that alter the glycosylation of a protein comprising the IL-2 mutein when expressed in a mammalian cell.

33. An expression vector comprising the isolated nucleic acid of any one of claims 20-32 operably linked to a promoter.

34. A host cell comprising the isolated nucleic acid of any one of claims 20-32.

35. The host cell of claim 34, wherein the isolated nucleic acid is operably linked to a promoter.

36. The host cell of claim 35, wherein the host cell is a prokaryotic cell.

37. The host cell of claim 36, wherein the host cell is Escherichia coli.

38. The host cell of claim 35, wherein the host cell is a eukaryotic cell.

39. The host cell of claim 38, wherein the host cell is a mammalian cell.

40. The host cell of claim 39, wherein the host cell is a Chinese Hamster Ovary (CHO) cell line.

41. A method for preparing a human IL-2 mutant protein, the method comprising culturing the host cell according to any one of claims 35 to 40 under conditions wherein the promoter is expressed, and harvesting the human IL-2 mutant protein from the culture.

42. A method for preparing an Fc fusion protein, the method comprising culturing the host cell of any one of claims 35 to 40 under conditions wherein the promoter is expressed, and harvesting the Fc fusion protein from the culture.

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