Interleukin-2 mutant protein for regulatory T cell proliferation

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 of related applications This application claims the interests of U.S. Patent Application No. 62 / 886,283, filed on 13 August 2019, which is incorporated herein by reference in its entirety. [Background technology]

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

[0003] T cells typically require CD25 expression to respond to low concentrations of IL-2 present in tissues. CD25-expressing T cells include both FOXP3+ regulatory T cells (Treg cells), essential for suppressing autoimmune inflammation, and FOXP3- T cells activated to express CD25. FOXP3-CD25+ effector T cells (Teffs) can be either CD4+ 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 crucial for normal Treg cell proliferation and survival, as well as for high FOXP3 expression.

[0004] In their jointly owned international publication brochure No. 2010 / 085495, the inventors describe the use of IL-2 mutant proteins to preferentially proliferate or stimulate Treg cells. When administered to subjects, the effects on Treg cells are useful for treating inflammatory and autoimmune diseases. While the IL-2 mutant proteins described in that specification are useful for in vivo proliferation of Treg cells more than Teff cells, it was desired to generate IL-2 mutant proteins with attributes optimal for human therapeutics. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2010 / 085495 [Overview of the Initiative] [Means for solving the problem]

[0006] This specification describes IL-2 mutant proteins that are suitable for high-yield production and possess pharmacological activity. In particular, the IL-2 mutant proteins of the present invention improve the Treg:Teff selectivity window. In attempting to produce molecules that can be used as human therapeutics, several unexpected and unpredictable observations were made. The compositions and methods described herein are the result of such efforts.

[0007] The IL-2 mutant proteins described herein are less likely to trigger an immune response to the IL-2 mutant protein and / or endogenous IL-2, thereby enabling preferential proliferation and activation of Tregs. Furthermore, in certain embodiments, the IL-2 mutant protein is fused to a molecule, such as an antibody Fc, that increases the serum half-life when administered to a subject. The IL-2 mutant protein has a short serum half-life (3-5 hours for subcutaneous injection). The exemplary IL-2 mutant protein Fc fusions described herein have half-lives in humans 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. This effect on the pharmacokinetics of the IL-2 mutant protein allows for reduced or infrequent dosing of IL-2 mutant protein therapeutics.

[0008] Furthermore, when creating large pharmaceutical molecules, it is necessary to consider the ability to generate large quantities of these molecules while minimizing molecular aggregation and maximizing molecular stability. 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 disable the effector function of IgG1 (e.g., ADCC activity), it has been found that a mutation of asparagine at position 297 to glycine (N297G; EU numbering scheme) results in greater purification efficiency and biophysical properties than other mutations that result in non-glycosylated IgG1 Fc. In preferred embodiments, cysteine ​​is manipulated to enter the Fc molecule via a disulfide bond, thereby increasing the stability of the non-glycosylated Fc-containing molecule. The usefulness of non-glycosylated Fc extends beyond the context of the IL-2 mutant protein Fc fusion. Therefore, provided herein are Fc-containing molecules, Fc fusions, and antibodies, including the N297G substitution and, optionally, the substitution of one or more additional residues to cysteine.

[0010] In one embodiment, the present invention provides a human interleukin-2 (IL-2) mutant protein having an amino acid sequence that is at least 90% identical to the amino acid sequence described in Sequence ID No. 1, wherein the IL-2 mutant protein is 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;H16 E,V91H,M104V;V91H,D20A,E61Q,M104T;V91H,H16R,E16Q;V91K,D20A,M10 4V;H16E,V91H;V91H,D20A,M104V;H16E,V91H,M104T;H16E,V91H,E61Q,M1 04T;V91K,E61Q,H16E;V91K,H16R,M104L;H16E,V91H,E16Q;V91K,E61Q,H1 6R;D20W,V91K,E61Q;V91H,H16R;V91K,H16R;D20W,V91K,E61Q,M104T;V91 K,D20A;V91H,D20A,E16Q;V91K,D20A,M104L;V91H,D20A;V91K,E61Q,D20A ;V91H,M104T;V91H,M104V;V91K,E61Q;V91K,N88K,E61Q,M104T;V91K,N88 K,E61Q;V91H,E61Q;V91K,N88K;D20A,H16E,M104T;D20A,M104T;H16E,N88 K;D20A,M104V;D20A,M104L;H16E,M104T;H16E,M104V;N88K,M104V;N88K, E61Q;D20A,E61Q;H16R,D20A;D20W,E61Q;H16E,E61Q;H16E,M104L;N88K,M 104T;D20A,H16E;D20A,H16E,E16Q;D20A,H16R,E16Q;V91K,D20W;V91A,H1 6A;V91A,H16D;V91A,H16E;V91A,H16S;V91E,H16A;V91E,H16D;V91E,H16E;The mutant protein has at least one mutation selected from V91E,H16S;V91K,H16A;V91K,H16D;V91K,H16S; and V91S,H16E, and preferentially stimulates regulatory T cells compared to other T cells or NK cells in both in vitro assays and in humanized mice (NSG mice reconstituted with CD34+ hematopoietic stem cells). In one embodiment, the mutant protein is at least 95% identical to the amino acid sequence described in SEQ ID NO: 1. In another embodiment, the mutant protein is at least 97% identical to the amino acid sequence described in SEQ ID NO: 1. In another embodiment, the amino acid sequence of the mutant protein is, with respect to C125A, 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,D2 0A,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;N 88K,M104T;D20A,H16E;D20A,H16E,E16Q;D20A,H16R,E16Q;V91K,D20W;V91A,H16A;V91A The amino acid sequence differs from that described in Sequence ID No. 1 only for one position selected from ,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 embodiment, the present invention provides an Fc fusion protein comprising Fc and a human IL-2 mutant protein described above. In one embodiment, Fc is human IgG1 Fc. In another embodiment, human IgG1 Fc comprises one or more mutations that alter the effector function of the Fc. In another embodiment, 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 of the protein to the human IL-2 mutant protein portion. 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 includes the addition, substitution, or deletion of amino acids that alter the glycosylation of the Fc fusion protein when expressed in mammalian cells. In another embodiment, the IL-2 mutant protein includes a T3 substitution. In another embodiment, the IL-2 mutant protein includes a T3N substitution or a T3A substitution. In another embodiment, the IL-2 mutant protein includes a T3N substitution. In another embodiment, the IL-2 mutant protein further includes an S5 mutation. In another embodiment, the IL-2 mutant protein further includes an S5T mutation. In another embodiment, the Fc fusion protein includes an Fc dimer. In another embodiment, the Fc fusion protein includes two IL-2 mutant proteins. In another embodiment, the Fc fusion protein includes a single IL-2 mutant protein.

