Interleukin-2 muteins for expansion of t-regulatory cells

Abstract

To provide IL-2 muteins and IL-2 mutein Fc fusion molecules that preferentially expand and activate cells and are amenable to large scale production.SOLUTION: Disclosed are human IL-2 muteins having specific mutations. The IL-2 muteins have a relatively low likelihood of inducing an immune response against the IL-2 mutein and / or endogenous IL-2, and enable preferential proliferation and activation of Treg. Also disclosed are methods for producing and using the compositions of the present invention.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of U.S. Patent Application No. 62 / 886,283, filed on August 13, 2019, which is hereby incorporated by reference in its entirety.

Background Art

[0002] IL - 2 binds to three transmembrane receptor subunits: IL - 2Rβ and IL - 2Rγ, which co - activate intracellular signaling events immediately upon IL - 2 binding, and CD25 (IL - 2Rα), which functions to stabilize the interaction between IL - 2 and IL - 2Rβγ. Signals delivered by IL - 2Rβγ include signals of the PI3 - kinase, Ras - MAP - kinase, and STAT5 pathways.

[0003] T cells typically require the expression of CD25 to respond to low concentrations of IL - 2 present in tissues. T cells expressing CD25 include both FOXP3 + regulatory T cells (Treg cells), which are essential for the suppression of autoimmune inflammation, and FOXP3 - T cells activated to express CD25. FOXP3 - CD25 + effector T cells (Teff) can be either CD4 + cells or CD8 + cells, both of which may contribute to inflammation, autoimmunity, organ transplant rejection, or graft - versus - host disease. IL - 2 - stimulated STAT5 signaling is very important for the proliferation and survival of normal Treg cells and for high FOXP3 expression.

[0004] In the jointly-owned International Publication No. WO 2010 / 085495 pamphlet, the inventors describe the use of IL-2 mutant proteins for preferentially expanding or stimulating Treg cells. When administered to a subject, the effect on Treg cells is useful for treating inflammatory and autoimmune diseases. The IL-2 mutant proteins described in that specification are useful for in vivo expansion of more Treg cells than Teff cells, but it was desired to generate IL-2 mutant proteins having optimal attributes for human therapeutic agents.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Means for Solving the Problems

[0006] Described herein are IL-2 mutant proteins that are suitable for high-yield production and have pharmacological activity. In particular, the IL-2 mutant proteins of the present invention improve the Treg:Teff selectivity window. There were several unexpected and unpredictable observations in attempting to generate molecules such as those used as human therapeutic agents. The compositions and methods described herein are the result of that effort.

[0007] The IL-2 mutant proteins described herein are relatively less likely to elicit an immune response against the IL-2 mutant protein and / or endogenous IL-2, and achieve preferential proliferation and activation of Tregs. Further, in certain embodiments, the IL-2 mutant protein is fused to a molecule that increases serum half-life, such as an antibody Fc, when administered to a subject. The IL-2 mutant protein has a short serum half-life (3-5 hours for subcutaneous injection). Exemplary IL-2 mutant protein Fc fusions 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 humans. This effect on the pharmacokinetics of the IL-2 mutant protein enables reduced dosing or less frequent dosing of the IL-2 mutant protein therapeutic agent.

[0008] Furthermore, when creating large pharmaceutical molecules, the ability to produce large amounts of the molecule while minimizing molecular aggregation and maximizing molecular stability must be considered. IL-2 mutant protein Fc fusion molecules demonstrate such attributes.

[0009] In addition, in certain embodiments, the IL-2 mutant protein Fc fusion protein contains an IgG1 Fc region. When it is desirable to inactivate the effector functions of IgG1 (e.g., ADCC activity), the mutation of asparagine at position 297 to glycine (N297G; EU numbering scheme) has been found to achieve a greater improvement in purification efficiency and biophysical properties than other mutations that result in non-glycosylated IgG1 Fc. In a preferred embodiment, cysteine is engineered into the Fc to form disulfide bonds, thereby increasing the stability of the non-glycosylated Fc-containing molecule. The utility of non-glycosylated Fc extends beyond the context of IL-2 mutant protein Fc fusions. Accordingly, provided herein are Fc-containing molecules, Fc fusions, and antibodies that include the N297G substitution and, optionally, the substitution of one or more additional residues to cysteine.

[0010] In one aspect, the present invention provides a human interleukin-2 (IL-2) mutant protein comprising an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1, wherein the IL-2 mutant protein has one or more of the following mutations: 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; 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; V91H, D20A, E16Q; V91K, D20A, M104L; V91H, D20A; V91K, 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;Having at least one mutation selected from V91E,H16S; V91K,H16A; V91K,H16D; V91K,H16S; and V91S,H16E, preferentially stimulates regulatory T cells as compared to other T cells or NK cells in both in vitro assays and humanized mice (NSG mice reconstituted with CD34+ hematopoietic stem cells). In one embodiment, the mutant protein is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:1. In another embodiment, the mutant protein is at least 97% identical to the amino acid sequence set forth in SEQ ID NO:1. In another embodiment, the amino acid sequence of the mutant protein is for C125A, and for 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; 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; V91H,D20A,E16Q; V91K,D20A,M104L; V91H,D20A; V91K,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;It differs from the amino acid sequence set forth in SEQ ID NO: 1 only at one position selected from 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 the human IL-2 mutant protein described above. In one embodiment, the Fc is human IgG1 Fc. In another embodiment, the human IgG1 Fc comprises one or more mutations that alter 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 portion and the human IL-2 mutant protein portion of the protein. In another embodiment, the linker is GGGGS (SEQ ID NO: 5), GGNGT (SEQ ID NO: 6), or YGNGT (SEQ ID NO: 7). In another embodiment, the linker is GGGGS (SEQ ID NO: 5). In another embodiment, the IL-2 mutant protein further comprises an addition, substitution, or deletion of an amino acid that alters the glycosylation of the Fc fusion protein when expressed in mammalian cells. In another embodiment, the IL-2 mutant protein comprises a T3 substitution. In another embodiment, the IL-2 mutant protein comprises a T3N substitution or a 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 the human IL-2 mutant protein described above.

[0013] In another aspect, the present invention provides an isolated nucleic acid encoding the Fc portion of an antibody and the human IL-2 mutant protein described above. In one embodiment, the Fc portion of the antibody and the human IL-2 mutant protein are encoded within a single open reading frame. In another embodiment, the Fc is human IgG1 Fc. In another embodiment, the human IgG1 Fc contains one or more mutations that alter the effector function of the Fc. In another embodiment, the human IgG1 contains 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 that links the Fc portion of the antibody and the human IL-2 mutant protein. In another embodiment, the linker is GGGGS (SEQ ID NO: 5), GGNGT (SEQ ID NO: 6), or YGNGT (SEQ ID NO: 7). In another embodiment, the linker is GGGGS (SEQ ID NO: 5). In another embodiment, the IL-2 mutant protein further contains an amino acid addition, substitution, or deletion that alters the glycosylation of the protein containing the IL-2 mutant protein when expressed in mammalian cells. In another embodiment, the IL-2 mutant protein contains a T3 substitution. In another embodiment, the IL-2 mutant protein contains a T3N substitution or a T3A substitution. In another embodiment, the IL-2 mutant protein contains a T3N substitution. In another embodiment, the IL-2 mutant protein further contains an S5 mutation. In another embodiment, the IL-2 mutant protein further contains 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 present 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 Escherichia coli (E. 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 forming a human IL-2 mutant protein, comprising culturing the host cell described above under conditions in which 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 forming an Fc fusion protein, comprising culturing the host cell described above under conditions in which the promoter is expressed, and harvesting the Fc fusion protein from the culture.

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

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

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

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

[0022] In another aspect, the present invention provides a method for increasing the ratio of regulatory T cells (Tregs) to natural killer (NK) cells in the peripheral blood of a subject, the method comprising administering an effective amount of the human IL-2 mutant protein 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%.

[0023] In another aspect, the present invention provides a method for increasing the ratio of regulatory T cells (Tregs) to natural killer (NK) cells in the peripheral blood of a subject, the method comprising administering an effective amount of the Fc fusion protein 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 present invention provides a method of treating a subject suffering from an inflammatory disease or an autoimmune disease, the method comprising administering to the subject a therapeutically effective amount of the IL-2 mutant protein described above, or a therapeutically effective amount of the Fc fusion protein described above. In one embodiment, administration causes a reduction in at least one symptom of the disease. In another embodiment, the ratio of regulatory T cells (Tregs) to non-regulatory T cells in the peripheral blood of the subject increases after administration. In another embodiment, the ratio of regulatory T cells (Tregs) to non-regulatory T cells in the peripheral blood of the subject remains essentially the same after administration. 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, Behçet's disease, spontaneous abortion, atopic disease, asthma, or inflammatory bowel disease.

Brief Description of the Drawings

[0025]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0026] The headings of sections used in this specification are for organizational purposes only and should not be construed as limiting the subject matter described. All references cited within the text of this specification are hereby expressly incorporated by reference in their entirety.

[0027] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, tissue culture and transformation, protein purification, etc. Enzyme reactions and purification techniques may be performed according to the manufacturer's specifications, or as commonly achieved in the art, or as described herein. The following procedures and techniques generally may be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. For example, see Sambrook et al., 2001, Molecular Cloning: A Laboratory Manuel, 3 rd ed., Cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y. This reference is hereby incorporated by reference into this specification for all purposes. Unless otherwise defined, the nomenclature used in connection with analytical chemistry, organic chemistry, and medicinal and pharmaceutical chemistry described herein, and these laboratory procedures and techniques are well known and commonly used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparations, formulations, and delivery, and the treatment of patients.

[0028] IL-2 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" shall mean a polypeptide having the following amino acid sequence: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFXQSIISTLT In the sequence, X is C, S, V, or A (SEQ ID NO: 2).

[0029] The variant may contain one or more substitutions, deletions, or insertions within the wild-type IL-2 amino acid sequence. Residues are designated herein by the IL-2 amino acid position following the one-letter amino acid code, e.g., K35 is the lysine residue at position 35 of SEQ ID NO: 2. Substitutions are designated herein by the IL-2 amino acid position following the one-letter amino acid code, followed by the one-letter amino acid code of the substituting residue, e.g., K35A is the substitution of the lysine residue at position 35 of SEQ ID NO: 2 with an alanine residue.

[0030] IL-2 mutant protein Provided herein are human IL-2 mutant proteins and anti-IL-2 antibodies that preferentially stimulate regulatory T (Treg) cells. As used herein, "preferentially stimulate regulatory T cells" means that the mutant protein or antibody promotes the proliferation, survival, activation, and / or function of CD3+FoxP3+ T cells over CD3+FoxP3- T cells. The ability to measure the preferential stimulation of Treg can be measured by flow cytometry of peripheral blood leukocytes, 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 in the percentage of FOXP3+ T cells compared to NK cells, and / or a greater increase in the expression level of CD25 on the surface of FOXP3+ T cells compared to an increase in CD25 expression on other T cells. Also, preferential proliferation of Treg cells can be detected as an increase in the expression of demethylated FOXP3 promoter DNA (i.e., the Treg-specific demethylated region, or TSDR) compared to demethylated CD3 gene in DNA extracted from whole blood, as detected by sequencing of polymerase chain reaction (PCR) products derived from bisulfite-treated genomic DNA (J. Sehouli, et al. 2011. Epigenetics 6:2, 236-246). In particular, the IL-2 mutant protein of the present invention enhances the Treg:Teff window, i.e., retains a high level of activity in Treg cells while showing significant attenuation in Teff cells. Activated Teff cells express high levels of CD25, and patients suffering from autoimmune and inflammatory diseases have an elevated number of such cells, so the inventors used CD25+-gating on Teff cells to mimic a more realistic differentiation of CD25 expression in patients.

[0031] An IL-2 mutant protein or an Fc fusion protein thereof that preferentially stimulates Treg cells increases the ratio of CD3+FoxP3+ T cells to CD3+FoxP3- T cells in a subject or 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%.

[0032] In some embodiments, the IL-2 mutant protein or an Fc fusion protein thereof has less than 50% pSTAT activation of effector T cells upon treatment with 1 nM IL-2 using the protocol of Example 1. In some embodiments, the IL-2 mutant protein or an Fc fusion protein thereof has less than 40% pSTAT activation of effector T cells upon treatment with 1 nM IL-2 using the protocol of Example 1. In some embodiments, the IL-2 mutant protein or an Fc fusion protein thereof has less than 30% pSTAT activation of effector T cells upon treatment with 1 nM IL-2 using the protocol of Example 1. In some embodiments, the IL-2 mutant protein or an Fc fusion protein thereof has less than 20% pSTAT activation of effector T cells upon treatment with 1 nM IL-2 using the protocol of Example 1. In some embodiments, the IL-2 mutant protein or an Fc fusion protein thereof has less than 10% pSTAT activation of effector T cells upon treatment with 1 nM IL-2 using the protocol of Example 1. Also, in some embodiments, the IL-2 mutant protein or an Fc fusion protein thereof has a stability of more than 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% over 10 days using the protocol of Example 2.

[0033] In some embodiments, the IL-2 mutant protein or its Fc fusion protein has less than 50% pSTAT activation of effector T cells upon treatment with 200 nM IL-2 using the protocol of Example 1. In some embodiments, the IL-2 mutant protein or its Fc fusion protein has less than 40% pSTAT activation of effector T cells upon treatment with 200 nM IL-2 using the protocol of Example 1. In some embodiments, the IL-2 mutant protein or its Fc fusion protein has less than 30% pSTAT activation of effector T cells upon treatment with 200 nM IL-2 using the protocol of Example 1. In some embodiments, the IL-2 mutant protein or its Fc fusion protein has less than 20% pSTAT activation of effector T cells upon treatment with 200 nM IL-2 using the protocol of Example 1. In some embodiments, the IL-2 mutant protein or its Fc fusion protein has less than 10% pSTAT activation of effector T cells upon treatment with 200 nM IL-2 using the protocol of Example 1. Also, in some embodiments, the IL-2 mutant protein or its Fc fusion protein has a 10-day stability greater than 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% using the protocol of Example 2.

[0034] In some embodiments, the IL-2 mutant protein or its Fc fusion protein has a pSTAT activation in regulatory T cells of more than 30% upon treatment with 1 nM IL-2 using the protocol of Example 1. In some embodiments, the IL-2 mutant protein or its Fc fusion protein has a pSTAT activation in regulatory T cells of more than 40% upon treatment with 1 nM IL-2 using the protocol of Example 1. In some embodiments, the IL-2 mutant protein or its Fc fusion protein has a pSTAT activation in regulatory T cells of more than 50% upon treatment with 1 nM IL-2 using the protocol of Example 1. In some embodiments, the IL-2 mutant protein or its Fc fusion protein has a pSTAT activation in regulatory T cells of more than 60% upon treatment with 1 nM IL-2 using the protocol of Example 1. In some embodiments, the IL-2 mutant protein or its Fc fusion protein has a pSTAT activation in regulatory T cells of more than 70% upon treatment with 1 nM IL-2 using the protocol of Example 1. Also, in some embodiments, the IL-2 mutant protein or its Fc fusion protein has a 10-day stability of more than 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% using the protocol of Example 2.

[0035] In some embodiments, the IL-2 mutant protein or its Fc fusion protein has a pSTAT activation in regulatory T cells of more than 30% upon treatment with 200 nM IL-2 using the protocol of Example 1. In some embodiments, the IL-2 mutant protein or its Fc fusion protein has a pSTAT activation in regulatory T cells of more than 40% upon treatment with 200 nM IL-2 using the protocol of Example 1. In some embodiments, the IL-2 mutant protein or its Fc fusion protein has a pSTAT activation in regulatory T cells of more than 50% upon treatment with 200 nM IL-2 using the protocol of Example 1. In some embodiments, the IL-2 mutant protein or its Fc fusion protein has a pSTAT activation in regulatory T cells of more than 60% upon treatment with 200 nM IL-2 using the protocol of Example 1. In some embodiments, the IL-2 mutant protein or its Fc fusion protein has a pSTAT activation in regulatory T cells of more than 70% upon treatment with 200 nM IL-2 using the protocol of Example 1. Also, in some embodiments, the IL-2 mutant protein or its Fc fusion protein has a 10-day stability of more than 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% using the protocol of Example 2.

[0036] Examples of the IL-2 mutant proteins include, but are not limited to, the following IL-2 mutant proteins: V91K, D20L in the amino acid sequence shown in SEQ ID NO: 2; 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; 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; V91H, D20A, E16Q; V91K, D20A, M104L; V91H, D20A; V91K, 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 substitution. The IL-2 mutant proteins of the present invention may optionally include C125A substitution. Also, while it may be advantageous to reduce the number of further mutations to the wild-type IL-2 sequence, the present invention also includes IL-2 mutant proteins that include truncation and / or additional insertions, deletions, and / or substitutions: 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; 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; V91H, D20A, E16Q; V91K, D20A, M104L; V91H, D20A; V91K, 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 substitution. Provided that this is limited to the case where the mutant protein maintains the activity of preferentially stimulating Tregs. Therefore, as an embodiment, it preferentially stimulates Treg cells and has 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; 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; V91H, D20A, E16Q; V91K, D20A, M104L; V91H, D20A; V91K, 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;An amino acid sequence having a 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 substitution, and an IL-2 mutant protein 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 to the amino acid sequence shown in SEQ ID NO: 2. In a particularly preferred embodiment, such an IL-2 mutant protein comprises an amino acid sequence 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 to the amino acid sequence shown in SEQ ID NO: 2.;

[0037] For amino acid sequences, sequence identity and / or similarity are determined by standard techniques known in the art, such as, 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 for similarity methods of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. U.S.A. 85:2444, the computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), by using the Best Fit sequence program described by Devereux et al., 1984, Nucl. Acid Res. 12:387-395, preferably with default settings, or by visual inspection. Preferably, the percent identity is calculated by FastDB based on the following parameters: a mismatch penalty of 1; a gap penalty of 1; a gap size penalty of 0.33; and a joining penalty of 30, "Current Methods in Sequence Comparison and Analysis", Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.

[0038] An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive pairwise alignment. This also allows plotting a tree that shows the clustering relationships used to create the alignment. PILEUP uses a simplified version of the progressive alignment method of Feng & Doolittle, 1987, J. Mol. Evol. 35:351 - 360; this 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 a weighted end gap.

[0039] 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. U.S.A. 90:5873 - 5787. A particularly useful BLAST program is the WU - BLAST - 2 program obtained from Altschul et al., 1996, Methods in Enzymology 266:460 - 480. WU - BLAST - 2 uses several search parameters, most of which are set to default values. Tunable parameters are set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T)=11. The HSP S parameter and the HSP S2 parameter are dynamic values that are established by the program itself depending on the composition of the particular sequences being searched and the composition of the particular database; however, the values may be adjusted to increase sensitivity.

[0040] An additional useful algorithm is gapped BLAST as reported by Altschul et al., 1993, Nucl. Acids Res. 25: 3389-3402. Gapped BLAST uses a BLOSUM-62 substitution score; a threshold T parameter set to 9; a two-hit method to trigger gapless extension, charging a cost of 10 + k for a gap length of k; an X set to 16 u , and an X set to 40 for the database search stage and set to 67 for the output stage of the algorithm. Gapped alignment is triggered by a score corresponding to approximately 22 bits. g

[0041] The site or region for introducing amino acid sequence variation may be predetermined, but the variation itself need not be predetermined. For example, to optimize the performance of a mutation at a given site, random mutagenesis may be performed at the target codon or target region, and the expressed IL-2 mutant protein may be screened for the optimal combination of desired activities. Techniques for forming substitution mutations at a predetermined site within a DNA having a known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis. Screening of mutants may be performed, for example, using the assays described herein.

[0042] Amino acid substitutions are typically of a single residue; insertions will usually be of the order of about 1 to about 20 amino acid residues, although much larger insertions may be tolerated. Deletions range from about 1 to about 20 amino acid residues, but in some cases may be much larger.

[0043] Substitutions, deletions, insertions, or any combination thereof may be used to reach the final derivative or variant. Generally, these changes are made to a few amino acids in order to minimize alterations in the immunogenicity and specificity of the molecule, particularly the antigen-binding protein. However, in certain situations, larger changes may be tolerated. Conservative substitutions are generally made according to the following chart shown as Table 1.

[0044]

Table 1

[0045] Substantial changes in function or immunological identity are formed by selecting substitutions that are less conservative than those shown in Table 1. For example: substitutions that have a greater impact on the structure of the polypeptide backbone within the region of the change, such as an alpha-helix structure or a beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain may be formed. Substitutions that are generally expected to have the greatest change on the properties of the polypeptide are those in which (a) a hydrophilic residue, such as seryl or threonyl, is substituted with (or by) a hydrophobic residue, such as leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) cysteine or proline is substituted with (or by) any other residue; (c) a residue with an electropositive side chain, such as lysyl, arginyl, or histidyl, is substituted with (or by) an electronegative residue, such as glutamyl or aspartyl; or (d) a residue with a bulky side chain, such as phenylalanine, is substituted with (or by) a residue without a side chain, such as glycine.

[0046] Variants are also selected to modify the characteristics of the IL-2 mutant protein as needed, but the variants will typically exhibit the same qualitative biological activity as the naturally occurring analogs and will elicit the same immune response. Additionally, variants may be designed such that the biological activity of the IL-2 mutant protein is altered. For example, glycosylation sites may be altered or removed as described herein.

[0047] IL-2 mutant protein with an extended serum half-life The IL-2 mutant proteins provided herein preferentially expand Treg cells over, for example, Teff cells or NK cells, and thus the safety profile when administered to a patient is expected to be different from that of wild-type IL-2 or PROLEUKIN® (aldesleukin; Novartis, Basel, Switzerland). Side effects associated with wild-type IL-2 or PROLEUKIN® include influenza-like symptoms, chills / rigors, arthralgia, fever, rash, pruritus, injection site reactions, hypotension, diarrhea, nausea, anxiety, confusion, and depression. The IL-2 mutant proteins provided herein may be modified to include or fused to a molecule that extends the serum half-life of the mutant protein, and such an extension of the half-life does not increase the risk of increasing the likelihood or intensity of side effects or adverse events in the patient. Subcutaneous dosing of such a mutant protein with an extended serum half-life may allow for a longer target range with a lower maximum systemic exposure (C max ). An extended serum half-life may allow for a dosing regimen of the mutant protein that is less frequent or less frequent.

[0048] The serum half-life of the IL-2 variant proteins provided in this specification may be extended by essentially any method known in the art. Such methods include modifying the sequence of the IL-2 variant protein to include a peptide that binds to the neonatal Fcγ receptor, or to bind to a protein with an extended serum half-life, such as IgG or human serum albumin. In other embodiments, the IL-2 variant protein is fused to a polypeptide that confers an extended half-life to the fusion molecule. Such polypeptides include IgG Fc or other polypeptides that bind to the neonatal Fcγ receptor, human serum albumin, or polypeptides that bind to a protein with an extended serum half-life. In a preferred embodiment, the IL-2 variant protein is fused to an IgG Fc molecule.

[0049] The IL-2 variant protein may 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 significantly more IL-2 variant protein activity than when fused to the N-terminus of IgG Fc.