[0012] In another embodiment, the present invention provides an isolated nucleic acid encoding the previously described human IL-2 mutant protein.

[0013] In another embodiment, the present invention provides an isolated nucleic acid encoding an antibody Fc portion and a human IL-2 mutant protein as 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, Fc is human IgG1 Fc. In another embodiment, human IgG1 Fc includes one or more mutations that alter the effector function of the Fc. In another embodiment, human IgG1 includes 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 connects the antibody Fc portion 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 includes the addition, substitution, or deletion of amino acids that alter the glycosylation of the protein containing the IL-2 mutant protein when expressed in mammalian cells. In another embodiment, the IL-2 mutant protein includes a T3 substitution. In another embodiment, the IL-2 mutant protein includes a T3N substitution or a T3A substitution. In another embodiment, the IL-2 mutant protein includes a T3N substitution. In another embodiment, the IL-2 mutant protein further includes an S5 mutation. In another embodiment, the IL-2 mutant protein further includes an S5T mutation.

[0014] In another embodiment, the present invention provides an expression vector comprising the previously described isolated nucleic acid, operably coupled 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 within 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 is increased. In another embodiment, the ratio of CD3+FoxP3+ cells to CD3+FoxP3− cells is increased 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 is increased. In another embodiment, the ratio of CD3+FoxP3+ cells to CD3+FoxP3− cells is increased 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 is increased. In another embodiment, the ratio of CD3+FoxP3+ cells to CD3+FoxP3− cells is increased by at least 50%.

[0022] In another embodiment, 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, comprising administering an effective amount of the previously described human IL-2 mutant protein. In one embodiment, the ratio of CD3+FoxP3+ cells to CD3-CD19- lymphocytes expressing CD56 and / or CD16 is increased. In another embodiment, the ratio of CD3+FoxP3+ cells to CD3-CD19- lymphocytes expressing CD56 and / or CD16 is increased by at least 50%.

[0023] In another embodiment, 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, 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 is increased. In another embodiment, the ratio of CD3+FoxP3+ cells to CD3-CD19- lymphocytes expressing CD56 and / or CD16 is increased by at least 50%.

[0024] In another embodiment, the present invention provides a method for treating a subject suffering from an inflammatory disease or autoimmune disease, comprising administering to the subject a therapeutically effective amount of the previously described IL-2 mutant protein or a therapeutically effective amount of the previously described Fc fusion protein. In one embodiment, the administration causes a reduction in at least one symptom of the disease. In another embodiment, the ratio of regulatory T cells (Tregs) to unregulatory T cells in the subject's peripheral blood increases after administration. In another embodiment, the ratio of regulatory T cells (Tregs) to unregulatory T cells in the subject's peripheral blood 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 1 diabetes, type 2 diabetes, multiple sclerosis, rheumatoid arthritis, alopecia areata, atherosclerosis, psoriasis, organ graft rejection, Sjögren's syndrome, Behçet's disease, spontaneous abortion, atopic disease, asthma, or inflammatory bowel disease. [Brief explanation of the drawing]

[0025] [Figure 1] The results of DSC Tm measurement of IL-2 mutant proteins are shown along with their stability at 40°C for 10 days, as measured by SEC chromatography. [Figure 2] The dose titration curves for three attenuated mutant proteins—H16R, V91K D20A M104V, and D20W—as well as the control, wild-type human IL-2.Fc, recombinant human IL-2, and recombinant mouse IL-2 in an in vitro pSTAT5 assay are shown. [Figure 3] This paper presents the evaluation of in vivo activity in mice of human IL-2 mutant proteins V91K D20A M104V, H16R, D20W, and the control wild-type IL-2-Fc. [Modes for carrying out the invention]

[0026] The section headings used in this specification are for organizational purposes only and should not be interpreted as limiting the subject matter discussed. All references cited within the text of this specification are explicitly incorporated in their entirety by reference.

[0027] Standard techniques can be used for recombinant DNA and oligonucleotide synthesis, tissue culture and transformation, protein purification, etc. Enzyme reactions and purification techniques may be carried out according to the manufacturer's specifications, as commonly achieved in such techniques, or as described herein. The following procedures and techniques may generally be carried out according to conventional methods well known in such techniques, and as described in the various general and more specific references cited and discussed throughout this specification. For example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3 rd See ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. This document is incorporated herein by reference for all purposes. Unless otherwise specified, the nomenclature, laboratory procedures, and techniques used in relation to analytical chemistry, organic chemistry, and medicinal chemistry and pharmaceutical chemistry described herein are well known and commonly used in those fields. Standard techniques can be used in chemical synthesis, chemical analysis, pharmaceutical preparations, formulations, and delivery, as well as in patient care.

[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" refers to polypeptides having the following amino acid sequence: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFXQSIISTLT In the array, X is C, S, V, or A (sequence ID 2).