[0050] One embodiment of the invention relates to a dimer comprising two Fc fusion polypeptides created by fusing an IL-2 variant protein to the Fc region of an antibody. The dimer can be formed, for example, by inserting a gene fusion encoding the fusion protein into a suitable expression vector, expressing the gene fusion in a host cell transformed with the recombinant expression vector, and assembling the expressed fusion protein to be very similar to an antibody molecule, such that an interchain bond is formed between the Fc portions, resulting in a dimer.

[0051] As used herein, the term "Fc polypeptide" or "Fc region" includes the native and mutant protein forms of a polypeptide derived from the Fc region of an antibody and can be part of either the IL-2 mutant protein fusion protein or the anti-IL-2 antibody of the present invention. Also included are truncated forms of such polypeptides containing a hinge region that promotes dimerization. In certain embodiments, the Fc region includes the CH2 and CH3 domains of an antibody. Along with the extended serum half-life, fusion proteins containing the Fc portion (and oligomers formed therefrom) offer the advantage of being easily purified by affinity chromatography on a protein A column or a protein G column. Preferred Fc regions are derived from human IgG, including IgG1, IgG2, IgG3, and IgG4. In this specification, certain residues within the Fc are identified by position. All Fc positions are based on the EU numbering scheme.

[0052] One function of the Fc portion of an antibody is to communicate with the immune system when the antibody binds to its target. This is regarded as an "effector function". The communication leads to antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). ADCC and ADCP are mediated through the binding of the Fc to Fc receptors on the surface of cells of the immune system. CDC is mediated by the binding of the Fc to a complement system protein, such as C1q.

[0053] IgG subclasses differ in their ability to mediate effector functions. For example, IgG1 is much superior to IgG2 and IgG4 in mediating ADCC and CDC. Thus, in embodiments where effector function is not desired, IgG2 Fc would be preferred. However, IgG2 Fc-containing molecules are known to have less attractive biophysical properties such as being more difficult to manufacture and having a shorter half-life compared to IgG1 Fc-containing molecules.

[0054] The effector function of an antibody can be increased or decreased by introducing one or more mutations into the Fc. As an embodiment of the present invention, there is provided an IL-2 mutant protein Fc fusion protein having an Fc engineered to increase the effector function (U.S. Patent No. 7,317,091 and Strohl, Curr. Opin. Biotech., 20:685-691, 2009; both of which are incorporated herein by reference in their entirety). Exemplary IgG1 Fc molecules with increased effector function include those having the following substituents: 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.

[0055] Another way to enhance the effector function of IgG Fc-containing proteins is by reducing the fucosylation of Fc. Removal of core fucose from the bisecting complex-type oligosaccharides attached to Fc significantly increases the ADCC effector function without changing the antigen-binding or CDC effector functions. Several methods are known for reducing or abolishing the fucosylation of Fc-containing molecules, such as antibodies. These methods include recombinant expression in specific mammalian cell lines, including FUT8 knockout cell lines, mutant CHO cell line Lec13, rat hybridoma cell line YB2 / 0, cell lines containing small interfering RNAs specific for the FUT8 gene, and cell lines co-expressing β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II. In addition, Fc-containing molecules may be expressed in non-mammalian cells such as plant cells, yeast, or prokaryotic cells such as Escherichia coli (E. coli).

[0056] In certain embodiments, the IL-2 mutant protein Fc fusion protein or anti-IL-2 antibody of the present invention comprises an Fc engineered to have a reduced effector function. Exemplary Fc molecules with reduced effector function include those having 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).

[0057] Human IgG1 has a glycosylation site at N297 (EU numbering system), and glycosylation is known to contribute to the effector function of IgG1 antibodies. An exemplary IgG1 sequence is set forth in SEQ ID NO: 3:

Chemical Formula

[0058] The base has N297 mutated to form a non-glycosylated antibody. The mutation focuses on substituting N297 with an amino acid whose physicochemical properties are similar to asparagine, such as glutamine (N297Q), or alanine that mimics asparagine without a polar group (N297A).

[0059] As used herein, "non-glycosylated antibody" or "non-glycosylated 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 non-glycosylated antibody or non-glycosylated Fc fusion protein.

[0060] In an attempt to form an effector functionless IgG1 Fc, it has been discovered that the mutation of amino acid N297 of human IgG1 to glycine, i.e., N297G, provides much better purification efficiency and biophysical properties than other amino acid substitutions at that residue. See Example 8. Thus, in a preferred embodiment, the IL-2 mutant protein Fc fusion protein comprises a human IgG1 Fc having an N297G substitution. The Fc containing the N297G substitution is useful in any context where the molecule contains a human IgG1 Fc and is not limited to use in the context of an IL-2 mutant protein Fc fusion. In a particular embodiment, the antibody comprises an Fc having an N297G substitution.

[0061] In addition, the Fc containing human IgG1 Fc having the N297G mutation may further include insertions, deletions, and substitutions. In certain embodiments, the human IgG1 Fc includes the N297G substitution and is at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 3. In particularly preferred embodiments, the C-terminal lysine residue is substituted or deleted. The amino acid sequence of human IgG1 containing the N297G substitution and deletion of the C-terminal lysine is set forth in SEQ ID NO: 4.

[0062] Glycosylated IgG1 Fc-containing molecules have been shown to be less stable than glycosylated IgG1 Fc-containing molecules. The Fc region may further be engineered to increase the stability of non-glycosylated molecules. In some embodiments, one or more amino acids are substituted with cysteine to form disulfide bonds in the dimeric state. Residues V259, A287, R292, V302, L306, V323, or I332 of the amino acid sequence set forth in SEQ ID NO: 3 may be substituted with cysteine. In preferred embodiments, certain residue pairs are substitutions that preferentially form disulfide bonds with each other to limit or prevent scrambling of disulfide bonds. Preferred pairs include, but are not limited to, A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C.

[0063] Provided herein are Fc-containing molecules in which one or more of residues V259, A287, R292, V302, L306, V323, or I332 are substituted with cysteine, examples of which include those containing the substitutions of A287C and L306C, V259C and L306C, R292C and V302C, or V323C and I332C.

[0064] Additional mutations that may be made to IgG1 Fc include those that promote heterodimer formation among Fc-containing polypeptides. In some embodiments, the Fc region is engineered to create a "knob" and a "hole" that promote heterodimer formation of two different Fc-containing polypeptide chains when co-expressed in a cell. U.S. Patent No. 7,695,963. In other embodiments, the Fc region is modified to promote heterodimer formation while preventing homodimer formation of two different Fc-containing polypeptides when co-expressed in a cell using electrostatic steering. International Publication No. 09 / 089,004 (which is incorporated herein by reference in its entirety). Preferred heterodimeric Fc includes one in which one chain of the Fc contains the substitutions D399K and E356K and the other chain of the Fc contains the substitutions K409D and K392D. In other embodiments, one chain of the Fc contains the substitutions D399K, E356K, and E357K and the other chain of the Fc contains the substitutions K409D, K392D, and K370D.

[0065] In certain embodiments, it may be advantageous for the IL-2 mutant protein Fc fusion protein to be monomeric, i.e., to contain only a single IL-2 mutant protein molecule. Similarly, bispecific, trispecific, or tetravalent antibodies that can specifically bind to one or more additional targets may be desirable. 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 has reciprocal mutations relative to the Fc region within the IL-2 mutant protein Fc fusion polypeptide and is co-expressed with an Fc region lacking the IL-2 mutant protein or the anti-IL-2 heavy chain variable domain. When a heterodimer of two Fc-containing polypeptides is formed, the resulting protein contains only a single IL-2 mutant protein or anti-IL-2 binding domain.

[0066] As another method of making a monomeric IL-2 mutant protein Fc fusion protein, there is one in which the IL-2 mutant protein is fused to a monomeric Fc, that is, one in which the Fc region does not dimerize. Stable monomeric Fc contains mutations that prevent dimerization and stabilize the molecule in the monomeric form. Preferred monomeric Fc is disclosed in WO 2011 / 063348 pamphlet, which is hereby incorporated by reference in its entirety. In certain embodiments, the IL-2 mutant protein Fc fusion protein contains a charged amino acid at positions 392 and 409, along with a threonine substitution at Y349, L351, L368, V397, L398, F405, or Y407 in the Fc.

[0067] In certain embodiments, the IL-2 mutant protein Fc fusion protein contains a linker between the Fc and the IL-2 mutant protein. A number of different linker polypeptides are known in the art and may be used in the context of the IL-2 mutant protein Fc fusion protein. In preferred embodiments, the IL-2 mutant protein Fc fusion protein contains one or more copies of a peptide consisting of GGGGS (SEQ ID NO: 5), GGNGT (SEQ ID NO: 6), or YGNGT (SEQ ID NO: 7) between the Fc and the IL-2 mutant protein. In some embodiments, the polypeptide region between the Fc region and the IL-2 mutant protein region contains a single copy of GGGGS (SEQ ID NO: 5), GGNGT (SEQ ID NO: 6), or YGNGT (SEQ ID NO: 7). As shown herein, the linker GGNGT (SEQ ID NO: 6) or YGNGT (SEQ ID NO: 7) is glycosylated when expressed in appropriate cells, and such glycosylation can help stabilize the protein when administered in solution and / or in vivo. Thus, in certain embodiments, the IL-2 mutant protein fusion protein contains a glycosylated linker between the Fc region and the IL-2 mutant protein region.

[0068] Glycosylation linkers are thought to be useful when placed in the context of a polypeptide. Provided herein are polypeptides comprising GGNGT (SEQ ID NO:6) or YGNGT (SEQ ID NO:7) that are inserted into the amino acid sequence of a polypeptide or that substitute for one or more amino acids within the amino acid sequence of a polypeptide. In preferred embodiments, GGNGT (SEQ ID NO:6) or YGNGT (SEQ ID NO:7) is inserted into a loop of the polypeptide tertiary structure. In other embodiments, one or more amino acids of the loop are substituted with GGNGT (SEQ ID NO:6) or YGNGT (SEQ ID NO:7).

[0069] The C-terminal portion of Fc and / or the N-terminal portion of the IL-2 mutant protein may contain one or more mutations that alter the glycosylation profile of the IL-2 mutant protein Fc fusion protein when expressed in mammalian cells. In certain embodiments, the IL-2 mutant protein further comprises a T3 substitution, such as T3N or T3A. The IL-2 mutant protein may further comprise an S5 substitution such as S5T.

[0070] Covalent modifications of the IL-2 mutant protein and the IL-2 mutant protein Fc fusion protein, as well as anti-IL-2 antibodies, are included within the scope of the invention and generally occur post-translationally, but not necessarily so. For example, some types of covalent modifications are introduced by reacting the molecule with an organic derivatizing agent capable of reacting some of its amino acid residues with a selected side chain or N-terminal or C-terminal residue.

[0071] Cysteinyl residues are most commonly derivatized by reaction with α-haloacetates (and the corresponding amines), such as chloroacetic acid or chloroacetamide, to yield carboxymethyl or carboxamidomethyl derivatives. Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidazolyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoic acid, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0072] Histidyl residues are derivatized by reaction with diethyl pyrocarbonate at pH 5.5 - 7.0 because the agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful; the reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0.

[0073] Lysinyl and amino-terminal residues react with succinic anhydride or other carboxylic acid anhydrides. Derivatization with these agents has the effect of inverting the charge of the lysinyl residue. Other reagents suitable for derivatizing alpha-amino-containing residues include imidoesters, such as methyl picolinate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and the transaminase-catalyzed reaction with glyoxylate.

[0074] Arginyl residues are modified by reaction with one or several conventional reagents, especially phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be carried out under alkaline conditions because of the high pK of the guanidine functional group. Furthermore, the reagent may react with the lysine group and the arginine epsilon-amino group. a This is because of its high value.

[0075] Specific modification of tyrosine residues may be carried out, in particular, for the purpose of introducing spectral labels into tyrosine residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. The tyrosine residue is 125 I or 131 iodinated using I to prepare a labeled protein for use in radioimmunoassay, and the previously described chloramine T method is suitable.

[0076] Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with a carbodiimide (R'-N=C=N--R'), where R and R' are, in some cases, different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl residues and glutamyl residues are converted to asparaginyl residues and glutaminyl residues by reaction with ammonium ions.

[0077] Derivatization with bifunctional agents is useful for crosslinking antigen-binding proteins to water-insoluble support matrices or surfaces used in various methods. Commonly used crosslinking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters such as esters with 4-azidosalicylic acid, homobifunctional imide esters (such as disuccinimidyl esters including 3,3'-dithiobis(succinimidyl propionate), etc.), and bifunctional maleimides such as bis-N-maleimide-1,8-octane. Photochemically activatable intermediates capable of forming crosslinks in the presence of light can be obtained with derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propionimidate. In addition, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and reactive substrates described in U.S. Patent No. 3,969,287; U.S. Patent No. 3,691,016; U.S. Patent No. 4,195,128; U.S. Patent No. 4,247,642; U.S. Patent No. 4,229,537; and U.S. Patent No. 4,330,440 are used for protein immobilization.

[0078] Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. In addition, these residues are deamidated under weakly acidic conditions. Any form of these residues is included within the scope of the present invention.

[0079] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of a seryl or threonyl residue, methylation of the α-amino group of the lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, pp. 79-86), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0080] Another type of covalent modification of the IL-2 mutant protein, IL-2 mutant protein Fc fusion, or anti-IL-2 antibody that is included within the scope of the present invention involves changing the glycosylation pattern of the protein. As is known in the art, the glycosylation pattern can be determined by both the protein sequence (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.

[0081] Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of a 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 other than proline) are recognition sequences for the enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of either 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.

[0082] Addition of glycosylation sites to an IL-2 mutant protein, an IL-2 mutant protein Fc fusion, or an anti-IL-2 antibody can be successfully achieved by altering the amino acid sequence such that it contains one or more of the above tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by addition of one or more serine or threonine residues to the starting sequence, or substitution therewith, for O-linked glycosylation sites. For ease, the amino acid sequence of the IL-2 mutant protein, the IL-2 mutant protein Fc fusion, or the anti-IL-2 antibody is preferably altered by mutating the DNA encoding the target polypeptide with a preselected base so as to generate a codon that will translate into the desired amino acid, via a change at the DNA level.

[0083] As another means of increasing the number of carbohydrate moieties on an IL-2 mutant protein, an IL-2 mutant protein Fc fusion, or an anti-IL-2 antibody, there is chemical or enzymatic coupling of a glycoside to the protein. This procedure is advantageous in that it does not require production of the protein in a glycosylation-competent host cell for either N-linked or O-linked glycosylation. Depending on the coupling mode used, the sugar may be linked to (a) arginine and histidine, (b) a free carboxyl group, (c) a free sulfhydryl group, such as that of cysteine, (d) a free hydroxyl group, such as that of serine, threonine, or hydroxyproline, (e) an aromatic residue, such as that of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in International Publication No. 87 / 05330 pamphlet published on September 11, 1987 and Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306.

[0084] Removal of carbohydrate moieties present on starting IL-2 mutant proteins, IL-2 mutant protein Fc fusions, or anti-IL-2 antibodies may be accomplished either chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment cleaves most or all of the sugars, leaving the polypeptide intact, except for the attached sugar (N-acetylglucosamine or N-acetylgalactosamine). Chemical deglycosylation is described by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and Edge et al., 1981, Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties on polypeptides can be accomplished by use of various endoglycosidases and exoglycosidases, as described by Thotakura et al., 1987, Meth. Enzymol. 138:350. Glycosylation at potential glycosylation sites can be inhibited by use of the compound tunicamycin, described by Duskin et al., 1982, J. Biol. Chem. 257:3105. Tunicamycin blocks the formation of protein-N-glycoside bonds.

[0085] Another type of covalent modification of the IL-2 mutant protein, the IL-2 mutant protein Fc fusion, or the anti-IL-2 antibody involves binding the protein to various non-proteinaceous polymers including, but not limited to, various polyols such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene, by the techniques described in U.S. Patent No. 4,640,835; U.S. Patent No. 4,496,689; U.S. Patent No. 4,301,144; U.S. Patent No. 4,670,417; U.S. Patent No. 4,791,192, or U.S. Patent No. 4,179,337. In addition, amino acid substitutions may be made at various positions within the IL-2 mutant protein, the IL-2 mutant protein Fc fusion, or the anti-IL-2 antibody to facilitate the addition of polymers such as PEG. Thus, embodiments of the invention include PEGylated IL-2 mutant proteins, IL-2 mutant protein Fc fusions, or anti-IL-2 antibodies. Such PEGylated proteins may have an increased half-life and / or a decreased immunogenicity compared to their non-PEGylated forms.

[0086] Polynucleotides encoding an IL-2 mutant protein and an IL-2 mutant protein Fc fusion protein Included within the present invention are nucleic acids encoding an IL-2 mutant protein, an IL-2 mutant protein Fc fusion, or an anti-IL-2 antibody. Aspects of the invention include polynucleotide variants (e.g., due to degeneracy) encoding the amino acid sequences described herein.

[0087] The nucleotide sequences corresponding to the amino acid sequences described herein, which are to be used as probes or primers for nucleic acid isolation or as query sequences for database searches, can be obtained by "reverse translation" from the amino acid sequences. Using well-known polymerase chain reaction (PCR) procedures, DNA sequences encoding the IL-2 mutant protein and the IL-2 mutant protein Fc fusion protein can be isolated and amplified. Oligonucleotides that define the desired ends of the DNA fragment combinations are used as 5' primers and 3' primers. The oligonucleotides can additionally contain recognition sites for restriction endonucleases to facilitate the insertion of the amplified combinations of DNA fragments into expression vectors. The PCR technique 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).

[0088] The nucleic acid molecules of the present invention include DNA and RNA in both single-stranded and double-stranded forms, as well as corresponding complementary sequences. An "isolated nucleic acid" is, in the case of a nucleic acid isolated from its natural source, a nucleic acid that is separated from the adjacent gene sequences present in the genome of the organism from which the nucleic acid was isolated. In the case of a nucleic acid enzymatically or chemically synthesized from a template, such as a PCR product, a cDNA molecule, or an oligonucleotide, a nucleic acid derived from such a process is understood to be an isolated nucleic acid. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. In a preferred embodiment, the nucleic acid is substantially free of contaminating endogenous substances. The nucleic acid molecule is preferably in a substantially pure form and, by standard biochemical methods (e.g., those outlined in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)), is derived from DNA or RNA that has been isolated at least once in an amount or concentration that allows for the identification, manipulation, and recovery of its component nucleotide sequences. Such sequences are preferably provided and / or constructed in the form of an open reading frame that is typically not interrupted by internal non-translated sequences or introns that are present within eukaryotic genes. The sequences of non-translated DNA can be present 5' or 3' to the open reading frame, in which case this does not interfere with the manipulation or expression of the coding region.

[0089] The IL-2 mutant proteins according to the present invention are typically prepared by site-directed mutagenesis of nucleotides in the DNA encoding the IL-2 mutant protein or IL-2 mutant protein Fc fusion protein using cassette or PCR mutagenesis, or other techniques well known in the art, to generate DNA encoding the variant, and then expressing the recombinant DNA in cell culture as outlined herein. However, the IL-2 mutant proteins and IL-2 mutant protein Fc fusions may also be prepared by in vitro synthesis using established techniques. Variants typically exhibit the same qualitative biological activity as naturally occurring analogs, e.g., Treg proliferation, but variants with modified characteristics can also be selected as described in more detail below.

[0090] 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 formed using standard techniques. Thus, given a particular amino acid sequence, one of ordinary skill in the art can form any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the encoded protein.

[0091] The present invention also provides expression systems and constructs in the form of plasmids, expression vectors, transcription or expression cassettes containing at least one of the foregoing polynucleotides. In addition, the present invention provides host cells containing such expression systems or constructs.

[0092] Typically, an expression vector used in any host cell will contain sequences for maintaining the plasmid, as well as sequences for cloning and expressing exogenous nucleotide sequences. Such sequences, collectively referred to as "flanking sequences", in certain embodiments will typically include 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 the polypeptide to be expressed, and one or more of selectable marker elements. Each of these sequences will be discussed below.

[0093] In some cases, the vector may contain a "tag" coding sequence, i.e., an oligonucleotide molecule located at the 5' or 3' end of an IL-2 mutant protein, an IL-2 mutant protein Fc fusion, or an anti-IL-2 antibody coding sequence; the oligonucleotide sequence encodes a polyHis (such as hexahistidine (SEQ ID NO: 8)), or another "tag", e.g., FLAG, HA (hemagglutinin influenza virus), or myc, for which a commercially available antibody exists. This tag is typically fused to the polypeptide immediately after expression of the polypeptide and can function as a means for affinity purification or detection of the polypeptide from the host cell. Affinity purification can be achieved, for example, by column chromatography using an antibody against the tag as an affinity matrix. In some cases, the tag can then be removed by various means, e.g., using a specific peptidase for cleavage.

[0094] 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 two or more sources), synthetic, or natural. Thus, the source of the flanking sequence may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence is functional in the host cell machinery and can be activated by that machinery.

[0095] The flanking sequences useful in the vectors of the present invention can be obtained by any of several methods well known in the art. Typically, since the flanking sequences useful herein will have been previously identified by mapping and / or restriction endonuclease digestion, they can be isolated from appropriate tissue sources using appropriate restriction endonucleases. Optionally, the entire nucleotide sequence of the flanking sequence may be known. In this case, the flanking sequence can be synthesized using the methods described herein for nucleic acid synthesis or cloning.

[0096] Regardless of whether all or only a portion of the flanking sequence is known, the flanking sequence can be obtained by screening a genomic library using the polymerase chain reaction (PCR) and / or appropriate probes such as oligonucleotides and / or flanking sequence fragments from the same or a different species. If the flanking sequence is unknown, a fragment of DNA containing the flanking sequence can be isolated from a larger DNA fragment that may contain, for example, the coding sequence or even one or more other genes. Isolation can be accomplished by generating an appropriate DNA fragment by digestion with a restriction endonuclease and then isolating it using agarose gel purification, Qiagen® column chromatography (Chatsworth, CA), or other methods known to those skilled in the art. The selection of an enzyme suitable for this purpose will be readily apparent to those skilled in the art.

[0097] The origin of replication is typically part of a commercially available prokaryotic expression vector, and this origin aids in the amplification of the vector within the host cell. If the selected vector does not contain an origin of replication site, the site can be chemically synthesized based on known sequences and ligated into the vector. For example, the origin of replication derived from plasmid pBR322 (New England Biolabs, Beverly, MA) is suitable for most Gram-negative bacteria, and various viral origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV), or papillomavirus, such as HPV or BPV) are useful for cloning vectors in mammalian cells. Generally, an origin of replication component is not necessary for mammalian expression vectors (e.g., the SV40 origin is often used only because it also contains the viral early promoter in many cases).

[0098] The transcription termination sequence is typically located 3' to the end of the polypeptide coding region and functions to terminate transcription. Usually, the transcription termination sequence in prokaryotic cells is a G-C rich fragment followed by a poly T sequence. Such sequences can be easily cloned from a library or even commercially purchased as part of a vector, and can also be easily synthesized using nucleic acid synthesis methods such as those described herein.