[0029] A variant may contain one or more substitutions, deletions, or insertions within the wild-type IL-2 amino acid sequence. A residue is specified herein by a single-letter amino acid code followed by the IL-2 amino acid position; for example, K35 is the lysine residue at position 35 of SEQ ID NO: 2. A substitution is specified herein by a single-letter amino acid code followed by the IL-2 amino acid position, followed by the single-letter amino acid code to be substituted; for example, 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 stimulates regulatory T cells" means that the mutant protein or antibody promotes the proliferation, survival, activation, and / or function of CD3+FoxP3+ T cells more than CD3+FoxP3- T cells. The ability to preferentially stimulate Tregs can be measured by flow cytometry of peripheral blood leukocytes, and a greater increase in CD25 expression levels on the surface of FOXP3+ T cells is observed compared to the increase in the percentage of FOXP3+CD4+ T cells among total CD4+ T cells, the increase in the percentage of FOXP3+CD8+ T cells among total CD8+ T cells, the increase in the percentage of FOXP3+ T cells compared to NK cells, and / or the increase in CD25 expression on other T cells. Furthermore, preferential proliferation of Treg cells can be detected as increased expression of demethylated FOXP3 promoter DNA (i.e., Treg-specific demethylation regions, or TSDRs) compared to demethylated CD3 genes in DNA extracted from whole blood, as detected by sequencing of polymerase chain reaction (PCR) products derived from Bisulfit-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 high levels of activity in Treg cells while showing significant attenuation in Teff cells. Activated Teff cells express high levels of CD25, and the number of such cells increases in patients with autoimmune and inflammatory diseases; therefore, the inventors used CD25+-gating on Teff cells to mimic a more realistic differentiation of CD25 expression in patients.

[0031] IL-2 mutant proteins or their Fc fusion proteins that preferentially stimulate Treg cells increase the ratio of CD3+FoxP3+ T cells to CD3+FoxP3- T cells in a subject or 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 its Fc fusion protein results in less than 50% pSTAT activation of effector T cells upon 1nM IL-2 treatment using the protocol of Example 1. In some embodiments, the IL-2 mutant protein or its Fc fusion protein results in less than 40% pSTAT activation of effector T cells upon 1nM IL-2 treatment using the protocol of Example 1. In some embodiments, the IL-2 mutant protein or its Fc fusion protein results in less than 30% pSTAT activation of effector T cells upon 1nM IL-2 treatment using the protocol of Example 1. In some embodiments, the IL-2 mutant protein or its Fc fusion protein results in less than 20% pSTAT activation of effector T cells upon 1nM IL-2 treatment using the protocol of Example 1. In some embodiments, the IL-2 mutant protein or its Fc fusion protein results in less than 10% pSTAT activation of effector T cells upon 1nM IL-2 treatment using the protocol of Example 1. Furthermore, in some embodiments, the IL-2 mutant protein or its Fc fusion protein exhibits 10-day stability of 25%, 30%, 40%, 50%, 60%, 70%, 80%, or more than 90% using the protocol of Example 2.

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

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

[0036] Examples of IL-2 mutant proteins include, but are not limited to, the following: V91K,D20L;D84R,E61Q;V91K,D20A,E61Q,M104T;N88K,M104L;V91H,M104L;V91K,H16E,M104V;V91K,H16R,M104V;V91K,H16R,M104T;V91K,D20A,M104T;V91K,H16E,M104T;V91K,H16E,E61Q,M104T;V91K,H16R,E61 Q,M104T;V91K,H16E;V91H,D20A,M104T;H16E,V91H,M104V;V91H,D20A,E6 1Q,M104T;V91H,H16R,E16Q;V91K,D20A,M104V;H16E,V91H;V91H,D20A,M10 4V;H16E,V91H,M104T;H16E,V91H,E61Q,M104T;V91K,E61Q,H16E;V91K,H1 6R,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;D 20A,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,H1 6S;V91E,H16A;V91E,H16D;V91E,H16E;V91E,H16S;V91K,H16A;V91K,H16D;V91K,H16S; and / or V91S,H16E substitutions. The IL-2 mutant proteins of the present invention may include, in some cases, C125A substitutions. While it may be advantageous to further reduce the number of mutations from the wild-type IL-2 sequence, the present invention includes IL-2 mutant proteins comprising truncation and / or additional insertions, deletions, and / or substitutions, in addition to the following: V91K,D20L;D84R,E61Q;V91K,D20A,E61Q,M104T;N88K,M104L;V91H,M104L;V91K,H16E,M104V;V91K,H16R,M104V;V91K,H16R,M104T;V91K,D20 A,M104T;V91K,H16E,M104T;V91K,H16E,E61Q,M104T;V91K,H16R,E61Q,M104T;V91K,H16E;V91H,D20A,M104T;H16E,V91H,M104V;V91H,D20 A,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,N 88K,E61Q;V91H,E61Q;V91K,N88K;D20A,H16E,M104T;D20A,M104T;H1 6E,N88K;D20A,M104V;D20A,M104L;H16E,M104T;H16E,M104V;N88K,M 104V;N88K,E61Q;D20A,E61Q;H16R,D20A;D20W,E61Q;H16E,E61Q;H16 E,M104L;N88K,M104T;D20A,H16E;D20A,H16E,E16Q;D20A,H16R,E16Q;V91K,D20W;V91A,H16A;V91A,H16D;V91A,H16E;V91A,H16S;V91E,H16A;V91E,H16D;V91E,H16E;V91E,H16S;V91K,H16A;V91K,H16D;V91K,H16S; and / or V91S,H16E substitutions, provided that the mutant protein maintains activity that preferentially stimulates Treg. Therefore, as an embodiment, Treg cells are preferentially stimulated, 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,E 61Q,M104T;V91K,H16E;V91H,D20A,M104T;H16E,V91H,M104V;V91H,D20A,E61Q,M104T;V91H,H16R,E16Q;V91K,D20A,M104 V;H16E,V91H;V91H,D20A,M104V;H16E,V91H,M104T;H16E,V91H,E61Q,M104T;V91K,E61Q,H16E;V91K,H16R,M104L;H16E,V 91H,E16Q;V91K,E61Q,H16R;D20W,V91K,E61Q;V91H,H16R;V91K,H16R;D20W,V91K,E61Q,M104T;V91K,D20A;V91H,D20A,E1 6Q;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;H1 6E,M104V;N88K,M104V;N88K,E61Q;D20A,E61Q;H16R,D20A;D20W,E61Q;H16E,E61Q;H16E,M104L;N88K,M104T;D20A,H16E;Examples of IL-2 mutant proteins include those comprising an amino acid sequence having the substitution 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, and which are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in Sequence ID No. 2. In a particularly preferred embodiment, such an IL-2 mutant protein contains an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 2.