[0099] A selectable marker gene encodes a protein essential for the survival and growth of host cells grown in a selective medium. Typical selectable marker genes confer (a) resistance to antibiotics or other toxins, such as ampicillin, tetracycline, or kanamycin, on prokaryotic host cells; (b) complement auxotrophic deficiencies of the cells; or (c) encode a protein that supplies an essential nutrient not available from complex or defined media. Specific selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. Advantageously, the neomycin resistance gene can also be used for selection in both prokaryotic and eukaryotic host cells.

[0100] Other selectable genes can be used to amplify the gene to be expressed. Amplification is the process by which genes required for the production of proteins important for growth or cell survival are tandemly repeated within the chromosome of successive generations of recombinant cells. Examples of selectable markers suitable for mammalian cells include the dihydrofolate reductase (DHFR) gene and the promoterless thymidine kinase gene. Mammalian cell transformants are placed under a selection pressure uniquely adapted such that only the transformants survive by the selectable gene present in the vector. The selection pressure is imposed by culturing the transformed cells under conditions where the concentration of the selection agent in the medium is continuously increased, thereby leading to the amplification of both the selectable gene and, as a result, the gene encoding the desired polypeptide, e.g., an IL-2 mutant protein, an IL-2 mutant protein Fc fusion, or the heavy and / or light chains of an anti-IL-2 antibody. As a result, large amounts of polypeptide are synthesized from the amplified DNA.

[0101] The ribosome binding site is usually essential for the initiation of mRNA translation and is characterized by the Shine-Dalgarno sequence (for prokaryotes) or the Kozak sequence (for eukaryotes). This element is typically positioned 3' of the promoter and 5' of the coding sequence of the polypeptide to be expressed. In certain embodiments, one or more coding regions may be operably linked to an internal ribosome entry site (IRES), enabling translation of two open reading frames from a single RNA transcript.

[0102] In cases where glycosylation is desired, such as in a eukaryotic host cell expression system, various pre-sequences or pro-sequences may be manipulated to improve glycosylation or yield. For example, the peptidase cleavage site of a specific signal peptide may be altered or a pro-sequence may be added, which can also affect glycosylation. The final protein product may have one or more additional amino acids associated with expression at the -1 position (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 within the peptidase cleavage site attached to the amino terminus. Additionally, the use of some enzyme cleavage sites may result in a slightly cleaved form of the desired polypeptide if the enzyme cuts at such regions within the mature polypeptide.

[0103] The expression vectors and cloning vectors of the present invention typically contain a promoter that is recognized by the host organism and is operably linked to a molecule encoding an IL-2 mutant protein, an IL-2 mutant protein Fc fusion, or a heavy chain and / or light chain of an anti-IL-2 antibody. A promoter is a non-transcribed sequence that controls the transcription of a structural gene and is positioned upstream (i.e., on the 5' side) of the start codon of the structural gene (generally within about 100 to 1000 bp). Conventionally, promoters are grouped into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate an increase in the transcription level from DNA in response to some change in culture conditions, such as the presence or absence of nutrients or a change in temperature, under their control. On the other hand, constitutive promoters transcribe the gene to which they are operably linked uniformly, i.e., with little or no control over gene expression. A number of promoters recognized by various potential host cells are well known.

[0104] Promoters suitable for use in yeast hosts are also well known in the art. Advantageously, yeast enhancers are used in conjunction with yeast promoters. Promoters suitable for use in mammalian host cells are well known and include, but are not limited to, those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus type 2), bovine papilloma virus, Rous sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and most preferably simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters such as the heat shock promoter and the actin promoter.

[0105] Additional promoters that may be targeted 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. U.S.A. 81:659 - 663); the promoter contained in the long terminal repeat on the 3' side of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787 - 797); the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1444 - 1445); the promoter and regulatory sequences derived from the metallothionein gene (Prinster et al., 1982, Nature 296:39 - 42); and prokaryotic promoters such as the beta - lactamase promoter (Villa - Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727 - 3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21 - 25). Also targeted are the following animal transcriptional control regions that exhibit tissue specificity and are utilized in transgenic animals: the elastase I gene control region that is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639 - 646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399 - 409; MacDonald, 1987, Hepatology 7:425 - 515); the insulin gene control region that is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115 - 122); the immunoglobulin gene control region that is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647 - 658; Adames et al., 1985, Nature 318:533 - 538; Alexander et al., 1987, Mol. Cell. Biol.7:1436 - 1444); mouse mammary tumor virus control regions that are active in testicular cells, breast cells, lymphocyte - like cells, and mast cells (Leder et al., 1986, Cell 45:485 - 495); albumin gene control regions that are active in the liver (Pinkert et al., 1987, Genes and Devel. 1:268 - 276); alpha - fetoprotein gene control regions that are active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639 - 1648; Hammer et al., 1987, Science 253:53 - 58); alpha1 - antitrypsin gene control regions that are active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161 - 171); beta - globin gene control regions that are active in bone marrow cells (Mogram et al, 1985, Nature 315:338 - 340; Kollias et al, 1986, Cell 46:89 - 94); myelin basic protein gene control regions that are active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703 - 712); myosin light chain 2 gene control regions that are active in skeletal muscle (Sani, 1985, Nature 314:283 - 286); and gonadotropin - releasing hormone gene control regions that are active in the hypothalamus (Mason et al., 1986, Science 234:1372 - 1378).

[0106] An enhancer sequence may be inserted into a vector to increase transcription by higher eukaryotes. An enhancer is a cis-acting element of DNA, usually about 10 - 300 bp in length, which acts on a promoter to increase transcription. Enhancers are relatively independent of direction and position and are found at positions both 5' and 3' to the transcription unit. Several enhancer sequences available from mammalian genes are known (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). However, typically, enhancers derived from viruses are used. The SV40 enhancer, cytomegalovirus immediate early promoter enhancer, polyoma enhancer, and adenovirus enhancer known in the art are exemplary enhancer elements for the activation of eukaryotic promoters. The enhancer may be located either 5' or 3' to the coding sequence in the vector, but is typically positioned at a site 5' to the promoter. A sequence encoding an appropriate native or heterologous signal sequence (leader sequence or signal peptide) can be incorporated into the expression vector to facilitate the extracellular secretion of the IL-2 mutant protein, the IL-2 mutant protein Fc fusion, or the heavy and / or light chains of the anti-IL-2 antibody. The choice of signal peptide or leader depends on the type of host cell in which the protein is to be produced, and a heterologous signal sequence can replace the native signal sequence. Examples of signal peptides that function in mammalian host cells include: the signal sequence of interleukin-7 (IL-7) described in U.S. Patent No. 4,965,195; the signal sequence of the interleukin-2 receptor described in Cosman et al., 1984, 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 European Patent No. 0460846. In one embodiment, the IL-2 mutant protein Fc fusion of the present invention comprises a leader sequence as shown in Figure 24.

[0107] The vector may contain one or more elements that promote expression when the vector is integrated into the host cell genome. Examples include the EASE element (Aldrich et al. 2003 Biotechnol Prog. 19:1433-38) and the matrix attachment region (MAR). The MAR can protect the integrated vector from "position" effects by mediating the structural organization of chromatin. Thus, the MAR is particularly useful when the vector is used to generate stable transfectants. Several natural and synthetic MAR-containing nucleic acids are known in the art, for example, in U.S. Patent No. 6,239,328; U.S. Patent No. 7,326,567; U.S. Patent No. 6,177,612; U.S. Patent No. 6,388,066; U.S. Patent No. 6,245,974; U.S. Patent No. 7,259,010; U.S. Patent No. 6,037,525; U.S. Patent No. 7,422,874; U.S. Patent No. 7,129,062.

[0108] The expression vector of the present invention may be constructed from an initial vector such as a commercially available vector. Such a vector may or may not contain all of the desired flanking sequences. If one or more of the flanking sequences described herein are not initially present in the vector, they may be obtained individually and ligated into the vector. The methods used to obtain each of the flanking sequences are well known to those skilled in the art.

[0109] A vector is constructed such that a nucleic acid molecule encoding an IL-2 mutant protein, an IL-2 mutant protein Fc fusion, or a heavy chain and / or light chain of an anti-IL-2 antibody is inserted into an appropriate site of the vector, and then the complete vector may be inserted into a host cell suitable for amplification and / or polypeptide expression. Transformation of the expression vector into the selected host cell may be achieved by well-known methods such as transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran-mediated transfection, or other known techniques. The method selected will depend, in part, on the type of host cell to be used. These methods and other suitable methods are well known to those skilled in the art and are shown, for example, in the aforementioned Sambrook et al., 2001.

[0110] When cultured under appropriate conditions, the host cell synthesizes an IL-2 mutant protein, an IL-2 mutant protein Fc fusion, or a heavy chain and / or light chain of an anti-IL-2 antibody, which can then be collected from the medium (if the host cell secretes into the medium) or directly from the producing host cell (if not secreted). The selection of an appropriate host cell will depend on various factors such as the desired expression level, polypeptide modifications (such as glycosylation or phosphorylation) desirable or essential for activity, and ease of folding into a biologically active molecule. The host cell may be eukaryotic or prokaryotic.

[0111] Mammalian cell lines that can be used as hosts for expression 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 used in expression systems known in the art can be used to form the recombinant polypeptides of the present invention. Generally, host cells are transformed with a recombinant expression vector containing DNA encoding the desired IL-2 mutant protein, IL-2 mutant protein Fc fusion, or anti-IL-2 antibody. Host cells that may be used include prokaryotes, yeast, or higher eukaryotic cells. Examples of prokaryotes include gram-negative or gram-positive organisms, such as Escherichia coli (E. coli) or Bacilli. Examples of higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL1651) (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, such as Veggie CHO and related cell lines that grow in serum-free medium (Rasmussen et al., 1998, Cytotechnology 28:31), HeLa cells, BHK (ATCC CRL10) cell line, and the CVI / EBNA cell line derived from the African green monkey kidney cell line CVI (ATCC CCL70) as described in McMahan et al., 1991, EMBO J. 10:2821, human fetal kidney cells, such as 293, 293EBNA or MSR293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro cultures of primary tissues, primary explants, HL-60 cells, U937 cells, HaK cells, or Jurkat cells. In some cases, mammalian cell lines, such as HepG2 / 3B, KB, NIH3T3, or S49, may be used for polypeptide expression when it is desirable to use the polypeptide in various signal transduction assays or reporter assays.

[0112] In addition to this, it is possible to produce polypeptides in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Suitable yeasts include Saccharomyces cerevisiae, Schizosaccharomyces pombe, strains of the genus Kluyveromyces, the genus 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 formed within the yeast or bacteria, it may be desirable to modify the polypeptide produced within the yeast or bacteria, for example, by phosphorylation or glycosylation at appropriate sites, to obtain a functional polypeptide. Such covalent bonds can be achieved using known chemical or enzymatic methods.

[0113] Alternatively, the polypeptide can be produced by operably linking the isolated nucleic acid of the invention to suitable control sequences in one or more insect expression vectors and using an insect expression system. Materials and methods for baculovirus / insect cell expression systems are commercially available, for example, in kit form from Invitrogen, San Diego, Calif., U.S.A. (MaxBac® kit), and such methods are well known in the art as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), and Luckow and Summers, Bio / Technology 6:47 (1988). Cell-free translation systems can also be used to produce polypeptides using RNA derived from the nucleic acid constructs disclosed herein. Cloning vectors and expression vectors suitable for use in 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 the isolated nucleic acid of the invention, preferably operably linked to at least one expression control sequence, is a "recombinant host cell".

[0114] In certain embodiments, the present invention preferentially stimulates regulatory T cells and 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; 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; V91H, D20A, E16Q; V91K, D20A, M104L; V91H, D20A; V91K, 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 an isolated nucleic acid encoding a human IL-2 mutant protein comprising 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;

[0115] Also included is an isolated nucleic acid encoding any of the exemplary IL-2 mutant protein Fc fusion proteins described herein. In a preferred embodiment, the Fc portion of the antibody and the human IL-2 mutant protein are optionally encoded with a linker between the Fc region and the IL-2 mutant protein and are encoded within a single open reading frame.

[0116] In another aspect, provided herein is an expression vector comprising a nucleic acid encoding the above-described IL-2 mutant protein or IL-2 mutant protein Fc fusion protein operably linked to a promoter.

[0117] In another aspect, provided herein is a host cell comprising an isolated nucleic acid encoding the above-described IL-2 mutant protein, IL-2 mutant protein Fc fusion protein, or anti-IL-2 antibody. The host cell may be a prokaryotic cell such as E. coli or a eukaryotic cell such as a mammalian cell. In a particular embodiment, the host cell is a Chinese hamster ovary (CHO) cell line.

[0118] In another aspect, provided herein is a method of forming a human IL-2 mutant protein. The method includes culturing a host cell under conditions in which a promoter operably linked to the human IL-2 mutant protein is expressed. Thereafter, the human IL-2 mutant protein is harvested from the culture. The IL-2 mutant protein can be harvested from the culture medium and / or the host cell lysate.

[0119] In another aspect, provided herein is a method of forming 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 the human IL-2 mutant protein Fc fusion protein is expressed. Thereafter, 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 the culture medium and / or host cell lysates.

[0120] In another aspect, provided herein is a method of forming an anti-IL-2 antibody. The method includes culturing a host cell under conditions in which a promoter operably linked to the heavy and light chains of the anti-IL-2 antibody is expressed. Thereafter, the anti-IL-2 antibody is harvested from the culture. The anti-IL-2 antibody can be harvested from the culture medium and / or host cell lysates.

[0121] Pharmaceutical composition In some embodiments, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of an IL-2 mutant protein or 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 invention include, but are not limited to, liquid compositions, frozen compositions, and lyophilized compositions.

[0122] Preferably, the formulation materials are non-toxic to the recipient at the dosages and concentrations employed. In certain embodiments, a pharmaceutical composition is provided that comprises a therapeutically effective amount of a therapeutic molecule containing an IL-2 mutant protein, such as an IL-2 mutant protein Fc fusion.

[0123] In certain embodiments, the pharmaceutical composition may contain formulation materials for modifying, maintaining, or preserving one or more of the composition's properties, such as, for example, pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution rate or release rate, absorbability, or permeability. In such embodiments, suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine, proline, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); coloring agents, flavoring agents, and diluents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as polysorbates (such as pluronics, PEG, sorbitan esters, polysorbate 20), polysorbates, tritons, tromethamine, lecithin, cholesterol, tyloxapol); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; excipients, and / or pharmaceutical adjuvants, but are not limited thereto. See REMINGTON’S PHARMACEUTICAL SCIENCES, 18th Edition, (A.R. Genrmo, ed.), 1990, Mack Publishing Company.

[0124] In certain embodiments, the optimal pharmaceutical composition will be determined by one of ordinary skill in the art, for example, depending on the intended route of administration, delivery format, and desired dosage. See, e.g., REMINGTON’S PHARMACEUTICAL SCIENCES, supra. 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 major vehicle or carrier in the pharmaceutical composition can actually be either aqueous or non-aqueous. For example, suitable vehicles or carriers can be water for injection, physiological saline, or artificial cerebrospinal fluid, although other materials common in compositions for parenteral administration may be added. Neutral buffered saline, or saline mixed with serum albumin, are further exemplary vehicles. In certain embodiments, the pharmaceutical composition includes a Tris buffer at about pH 7.0 - 8.5, or an acetate buffer at about pH 4.0 - 5.5, and may further include sorbitol or a suitable alternative thereof. In certain embodiments of the present invention, the Il-2 mutant protein composition or anti-IL-2 antibody composition may be prepared for storage in the form of a lyophilized cake or an aqueous solution by mixing a selected composition having the desired purity with optional pharmaceutical agents (REMINGTON’S PHARMACEUTICAL SCIENCES, supra). Further, in certain embodiments, the IL-2 mutant protein or anti-IL-2 antibody product may be formulated as a lyophilized product using a suitable excipient such as sucrose.

[0125] The pharmaceutical compositions of the present invention can be selected for parenteral delivery. Additionally, the compositions may be selected for delivery by inhalation or via the gastrointestinal tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the scope of the art. The formulation components are preferably present at concentrations acceptable to the site of administration. In certain embodiments, buffers are used to maintain the composition within a physiological pH or slightly lower pH, typically in the pH range of about 5 to about 8.

[0126] When parenteral administration is intended, the therapeutic compositions used in the present invention may be provided in the form of a pyrogen-free parenterally acceptable aqueous solution containing the desired IL-2 mutant protein or anti-IL-2 antibody composition in a pharmaceutically acceptable vehicle. A vehicle particularly suitable for parenteral injection is sterile distilled water, in which the mutant protein or anti-IL-2 antibody composition is formulated as a sterile isotonic solution and properly retained. In certain embodiments, the preparation may include the formulation of the desired molecule with an agent capable of achieving controlled or sustained release of the product, which can be delivered via depot injection, such as injectable microspheres, biodegradable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes. In certain embodiments, hyaluronic acid having an effect of enhancing the duration in the circulatory system may be used. In certain embodiments, an implantable drug delivery device may be used to introduce the IL-2 mutant protein or anti-IL-2 antibody composition.

[0127] Additional pharmaceutical compositions will be apparent to those skilled in the art that include a formulation containing an IL-2 mutant protein composition or an anti-IL-2 antibody composition in a sustained release formulation or a controlled release formulation. Also, various other sustained or controlled delivery means, such as liposome carriers, biodegradable microparticles, or porous beads, and techniques for formulating depot injections are known to those skilled in the art. See, for example, International Application PCT / US93 / 00829 (incorporated by reference). This document describes the controlled release of porous polymer microparticles for delivering pharmaceutical compositions. Sustained release preparations may include a semipermeable polymer matrix in the form of a molded article, such as a film or microcapsule. Sustained release matrices can include polyesters, hydrogels, polylactides (disclosed in U.S. Patent No. 3,773,919 and European Patent Application Publication No. 058481, each of which is incorporated by reference), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinyl acetate (Langer et al., 1981, supra), or poly-D(-)-3-hydroxybutyric acid (European Patent Application Publication No. 133,988). Also, sustained release compositions can 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. U.S.A. 82:3688-3692; European Patent Application Publication No. 036,676; European Patent Application Publication No. 088,046, and European Patent Application Publication No. 143,949, each incorporated by reference.

[0128] Pharmaceutical compositions for in vivo administration are typically provided as sterile preparations. Sterilization can be achieved by filtration through a sterile filtration membrane. When the composition is lyophilized, sterilization using this method may be carried out either before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution. Parenteral compositions are generally placed in a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper penetrable by a hypodermic needle.

[0129] Aspects of the invention include self - buffering IL - 2 mutant protein formulations or anti - IL - 2 antibody formulations, which can be used as pharmaceutical compositions as described in International Publication No. WO 2006 / 138181 (A2) (PCT / US2006 / 022599), which is hereby incorporated by reference in its entirety.

[0130] As previously discussed, certain embodiments provide pharmaceutical Il - 2 mutant protein Fc fusion proteins that include an IL - 2 mutant protein composition or an anti - IL - 2 antibody composition, particularly in addition to the IL - 2 mutant protein or anti - IL - 2 antibody composition, one or more excipients such as those exemplified elsewhere in this section and in this specification. Excipients can be used in the present invention for a wide range of purposes, such as adjusting the physical, chemical, or biological properties of the formulation, such as adjusting viscosity, and / or improving efficacy and / or stabilizing such a formulation, as well as processes for degradation and spoilage due to stress occurring, for example, during manufacture, transportation, storage, preparation before use, administration, and after these.

[0131] Various explanations are possible for the stabilization of proteins and for pharmaceutical materials and methods useful in this regard. For example, Arakawa 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), and in particular, in the part regarding excipients for self-buffering protein formulations according to the present invention and its process, especially regarding protein pharmaceutical products and processes for veterinary and / or human medical use, various explanations are possible.

[0132] According to certain embodiments of the present invention, salts can be used to, for example, adjust the ionic strength and / or isotonicity of a formulation, and / or improve the solubility and / or physical stability of a protein or other component of a composition according to the present invention.

[0133] As is well known, ions can stabilize a protein in its native state by binding to charged residues on the surface of the protein and by shielding the charged and polar groups in the protein to reduce the strength of its electrostatic interactions, attractive forces, and repulsive interactions. Also, ions can stabilize a protein in its denatured state, particularly by binding to the denatured peptide bonds (--CONH) of the protein. Furthermore, ionic interactions with charged and polar groups in the protein can reduce intermolecular electrostatic interactions, thereby also preventing or reducing protein aggregation and insolubilization.

[0134] Ionic species have significantly different effects on proteins. Some rankings of ions and the effects on proteins have been developed and can be used in formulating the pharmaceutical compositions according to the present invention. One example is the Hofmeister series that ranks ionic solutes and polar non-ionic solutes according to their effects on the conformational stability of proteins in solution. Stabilizing solutes are termed "cosmotropic." Destabilizing solutes are termed "chaotropic." Cosmotropes are generally used at high concentrations (e.g., >1 molar ammonium sulfate) to precipitate ("salting out") proteins from solution. Chaotropes are generally used to denature and / or solubilize ("salting in") proteins. The relative effectiveness of ions for "salting in" and "salting out" defines the position of the ions in the Hofmeister series.

[0135] Free amino acids can be used in IL-2 mutant protein formulations or anti-IL-2 antibody formulations according to various embodiments of the present invention as bulking agents, stabilizers, and antioxidants and for other standard uses. Lysine, proline, serine, and alanine can be used to stabilize proteins in formulations. Glycine is useful in lyophilization to ensure the correct cake structure and properties. Arginine can be useful in inhibiting protein aggregation in both liquid and lyophilized formulations. Methionine is useful as an antioxidant.

[0136] Polyols include sugars such as mannitol, sucrose, and sorbitol, as well as polyhydric alcohols such as glycerol and propylene glycol, and for the purposes of discussion herein, polyethylene glycol (PEG) and related substances. Polyols are cosmotrophic. Polyols are useful stabilizers for protecting proteins from physical and chemical degradation processes in both liquid and lyophilized formulations. Also, polyols are useful for adjusting the tonicity of formulations.

[0137] Among the polyols, mannitol is useful in a selected embodiment of the present invention, which is generally used to ensure the structural stability of the cake in lyophilized formulations. Mannitol ensures the structural stability of the cake. Mannitol is generally used together with a lyoprotectant such as sucrose. Sorbitol and sucrose are preferred agents among the preferred agents as stabilizers for adjusting tonicity and protecting against freeze-thaw stress during transport or from the preparation of the bulk during the manufacturing process. Reducing sugars (containing free aldehyde or ketone groups), such as glucose and lactose, can glycosylate surface lysine and arginine residues. Therefore, reducing sugars generally do not enter into the preferred polyols for use according to the present invention. In addition, sugars such as sucrose that form such reactive species also do not enter into the preferred polyols of the present invention in that they are hydrolyzed to fructose and glucose under acidic conditions, resulting in glycosylation. PEG is useful for stabilizing proteins and as a cryoprotective substance and can be used in the present invention in this regard.