[0037] With respect to 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 similarity search of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. USA 85:2444, computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program, preferably with default settings, as described by Develeux et al., 1984, Nucl. Acid Res. 12:387-395, or by visual inspection. Preferably, the identity percentage is calculated by FastDB based on the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and join 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] One example of a useful algorithm is PILEUP. PILEUP uses progressive pairwise alignment to create a multisequence alignment from a group of related sequences. This also allows plotting a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplified version of the progressive alignment method described by 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.215:403410; Altschul et al., 1997, Nucleic Acids Res.25:33893402; and Karin et al., 1993, Proc.Natl.Acad.Sci.USA90:5873-5787. 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 their default values. The adjustable parameters are set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = II. The HSP S-parameter and HSP S2-parameter are dynamic values ​​established by the program itself, depending on the composition of the specific sequence being searched and the composition of the specific 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, with a cost charge of 10+k to a gap length of k; and X set to 16. u X is set to 40 for the database search stage and to 67 for the algorithm output stage. g This is used. Gap alignment is triggered by a score corresponding to approximately 22 bits.

[0041] The site or region for introducing amino acid sequence mutations may be predetermined, but the mutations themselves do not need to be predetermined. For example, to optimize the performance of mutations at a given site, random mutagenesis may be performed at a target codon or target region, and the expressed IL-2 mutant proteins may be screened for the optimal combination of desired activity. Techniques for forming substitutional mutations at predetermined sites in DNA with known sequences are well known, such as M13 primer mutagenesis and PCR mutagenesis. Screening of mutants may be performed, for example, using assays described herein.

[0042] Amino acid substitutions are typically single-residue substitutions; insertions are usually around 1 to 20 amino acid residues, but significantly larger insertions are permitted. Deletions range from approximately 1 to 20 amino acid residues, but in some cases, deletions may be much larger.

[0043] Substitutions, deletions, insertions, or any combination thereof may be used to arrive at the final derivative or variant. Generally, these changes are made to a few amino acids to minimize alterations to the immunogenicity and specificity of the molecule, particularly the antigen-binding protein. However, in certain circumstances, larger changes may be permissible. Conservative substitutions are generally performed 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 conserved than those shown in Table 1. For example, substitutions that have a greater effect on the structure of the polypeptide backbone in the region of modification, e.g., alpha-helix or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chains may be formed. Substitutions that are generally expected to result in the greatest change in polypeptide properties are: (a) a hydrophilic residue, e.g., ceryl or threonyl, being substituted with (or thereby) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) cysteine ​​or proline being substituted with (or thereby) any other residue; (c) a residue with an electronegative side chain, e.g., lysyl, arginyl, or histidyl, being substituted with (or thereby) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue with a bulky side chain, e.g., phenylalanine, being substituted with (or thereby) a residue without a side chain, e.g., glycine.

[0046] Variants may also be selected to modify the characteristics of the IL-2 mutant protein as needed, but typically the variants will exhibit the same qualitative biological activity and induce the same immune response as the naturally occurring analogues. In addition, variants may be designed to alter the biological activity of the IL-2 mutant protein. For example, glycosylation sites may be modified or removed as described herein.

[0047] IL-2 mutant protein with extended serum half-life The IL-2 mutant proteins provided herein, for example, preferentially promote the proliferation of Treg cells over Teff cells or NK cells, and therefore, when administered to patients, their safety profile is expected to differ from that of wild-type IL-2 or PROLEUKIN® (aldesleukin; Novartis, Basel, Switzerland). Side effects associated with wild-type IL-2 or PROLEUKIN® include flu-like symptoms, chills, arthralgia, fever, rash, itching, injection site reactions, hypotension, diarrhea, nausea, anxiety, confusion, and depression. The IL-2 mutant proteins provided herein may be modified to include a molecule that extends the serum half-life of the mutant protein, or may be fused to such a molecule, and such extension of the half-life does not increase the risk of increased likelihood or severity of side effects or adverse events in patients. Subcutaneous administration of mutant proteins with such extended serum half-lives may result in maximum systemic exposure (C). max This may allow for longer-term targeting of lower-level targets. Extending the serum half-life may enable drug regimens with fewer or less frequent mutant proteins.

[0048] The serum half-life of the IL-2 mutant protein provided herein may be extended by essentially any method known in the art. Such methods include modifying the sequence of the IL-2 mutant 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 mutant 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 mutant protein is fused to an IgG Fc molecule.

[0049] The IL-2 mutant 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 IL-2 mutant protein activity more significantly than fusion to the N-terminus of IgG Fc.

[0050] One embodiment of the present invention relates to a dimer comprising two Fc fusion polypeptides produced by fusing an IL-2 mutant 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 host cells transformed by a recombinant expression vector, and assembling the expressed fusion protein to be very similar to an antibody molecule, thereby forming an interchain bond between the Fc portions and generating a dimer.

[0051] As used herein, the terms “Fc polypeptide” or “Fc region” include the native and mutant protein forms of polypeptides derived from the Fc region of an antibody, which may be part of either the IL-2 mutant protein fusion protein or the anti-IL-2 antibody of the present invention. It also includes cleavage forms of such polypeptides containing a hinge region that promotes dimerization. In certain embodiments, the Fc region includes the CH2 and CH3 domains of the antibody. Along with an extended serum half-life, the fusion protein (and oligomers formed therefrom) containing the Fc portion offers the advantage of easy purification by affinity chromatography on a protein A or protein G column. Preferred Fc regions are derived from human IgG, including IgG1, IgG2, IgG3, and IgG4. In this specification, certain residues within Fc are identified by their 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 once the antibody binds to its target. This is considered an "effector function." This communication can lead to antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cell-mediated cytotoxicity (CDC). ADCC and ADCP are mediated by the binding of Fc to Fc receptors on the surface of immune system cells. CDC is mediated by the binding of Fc to complement system proteins, such as C1q.

[0053] IgG subclasses differ in their ability to mediate effector functions. For example, IgG1 is far superior to IgG2 and IgG4 in mediating ADCC and CDC. Therefore, in embodiments where effector function is undesirable, IgG2 Fc would be preferred. However, molecules containing IgG2 Fc are known to be more difficult to manufacture and have less attractive biophysical properties, such as a shorter half-life, compared to molecules containing IgG1 Fc.

[0054] The effector function of an antibody can be increased or decreased by introducing one or more mutations into the Fc. Embodiments of the present invention include IL-2 mutant protein Fc fusion proteins having an Fc manipulated to increase effector function (U.S. Patent No. 7,317,091 and Strohl, Curr. Opin. Biotech., 20:685-691, 2009; both 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 / A 339Q;D280H / K290S;D280H / K290S / S298D;D280H / K290S / S298V;F243L / R292P / Y300L;F243L / R292P / Y3 00L / 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 method to enhance the effector function of IgG Fc-containing proteins involves reducing the fucosylation of Fc. By removing core fucose from branched complex oligosaccharides bound to Fc, ADCC effector function is significantly increased without altering antigen-binding or CDC effector function. Several methods are known for reducing or inactivating the fucosylation of Fc-containing molecules, such as antibodies. These methods include recombinant expression in specific mammalian cell lines, such as FUT8 knockout cell lines, mutant CHO cell line Lec13, rat hybridoma cell line YB2 / 0, cell lines containing small interfering RNAs specific to 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 plant cells, yeast, or prokaryotic cells, such as non-mammalian cells like 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 that has been manipulated to reduce effector function. Examples of Fc molecules with reduced effector function include those having the following substituents: 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 shown in Sequence ID No. 3: [ka]

[0058] The group has a mutated N297 that forms a non-glycosylated antibody. The mutations focus on substituting N297 with an amino acid that has physiological and chemical properties similar to asparagine, such as glutamine (N297Q), or with alanine that mimics asparagine without a polar group (N297A).

[0059] As used herein, "non-glycosylated antibody" or "non-glycosylated fc" refers to the glycosylation status 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 a non-glycosylated Fc fusion protein.

[0060] In attempting to form effector-less IgG1 Fc, it was discovered that the mutation of amino acid N297 of human IgG1 to glycine, i.e., N297G, provides far superior purification efficiency and biophysical properties than other amino acid substitutions at that residue. See Example 8. Therefore, in a preferred embodiment, the IL-2 mutant protein Fc fusion protein contains human IgG1 Fc having the N297G substitution. Fc containing the N297G substitution is useful in any context in which the molecule contains human IgG1 Fc, and is not limited to use in the context of the IL-2 mutant protein Fc fusion. In a particular embodiment, the antibody contains Fc having the N297G substitution.

[0061] Furthermore, Fc containing the N297G mutation may also include insertions, deletions, and substitutions. In certain embodiments, human IgG1 Fc containing the N297G substitution is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence described 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 including the N297G substitution and the deletion of the C-terminal lysine is described in SEQ ID NO: 4.

[0062] It has been shown that glycosylated IgG1 Fc-containing molecules are less stable than glycosylated IgG1 Fc-containing molecules. The Fc region may be further manipulated to increase the stability of the non-glycosylated molecule. In some embodiments, one or more amino acids are substituted with cysteine ​​to form a disulfide bond in a dimeric state. Residues V259, A287, R292, V302, L306, V323, or I332 of the amino acid sequence described in SEQ ID NO: 3 may be substituted with cysteine. In preferred embodiments, certain residue pairs are substitutions that restrict or prevent disulfide bond scrambling by preferentially forming disulfide bonds with each other. 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 residues V259, A287, R292, V302, L306, V323, or I332 are substituted with cysteine, examples of which include substitutions of A287C and L306C, V259C and L306C, R292C and V302C, or V323C and I332C.

[0064] Additional mutations that may be formed on IgG1 Fc include those that promote heterodimerization in Fc-containing polypeptides. In some embodiments, the Fc region is manipulated to create “knobs” and “holes” that promote heterodimerization of two different Fc-containing polypeptide chains when co-expressed in cells. U.S. Patent No. 7,695,963. In other embodiments, the Fc region is modified to promote heterodimerization while inhibiting homodimerization of two different Fc-containing polypeptides when co-expressed in cells using electrostatic steering. International Publication No. 09 / 089,004 (which is incorporated herein by reference in its entirety). Preferred heterodimer Fc includes those in which one chain of Fc contains the substitutions D399K and E356K and the other chain of Fc contains the substitutions K409D and K392D. In another embodiment, one chain of Fc includes substitutions of D399K, E356K, and E357K, and the other chain of Fc includes substitutions of 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, a bi-, tri-, or quadri-specific antibody capable of specifically binding 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 heterodimerization. The fusion protein or antibody is co-expressed with an Fc region that has intermutations with the Fc region within the IL-2 mutant protein Fc fusion polypeptide but lacks the IL-2 mutant protein or anti-IL-2 heavy chain variable domain. When a heterodimer of the two Fc-containing polypeptides is formed, the resulting protein contains only the single IL-2 mutant protein or anti-IL-2 binding domain.

[0066] Another method for producing monomeric IL-2 mutant protein Fc fusion proteins involves fusing an IL-2 mutant protein to monomeric Fc, i.e., preventing the Fc region from dimerizing. Stable monomeric Fc contains mutations that inhibit dimerization and stabilize the molecule in monomeric form. Preferred monomeric Fc are disclosed in International Publication No. 2011 / 063348, which is incorporated herein by reference in its entirety. In certain embodiments, the IL-2 mutant protein Fc fusion protein contains Fc with loaded amino acids at positions 392 and 409, along with threonine substitutions at Y349, L351, L368, V397, L398, F405, or Y407.