[0138] Embodiments of the IL-2 variant protein formulation and / or the anti-IL-2 antibody formulation further include a surfactant. Protein molecules are susceptible to surface adsorption and the effects of denaturation and resultant aggregation at gas-liquid interfaces, solid-liquid interfaces, and liquid-liquid interfaces. These effects generally vary inversely with protein concentration. These detrimental interactions generally vary inversely with protein concentration and are typically exacerbated by physical agitation that occurs, for example, during product transport and handling.

[0139] Surfactants are routinely used to prevent, minimize, or reduce surface adsorption. In this regard, useful surfactants in the present invention include polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan polyethoxylate, and poloxamer 188.

[0140] Also, surfactants are generally used to control the conformational stability of proteins. In this regard, the use of surfactants is protein-specific since any given surfactant will typically stabilize some proteins and destabilize others.

[0141] Polysorbates are susceptible to oxidative degradation and often contain sufficient amounts of peroxide to cause oxidation of the side chains of protein residues, particularly methionine, if supplied. As a result, polysorbates should be used with caution and, when used, should be used at the lowest effective concentration. In this regard, polysorbates exemplify the principle that excipients should be used at the lowest effective concentration.

[0142] Embodiments of the IL-2 mutant protein preparation or anti-IL-2 antibody preparation further contain one or more antioxidants. The harmful oxidation of proteins in pharmaceutical preparations can be prevented to some extent by maintaining appropriate levels of ambient oxygen and ambient temperature and by avoiding exposure to light. Antioxidant excipients can likewise be used to prevent the oxidative degradation of proteins. Useful antioxidants in this regard include reducing agents, oxygen / free radical scavengers, and chelating agents. The antioxidants used in the therapeutic protein preparations 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.

[0143] Antioxidants can potentially damage proteins. For example, reducing agents such as glutathione can particularly disrupt intramolecular disulfide bonds. Therefore, the antioxidants used in the present invention are selected such that, inter alia, they rule out or sufficiently reduce the possibility of themselves damaging the proteins in the preparation.

[0144] The preparations according to the present invention may contain metal ions that are protein cofactors and are essential for forming protein coordination complexes, such as zinc that is essential for forming certain insulin suspensions. Also, metal ions can inhibit some processes that degrade proteins. However, metal ions can also catalyze physical and chemical processes that degrade proteins.

[0145] Isomerization of aspartic acid to isoaspartic acid can be inhibited using magnesium ions (10-120 mM). Ca +2 ions (up to 100 mM) can increase the stability of human deoxyribonuclease. However, Mg +2 , Mn +2 , and Zn +2 can destabilize rhDNase. Similarly, Ca +2 and Sr +2 can stabilize factor VIII, which is Mg+2 、 Mn +2 and Zn +2 、 Cu +2 and Fe +2 can be destabilized by, and its aggregation can be increased by, Al +3 ions.

[0146] Embodiments of the IL-2 mutant protein formulation or anti-IL-2 antibody formulation further comprise one or more preservatives. Preservatives are essential when developing multi-dose parenteral formulations involving more than one withdrawal from the same container. Their main function is to inhibit the growth of microorganisms throughout the shelf life or period of use of the drug product and to ensure the sterility of the product. Commonly used preservatives include benzyl alcohol, phenol, and m-cresol. Preservatives have a long history of use in low molecular weight parenteral drugs, but the development of protein formulations containing preservatives can be difficult. Preservatives almost always have a destabilizing effect (aggregation) on proteins, which is a major factor limiting their use in multi-dose protein formulations. To date, most protein drugs have been formulated only for single use. However, when multi-dose formulations are possible, there is the added advantage of increased patient convenience and marketability. A good example is the human growth hormone (hGH), where the development of a preserved formulation led to the commercialization of a more convenient multi-use injection pen. At least four such pen devices containing a preserved formulation of hGH are currently available on the market. Norditropin (liquid, Novo Nordisk), Nutropin AQ (liquid, Genentech), and Genotropin (lyophilized - dual chamber cartridge, Pharmacia & Upjohn) contain phenol, while Somatrope (Eli Lilly) is formulated with m-cresol.

[0147] In one embodiment, an IL-2 mutant protein, or an Fc-fusion of an IL-2 mutant protein, e.g., any of the IL-2 mutant proteins described herein, or an Fc-fusion of an IL-2 mutant protein, is formulated in 10 mM KPi, 161 mM L-arginine at pH 7.6.

[0148] Several aspects need to be considered during the formulation and development of the preservative type. The effective preservative concentration in the drug product must be optimized. For this, it is necessary to test a given preservative in the dosage form within the concentration range that confers antimicrobial effectiveness without compromising protein stability.

[0149] In another aspect, the present invention provides an IL-2 mutant protein, or an Fc-fusion of an IL-2 mutant protein, as a lyophilized formulation. The lyophilized product is lyophilized without a preservative and can be reconstituted with a diluent containing a preservative at the time of use. This shortens the time of contact of the preservative with the protein and significantly minimizes the associated stability risks. In the case of a liquid formulation, the effectiveness and stability of the preservative should be maintained throughout the product shelf life (about 18 - 24 months). An important point to note is that the effectiveness of the preservative should be demonstrated in the final formulation containing the active drug and all excipient components.

[0150] IL-2 mutant protein formulations will generally be designed for a particular route and method of administration, for a particular dosage and frequency of administration, for the treatment of a particular disease, especially within the range of bioavailability and persistence. Thus, the formulations can be designed in accordance with the present invention for delivery by any suitable route including, but not limited to, oral, otic, ophthalmic, rectal, and vaginal routes, and by parenteral routes including intravenous and intraarterial injection, intramuscular injection, and subcutaneous injection.

[0151] When the pharmaceutical composition is formulated, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated powder or lyophilized powder. Such formulations can be stored in a form ready for immediate use or in a form that is reconstituted prior to administration (e.g., lyophilized form). The present invention also provides a kit for generating single-dose administration units. The kits of the present invention may each contain both a first container having a dry protein and a second container having an aqueous formulation. In certain embodiments of the present invention, kits are provided that contain single-chamber and multi-chamber pre-filled syringes (e.g., liquid syringes and riosyringes).

[0152] The therapeutically effective amount of the IL-2 mutant protein pharmaceutical composition to be used will be determined, for example, by the context and purpose of the treatment. One of ordinary skill in the art will understand that the appropriate dosage level for treatment will vary in part depending on the molecule being delivered, the indication for which the IL-2 mutant protein or anti-IL-2 antibody is being used, the route of administration, and the size (body weight, body surface area, or organ size) and / or condition (age and health status) of the patient. In certain embodiments, the clinician may titrate the dosage and modify the route of administration to obtain an optimal therapeutic effect. Typical dosages can range from about 0.1 μg / kg to up to about 1 mg / kg or more, depending on the factors described above. In certain embodiments, the dosage can range from 0.5 μg / kg to up to about 100 μg / kg, and in some cases from 2.5 μg / kg to up to about 50 μg / kg.

[0153] A therapeutically effective amount of an IL-2 mutant protein 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-free periods, or the prevention of impairments or disabilities resulting from the pain of the disease.

[0154] The pharmaceutical composition may be administered using a medical device. Examples of medical devices for administering the pharmaceutical composition are described in U.S. Patent No. 4,475,196; U.S. Patent No. 4,439,196; U.S. Patent No. 4,447,224; U.S. Patent No. 4,447,233; U.S. Patent No. 4,486,194; U.S. Patent No. 4,487,603; U.S. Patent No. 4,596,556; U.S. Patent No. 4,790,824; U.S. Patent No. 4,941,880; U.S. Patent No. 5,064,413; U.S. Patent No. 5,312,335; U.S. Patent No. 5,312,335; U.S. Patent No. 5,383,851; and U.S. Patent No. 5,399,163 (all of which are incorporated herein by reference).

[0155] In one embodiment, a pharmaceutical composition comprising is provided.

[0156] Method for treating an autoimmune disorder or an inflammatory disorder In certain embodiments, the IL-2 mutant protein or anti-IL-2 antibody of the invention is used for treating an autoimmune disorder or an inflammatory disorder. In a preferred embodiment, an IL-2 mutant protein Fc fusion protein is used.

[0157] Disorders particularly suitable for treatment with the IL-2 mutant proteins or anti-IL-2 antibodies disclosed herein include inflammation, autoimmune diseases, atopic diseases, tumor-associated autoimmune diseases, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, pauciarticular 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, pauciarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic-onset rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myelitis, polymyositis, dermatomyositis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, rheumatoid polymyalgia, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, psoriasis vulgaris, guttate psoriasis, inverse psoriasis, pustular psoriasis, psoriatic erythroderma, 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, rhinosinusitis, rhinosinusitis with polyps, eosinophilic esophagitis, eosinophilic bronchitis, Guillain-Barre disease, type I diabetes, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD, transplant rejection, kidney disorders, hepatitis C-induced vasculitis, and spontaneous abortion, but are not limited thereto.

[0158] In a preferred embodiment, the autoimmune disorder or inflammatory disorder is lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type I diabetes, multiple sclerosis, spontaneous abortion, atopic disease, and inflammatory bowel disease.

[0159] In another embodiment, a patient suffering from or at risk of developing an autoimmune disorder or an inflammatory disorder is treated with an IL-2 variant protein or an anti-IL-2 antibody (e.g., an IL-2 variant protein disclosed herein, e.g., an IL-2 variant protein Fc fusion as disclosed herein, or another IL-2 variant protein or wild-type IL-2 known in the art as part of an Fc fusion molecule of the type described herein), and the patient's response to the treatment is monitored. The patient response to be monitored 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 a change in body temperature or fever, appetite, sweating, headache, nausea, fatigue, hunger, thirst, mental acuity, etc. In addition, the response can be, for example, a change in the amount of a cell type or gene product (e.g., a protein, peptide, or nucleic acid) in a sample of peripheral blood taken from the patient. In one embodiment, the patient's treatment regimen is changed if the patient has a detectable or measurable response to the treatment, or if such a response exceeds a specific threshold. This change can be a decrease or increase in the dosing frequency, or a decrease or increase in the amount of the IL-2 variant protein or anti-IL-2 antibody administered per single dose, or a "rest" of the dosing (i.e., a temporary interruption of the treatment for any of a specific period of time, or until the treating physician determines that the treatment should be continued, or until the monitored patient response indicates that the treatment should be resumed or can be resumed), or the end of the treatment. In one embodiment, the response is a change in the patient's body 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 particular embodiment, the patient's treatment is reduced, temporarily interrupted, or ended if 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 during the course of the treatment.In another specific embodiment, the treatment of the patient is reduced, interrupted, or terminated if 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 during the treatment process. Other patient responses that can be monitored and used in determining whether to modify, reduce, interrupt, or terminate the treatment include the onset or exacerbation of capillary leak syndrome (hypotension and cardiovascular instability), neutrophil dysfunction (e.g., the onset or exacerbation of an infectious disease occurs or is detected), thrombocytopenia, thrombotic angiopathy, injection site reactions, vasculitis (such as hepatitis C virus vasculitis), or inflammatory signs or diseases. Further patient responses that can be monitored and used in determining whether to modify, reduce, increase, interrupt, or terminate the treatment include NK cells, Treg cells, FOXP3. - CD4 T cells, FOXP3 + + includes an increase in the number of 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 the number of cells per milliliter of blood), or as a percentage increase of such cell types compared to another cell type in a blood sample. Another patient response that can be monitored is the increase in the amount of cell - surface - bound IL - 2 mutant protein or anti - IL - 2 antibody on CD25 + cells in a sample of the patient's peripheral blood.