[0067] In certain embodiments, the IL-2 mutant protein Fc fusion protein includes a linker between Fc and the IL-2 mutant protein. A variety of 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 includes 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 Fc and the IL-2 mutant protein. In some embodiments, the polypeptide region between the Fc region and the IL-2 mutant protein region includes 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 a suitable cell, and such glycosylation may help stabilize the protein when administered in solution and / or in vivo. Therefore, in certain embodiments, the IL-2 mutant protein fusion protein includes a glycosylated linker between the Fc region and the IL-2 mutant protein region.

[0068] Glycosylated linkers are considered to be useful when positioned within the context of a polypeptide. Provided herein are polypeptides comprising GGNGT (SEQ ID NO: 6) or YGNGT (SEQ ID NO: 7) which are inserted into or substitute for one or more amino acids in the amino acid sequence of the polypeptide. In preferred embodiments, GGNGT (SEQ ID NO: 6) or YGNGT (SEQ ID NO: 7) are inserted into a loop of the polypeptide tertiary structure. In other embodiments, one or more amino acids in 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 amino-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 may further include T3 substitutions, e.g., T3N or T3A. The IL-2 mutant protein may further include S5 substitutions, such as S5T.

[0070] The covalent modifications of IL-2 mutant proteins, IL-2 mutant protein Fc fusion proteins, and anti-IL-2 antibodies are within the scope of the present invention and are generally performed post-translation, but not necessarily. For example, several types of covalent modifications are introduced into a molecule by reacting some of its amino acid residues with an organic derivatizing agent that can react with selected side chains or N-terminal or C-terminal residues.

[0071] Cysteinyl residues most commonly react with α-haloacetates (and their corresponding amines), such as chloroacetic acid or chloroacetamide, to produce carboxymethyl or carboxyamidemethyl derivatives. Cysteinyl residues can also be derivatized by reactions with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercrinebenzoic acid, 2-chloromercrine-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0072] The histidyl residue is derivatized by reaction with diethyl pyrocarbonate at pH 5.5–7.0 because this agent is relatively specific to 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] The ricinyl and amino-terminal residues react with succinic anhydride or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the ricinyl residue. Other reagents suitable for derivatization of alpha-amino-containing residues include transaminase-catalyzed reactions with imide esters, such as methyl picolinimide; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and glyoxylate.

[0074] Arginyl residues are modified by reaction with one or more conventional reagents, particularly 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 pK of the guanidine functional group. a This is because the reaction rate is high. Furthermore, the reagent can react with lysine groups and arginine epsilon-amino groups.

[0075] Specific modification of tyrosyl residues may be carried out, in particular, for the purpose of introducing spectral labeling into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidisole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. 125 I or 131 The chloramine T method, described above, is suitable for preparing labeled proteins used in radioimmunoassays by iodizing I.

[0076] The carboxyl side group (aspartyl or glutamyl) is 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, the aspartyl and glutamyl residues are converted to asparaginyl and glutamyl residues by reaction with ammonium ions.

[0077] Derivatization with difunctional 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), homodifunctional imide esters (disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate)), and difunctional maleimides such as bis-N-maleimide-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that can form crosslinks in the presence of light. In addition, reactive, water-insoluble matrices, such as cyanide-activated carbohydrates and reaction substrates described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440, can be used for protein immobilization.

[0078] Glutaminyl and asparaginyl residues are frequently deamidated to their corresponding glutamyl and aspartyl residues, respectively. These residues can also be 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 hydroxyl groups of ceryl or threonyl residues, methylation of α-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, 1983, pp. 79-86), acetylation of N-terminal amines, and amidation of any C-terminal carboxyl group.

[0080] The covalent modifications of IL-2 mutant proteins, IL-2 mutant protein Fc fusions, or anti-IL-2 antibodies that fall within the scope of the present invention include altering the glycosylation pattern of a protein. As is known in the art, the glycosylation pattern can be determined by the protein sequence (e.g., the presence or absence of certain glycosylated amino acid residues, as discussed below) or by both the host cell or organism in which the protein is produced. Specific expression systems are discussed below.

[0081] Polypeptide glycosylation is typically either N-linked or O-linked. N-linked glycosylation refers to the binding of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for the enzymatic binding of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the binding 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] The addition of a glycosylation site to an IL-2 mutant protein, an IL-2 mutant protein Fc fusion, or an anti-IL-2 antibody can be successfully achieved by modifying the amino acid sequence so that the amino acid sequence contains one or more of the above-mentioned tripeptide sequences (of the N-linked glycosylation site). Alternatively, the modification may be made by adding or substituting one or more serine or threonine residues to the start sequence (for the O-linked glycosylation site). For ease of modification, the amino acid sequence of the IL-2 mutant protein, the IL-2 mutant protein Fc fusion, or an anti-IL-2 antibody is preferably modified via changes at the DNA level, particularly by mutating the DNA encoding the target polypeptide with pre-selected bases to generate codons that will be translated to the desired amino acids.

[0083] Another means of increasing the number of carbohydrate moieties on IL-2 mutant proteins, IL-2 mutant protein Fc fusions, or anti-IL-2 antibodies is by chemical or enzymatic coupling of glycosides with proteins. This procedure is advantageous for N-linked and O-linked glycosylation in that it does not require the production of proteins in glycosylated host cells. Depending on the coupling method used, the sugar may be bonded to (a) arginine and histidine, (b) a free carboxyl group, (c) a free sulfhydryl group, such as the group of cysteine, (d) a free hydroxyl group, such as the group of serine, threonine, or hydroxyproline, (e) an aromatic residue, such as a residue of phenylalanine, tyrosine, or tryptophan, or (f) a glutamine amide group. These methods are described in the international publication No. 87 / 05330, published on September 11, 1987, and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306.

[0084] The removal of the carbohydrate moiety present on the initiating IL-2 mutant protein, the IL-2 mutant protein Fc fusion, or the anti-IL-2 antibody may be achieved chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment cleaves almost all or all of the sugars except for the bound sugar (N-acetylglucosamine or N-acetylgalactosamine) while leaving the polypeptide intact. 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 the carbohydrate moiety on the polypeptide can be achieved using various endoglycosidases and exoglycosidases, as described by Thotakura et al., 1987, Meth. Enzymol. 138:350. Glycosylation at potential glycosylation sites can be inhibited by the use of the compound tunicamycin, described by Duskin et al., 1982, J. Biol. Chem. 257:3105. Tunicamycin inhibits the formation of protein-N-glycosidic bonds.

[0085] Another type of covalent modification of an IL-2 mutant protein, an IL-2 mutant protein Fc fusion, or an anti-IL-2 antibody involves conjugating the protein to various non-proteinaceous polymers, including, but not limited to, various polyols, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene, using methods 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 present invention include PEGylated IL-2 mutant proteins, IL-2 mutant protein Fc fusions, or anti-IL-2 antibodies. Such PEGylated proteins may have a longer half-life and / or reduced immunogenicity compared to their non-PEGylated forms.

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

[0087] Nucleotide sequences corresponding to the amino acid sequences described herein, which will be used as probes or primers for nucleic acid isolation or as query sequences for database searches, can be obtained by "back translation" from the amino acid sequences. DNA sequences encoding IL-2 mutant proteins and IL-2 mutant protein Fc fusion proteins can be isolated and amplified using well-known polymerase chain reaction (PCR) procedures. Oligonucleotides defining the desired ends of the DNA fragment combinations are used as 5' and 3' primers. The oligonucleotides may additionally contain recognition sites for restriction endonucleases to facilitate insertion of the amplified DNA fragment combinations into an expression vector. PCR techniques are 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 their corresponding complementary sequences. “Isolated nucleic acid” refers to a nucleic acid isolated from adjacent gene sequences within the genome of the organism from which the nucleic acid was isolated, in the case of nucleic acids isolated from naturally occurring sources. In the case of nucleic acids synthesized enzymatically or chemically from a template, such as PCR products, cDNA molecules, or oligonucleotides, nucleic acids derived from such processes are understood to be isolated nucleic acids. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a distinct fragment, or a nucleic acid molecule as a component of a larger nucleic acid construct. In one preferred embodiment, the nucleic acid is substantially free of contaminating endogenous material. The nucleic acid molecules were preferably derived from DNA or RNA that had been isolated at least once, in a substantially pure form and in an amount or concentration that allowed for the identification, manipulation, and recovery of its constituent nucleotide sequences 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)). Such sequences were preferably provided and / or constructed in the form of an open reading frame not interrupted by internal untranslated sequences or introns typically found within eukaryotic genes. The sequence of untranslated DNA may be located at the 5' or 3' end of the open reading frame, in which case this does not interfere with the manipulation or expression of the coding region.

[0089] IL-2 mutant proteins according to the present invention are typically prepared by generating variant-encoding DNA 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 known in the art, and then expressing the recombinant DNA in cell culture as outlined herein. However, 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 may also be selected, as outlined in more detail below.

[0090] As will be understood 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 falls within the scope of the present invention and can be formed using standard techniques. Therefore, once a specific amino acid sequence is identified, those skilled 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 alter 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, transcriptions, or expression cassettes containing at least one of the aforementioned 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 a sequence for maintaining the plasmid, as well as a sequence for cloning and expressing the exogenous nucleotide sequence. Such sequences, collectively referred to as “flanking sequences,” will, in certain embodiments, 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 the nucleic acid encoding the polypeptide to be expressed, and one or more selection 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 may encode polyHis (e.g., hexaHis (SEQ ID NO: 8)), for which commercially available antibodies exist, or another “tag,” such as FLAG, HA (hemagglutinin influenza virus), or myc. This tag is typically fused to the polypeptide immediately after polypeptide expression and can function as a means for affinity purification or detection of the polypeptide from host cells. 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, for example, using a specific peptidase for cleavage.

[0094] Flanking sequences may be homogeneous (i.e., derived from the same species and / or strain as the host cell), heterogeneous (i.e., derived 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 native. Therefore, the source of a flanking sequence may be any prokaryote or eukaryote, any vertebrate or invertebrate, or any plant, provided that the flanking sequence is functional in a host cellular mechanism and can be activated by such mechanism.

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

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

[0097] The origin of replication is typically a component of commercially available prokaryotic expression vectors, and this origin assists in the amplification of the vector within the host cell. If the selected vector does not contain an origin of replication site, it may be chemically synthesized based on a known sequence 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, varicella-stomatitis virus (VSV), or papillomavirus, e.g., HPV or BPV) are useful for cloning vectors in mammalian cells. In general, the origin of replication component is not required for mammalian expression vectors (for example, the SV40 origin is often used only because it also contains the viral initial promoter).

[0098] Transcription termination sequences are typically located 3' to the end of the polypeptide coding region and function to terminate transcription. In prokaryotic cells, the transcription termination sequence is usually a GC-rich fragment followed by a poly-T sequence. These sequences can be readily cloned from libraries or even commercially purchased as part of a vector, while they can also be readily synthesized using nucleic acid synthesis methods such as those described herein.

[0099] Selection marker genes encode proteins essential for the survival and proliferation of host cells grown in a selective medium. Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, tetracycline, or kanamycin, to prokaryotic host cells; (b) compensate for deficiencies in the cellular nutritional requirements; or (c) supply essential nutrients unavailable from complex or standard media. Specific selection markers include kanamycin resistance genes, ampicillin resistance genes, and tetracycline resistance genes. Advantageously, neomycin resistance genes can also be used for selection in both prokaryotic and eukaryotic host cells.