[0160] Method for proliferating Treg cells An IL-2 mutant protein, an anti-IL-2 antibody, or an IL-2 mutant protein Fc fusion protein may be used to expand Treg cells in a subject or sample. Provided herein is a method of increasing the ratio of Tregs to non-regulatory T cells. The method comprises contacting a population of T cells with an effective amount of a human IL-2 mutant protein, an anti-IL-2 antibody, or an IL-2 mutant protein Fc fusion. The ratio can be measured by determining the ratio of CD3+FOXP3+ cells to CD3+FOXP3− cells within the population of T cells. A typical Treg frequency in human blood is 5-10% of total CD4+CD3+ T cells. However, in the diseases enumerated above, this percentage may be lower or higher. In a preferred embodiment, the percentage of Tregs is increased 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%. The maximum expansion rate of Tregs can vary for each specific disease; however, the maximum Treg frequency that may be obtained by treatment with an IL-2 mutant protein is 50% or 60% of total CD4+CD3+ T cells. In certain embodiments, administration of an IL-2 mutant protein, an anti-IL-2 antibody, or an IL-2 mutant protein Fc fusion protein to a subject increases the ratio of regulatory T cells (Tregs) to non-regulatory T cells in the subject's peripheral blood.

[0161] In addition, an IL-2 mutant protein, an anti-IL-2 antibody, and an IL-2 mutant protein Fc fusion protein preferentially expand Tregs over other cell types and are thus useful for increasing the ratio of regulatory T cells (Tregs) to natural killer (NK) cells in a subject's peripheral blood. The ratio can be measured by determining the ratio of CD3+FOXP3+ cells to CD16+ and / or CD56+ lymphocytes that are CD19− and CD3−.

[0162] An IL-2 mutant protein, an anti-IL-2 antibody, or an IL-2 mutant protein Fc fusion protein may have a therapeutic effect on a disease or disorder in a patient without significantly increasing the ratio of Tregs in the patient's peripheral blood to unregulated T cells or NK cells. The therapeutic effect may be due to the localized activity of the IL-2 mutant protein, anti-IL-2 antibody, or IL-2 mutant protein Fc fusion protein at the site of inflammation or autoimmunity.

Examples

[0163] The following examples, both actual and predicted, are provided for the purpose of illustrating specific embodiments or features of the invention and are not intended to limit its scope.

[0164] Example 1 - pSTAT5 Signaling of IL-2 Mutant Protein IL-2 mutant proteins were investigated for relative pSTAT5 activation. An activity screen was designed to identify mutant proteins that enhance the Treg:Teff window, i.e., retain high levels of activity in Treg cells while showing significant attenuation in Teff cells. Activated Teff cells express high levels of CD25, and patients with autoimmune and inflammatory diseases have elevated numbers of such cells, so the inventors mimicked a more realistic differentiation of CD25 expression in patients using CD25+-gating on Teff cells. The activity of the IL-2 mutant proteins was evaluated by intracellular phospho-STAT5 responses measured by a FACS-based assay. Briefly, previously frozen human PBMCs were thawed and allowed to rest in complete medium for 0.5 - 2 hours. The cells were resuspended at 5 - 10 million cells / ml and aliquoted at 100 μl per well (500,000 - 1 million cells per well) into 96-well deep well plates. The cells were stimulated with IL-2 mutant proteins for 30 minutes in a 10× dose titration ranging from 1 nM to 200 nM in a final volume of 10 μl. The level of STAT5 phosphorylation was measured using the BD phosflow buffer kit. Briefly, 1 ml of BD lyse / fix phosflow buffer was added to stop the stimulation. The cells were fixed at 37°C for 10 - 15 minutes, permeabilized with 1× BD phosflow perm buffer on ice, and then stained for CD3, CD4, CD25, FOXP3, CD8, and pSTAT5. The results for two donors of PBMCs are shown in Table 2 below.

[0165]

Table 2

[0166]

Table 3

[0167] Example 2 - Stability of IL-2 Mutant Proteins The molecular evaluation assay for DSC Tm measurement was tested for the selection of molecules that were stable and had good potential for ease of manufacture, along with the 40°C 10-day stability measured by SEC chromatography. The results of the MP% after 10 days are shown in Figure 1.

[0168] Example 3 - Attenuation of Human IL-2 Mutant Protein The attenuated human IL-2 mutant protein was evaluated for activity in mouse splenocytes by an in vitro pSTAT5 assay. The inventors confirmed the activity of the selected mutant proteins on mouse immune cells by a mouse splenocyte pSTAT5 assay (Figure 2). Shown are the dose-titration curves for three attenuated mutant proteins, H16R, V91K D20A M104V, D20W, and the controls, wild-type human IL-2.Fc, recombinant human IL-2, and recombinant mouse IL-2. Mouse splenocytes were stimulated for 30 minutes in medium containing titrated concentrations of the mutant proteins and analyzed by FACS. The mutant proteins demonstrated similar rank-order activity in mouse Treg cells as demonstrated in human Treg cells (i.e., WT > H16R > V91K D20A M104V > D20W). The rank order was also similar for effector T cells.

[0169] Example 4 - In Vivo Activity of Human IL-2 Mutant Protein in Mice C57Bl6 mice were given a single dose of PBS (vehicle control), wild-type IL-2-Fc, V91K D20A M104V, H16R, or D20W on day 0, and on day 4, splenocytes were collected and analyzed for their effects on Treg cells, CD8 T cells, and NK cells. Except for D20W which was given at only 25 μg, three doses (1 μg, 5 μg, or 25 μg per mouse) were evaluated. The percentage of Treg cells defined by CD4+CD25+FoxP3+ cells in total gated live cells is shown in (A), and the calculated total numbers of Treg cells (B), CD8 T cells (C), and NK cells (D) are shown. As shown in Figure 3, wild-type IL-2, H16R, and V91K D20A M104V induced significant dose-dependent in vivo proliferation of Treg cells. Two mutant proteins, H16R and V91K D20A M104V, surprisingly showed robust activity on Treg cells similar to or to an even greater extent than wild-type IL-2.Fc. In contrast, neither H16R nor V91K D20A M104V demonstrated significant activity on CD8 T cells or NK cells compared to wild-type IL-2.Fc. These results demonstrate that, despite a significant attenuation of activity measured by in vitro pSTAT5 readout, the attenuated mutant proteins retain the ability to induce robust Treg responses in vivo while inducing minimal CD8 T and NK cell responses. Thus, attenuation disproportionately affects Treg:non-Treg in vivo selectivity.

[0170] Example 5 - pSTAT5 Signaling of IL-2 Mutant Proteins The IL-2 mutant proteins were investigated for relative pSTAT5 activation. An activity screen was designed to identify mutant proteins that enhance the Treg:Teff window, i.e., retain high levels of activity in Treg cells while showing significant attenuation in Teff cells. Since activated Teff cells express high levels of CD25 and patients with autoimmune and inflammatory diseases have elevated numbers of these cells, the inventors mimicked a more realistic differentiation of CD25 expression in patients using CD25+-gating on Teff cells. The activity of the IL-2 mutant proteins was evaluated by the intracellular phospho-STAT5 response measured by a FACS-based assay. Briefly, previously frozen human PBMCs were thawed and allowed to rest in complete medium for 0.5 - 2 hours. The cells were resuspended at 5 - 10 million cells / ml and aliquoted into 96-well deep-well plates at 100 μl per well (500,000 - 1 million cells per well). The cells were stimulated with the IL-2 mutant proteins at 10× dose titrations ranging from 0.4 nM to 25 nM in a final volume of 10 μl for 30 minutes. The level of STAT5 phosphorylation was measured using the BD phosflow buffer kit. Briefly, 1 ml of BD lyse / fix phosflow buffer was added to stop the stimulation. The cells were fixed at 37 °C for 10 - 15 minutes and permeabilized with 1× BD phosflow perm buffer on ice before staining for CD3, CD4, CD25, FOXP3, CD8, and pSTAT5. The results for two donors of PBMCs are shown in Table 3 below.

[0171]

Table 4

Claims

1. A human interleukin-2 (IL-2) mutein comprising an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 2, and which contains the following mutations relative to SEQ ID NO: 2: V91K, H16E; H16E, V91H; V91H, H16R; V91A, H16A; V91A, H16D; V91A, H16E; V91A, H16S; V91E, H16A; V91E, H16D; V91E, H16E; V91E, H16S; V91K, H16A; V91K, H16D; V91K, H16S; or V91S, H16E, and which preferentially stimulates regulatory T cells compared to other T cells.

2. The human IL-2 mutein of claim 1, which further preferentially stimulates regulatory T cells compared to NK cells.

3. A human IL-2 mutant protein as described in claim 1, wherein the amino acid sequence of the human IL-2 mutant protein includes an alanine at position 125 of the numbering of SEQ ID NO:

2.

4. The human IL-2 mutein of claim 1, wherein the amino acid sequence of the human IL-2 mutein further comprises a T3A mutation or a T3N mutation.

5. An Fc fusion protein comprising an Fc and a human IL-2 mutein according to any one of claims 1 to 4.

6. The Fc fusion protein of claim 5 , wherein the Fc is a human IgG1 Fc.

7. 7. The Fc fusion protein of claim 6, wherein the human IgG1 Fc comprises one or more mutations that alter the effector function of the Fc.

8. 7. The Fc fusion protein of claim 6, comprising a substitution or deletion of the C-terminal lysine of the human IgG1 Fc.

9. 6. The Fc-fusion protein of claim 5, wherein a linker connects the Fc portion and the human IL-2 mutein portion of the Fc-fusion protein.

10. 10. The Fc fusion protein of claim 9, wherein the linker is GGGGS (SEQ ID NO: 5), GGNGT (SEQ ID NO: 6), or YGNGT (SEQ ID NO: 7).

11. 6. The Fc-fusion protein of claim 5, wherein the human IL-2 mutein further comprises an amino acid addition, substitution, or deletion that alters glycosylation of the Fc-fusion protein when expressed in a mammalian cell.

12. The Fc fusion protein of claim 5 , comprising an Fc dimer.

13. 13. The Fc-fusion protein of claim 12, comprising two human IL-2 muteins.

14. 13. The Fc-fusion protein of claim 12, comprising a single human IL-2 mutein.

15. An isolated nucleic acid encoding a human IL-2 mutein according to any one of claims 1 to 4.

16. An isolated nucleic acid encoding the Fc fusion protein described in claim 5.

17. 10. A pharmaceutical composition comprising a human IL-2 mutein according to any one of claims 1 to 4 for use in increasing the ratio of regulatory T cells (Treg) to non-regulatory T cells within a population of T cells or in the peripheral blood of a subject.

18. 10. A pharmaceutical composition comprising the Fc-fusion protein of claim 5 for use in increasing the ratio of regulatory T cells (Treg) to non-regulatory T cells within a population of T cells or in the peripheral blood of a subject.

19. A pharmaceutical composition comprising a human IL-2 mutein according to any one of claims 1 to 4, for use in increasing the ratio of regulatory T cells (Treg) to natural killer (NK) cells in the peripheral blood of a subject.

20. A pharmaceutical composition comprising the Fc fusion protein of claim 5 for use in increasing the ratio of regulatory T cells (Treg) to natural killer (NK) cells in the peripheral blood of a subject.

21. A pharmaceutical composition comprising a human IL-2 mutein according to any one of claims 1 to 4 for use in treating a subject suffering from an inflammatory or autoimmune disease.

22. A pharmaceutical composition comprising the Fc-fusion protein of claim 5 for use in treating a subject suffering from an inflammatory disease or an autoimmune disease.

23. 23. The pharmaceutical composition of claim 21 or 22, wherein the inflammatory disease or the 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, Sjogren's syndrome, Behcet's disease, spontaneous abortion, atopic disease, asthma, or inflammatory bowel disease.

Images