[0100] The gene to be expressed may be amplified using other selected genes. Amplification is the process by which genes required for the production of proteins important for proliferation or cell survival are repeated in tandem within the chromosomes of successive generations of recombinant cells. Examples of suitable selection markers for mammalian cells include the dihydrofolate reductase (DHFR) gene and the promoter resthymidine kinase gene. Mammalian cell transformants are subjected to selective pressure such that only the transformant is uniquely adapted to survive by the selective gene present in the vector. Selective pressure is imposed by culturing the transformed cells under conditions of continuously increasing concentration of the selector in the culture medium, which leads to amplification of both the selectable gene and, consequently, the genes encoding the desired polypeptide, such as the heavy and / or light chain of the IL-2 mutant protein, the IL-2 mutant protein Fc fusion, or an anti-IL-2 antibody. As a result, large quantities of polypeptides are synthesized from the amplified DNA.

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

[0102] In cases where glycosylation is desired in eukaryotic host cell expression systems, various pre-sequences or pro-sequences may be manipulated to improve glycosylation or yield. For example, the peptidase cleavage site of a particular signal peptide may be altered, or a pro-sequence may be added, which may also affect glycosylation. The final protein product may have one or more additional amino acids associated with expression at position -1 (relative to the first amino acid of the mature protein), which may not be completely removed. For example, the final protein product may have one or two amino acid residues found within the peptidase cleavage site, attached to the amino terminus. In addition, the use of several enzyme cleavage sites may result in a slightly cleaved form of the desired polypeptide if the enzyme cleaves in such a region within the mature polypeptide.

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

[0104] Promoters suitable for use in yeast hosts are also well known in this 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 derived from the genomes of viruses such as polyomaviruses, fowlpox virus, adenoviruses (adenovirus type 2, etc.), bovine papillomavirus, aerosarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and most preferably Simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and actin promoters.

[0105] Additional promoters that may be targeted include, but are not limited to, the following: the SV40 early promoter (Benoist and Chambon, 1981, Nature 290:304-310); the CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad. USA 81:659-663); the promoter contained in the long terminal repeat on the 3' side of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797); the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1444-1445); the promoter and regulatory sequence derived from the metallothionein gene (Prinster et al., 1982, Nature 296:39-42); and prokaryotic promoters such as beta-lactamase promoters (Villa-Kamaroff et al.) Examples include al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731; or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Furthermore, the target areas are the following animal transcriptional regulatory regions that exhibit tissue specificity and are utilized in transgenic animals: the elastase I gene regulatory region 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 regulatory region active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122); and the immunoglobulin gene regulatory region active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al. al., 1987, Mol. Cell. Biol.7:1436-1444); Mouse mammary tumor virus regulatory region active in testicular cells, mammary cells, lymphoid cells and mast cells (Leder et al., 1986, Cell 45:485-495); Albumin gene regulatory region active in the liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); Alpha-fetoprotein gene regulatory region active in the liver (Krumlauf et al., 1985, Mol.Cell.Biol. 5:1639-1648; Hammer et al., 1987, Science 253:53-58); Alpha-1 antitrypsin gene regulatory region active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); Betaglobin gene regulatory region 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 regulatory region active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain 2 gene regulatory region active in skeletal muscle (Sani, 1985, Nature 314:283-286); and gonadotropin-releasing hormone gene regulatory region active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

[0106] Enhancer sequences may be inserted into vectors to increase transcription by higher eukaryotes. Enhancers are cis-acting elements of DNA, typically about 10–300 bp in length, and act on promoters to increase transcription. Enhancers are relatively independent of direction and position, and are found at both the 5' and 3' ends of the transcription unit. Several enhancer sequences are known to be available from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). However, viral enhancers are typically used. The SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, and adenovirus enhancer known in this technique are exemplary enhancing elements for the activation of eukaryotic promoters. Enhancers may be located in the vector at either the 5' or 3' end of the coding sequence, but are typically positioned at the 5' end of the promoter. By incorporating sequences encoding appropriate native or heterologous signal sequences (leader sequences or signal peptides) into expression vectors, extracellular secretion of IL-2 mutant proteins, IL-2 mutant protein Fc fusions, or the heavy and / or light chains of anti-IL-2 antibodies can be promoted. The choice of signal peptide or leader depends on the type of host cell in which the protein will be produced, and heterologous signal sequences may replace native signal sequences. Examples of signal peptides that function in mammalian host cells include: the interleukin-7 (IL-7) signal sequence described in U.S. Patent No. 4,965,195; the interleukin-2 receptor signal sequence 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; and 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 includes a leader sequence as shown in Figure 24.

[0107] A 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-binding region (MAR). The MAR can protect the integrated vector from "location" effects by mediating the structural organization of chromatin. Therefore, the MAR is particularly useful when the vector is used to produce a stable transfectant. Several natural and synthetic MAR-containing nucleic acids are known in the art, for example, U.S. Patent Nos. 6,239,328; 7,326,567; 6,177,612; 6,388,066; 6,245,974; 7,259,010; 6,037,525; 7,422,874; and 7,129,062.

[0108] The expression vector of the present invention may be constructed from an initiation 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. Methods used to obtain each of the flanking sequences are well known to those skilled in the art.

[0109] After the vector is constructed and nucleic acid molecules encoding an IL-2 mutant protein, an IL-2 mutant protein Fc fusion, or the heavy and / or light chain of an anti-IL-2 antibody are inserted into the appropriate sites in the vector, the complete vector may be inserted into a host cell suitable for amplification and / or polypeptide expression. Transformation of the expression vector into selected host cells may be achieved by well-known methods, e.g., translocation, infection, calcium phosphate coprecipitation, electroporation, microinjection, lipofection, DEAE-dextran-mediated translocation, 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 Sambrook et al., 2001.

[0110] When cultured under appropriate conditions, host cells synthesize IL-2 mutant proteins, IL-2 mutant protein Fc fusions, or heavy and / or light chains of anti-IL-2 antibodies, which can then be collected from the culture medium (if secreted by the host cells) or directly from the producing host cells (if not secreted). The selection of appropriate host cells will depend on various factors, including the desired expression level, polypeptide modifications (such as glycosylation or phosphorylation) desirable or essential for activity, and the ease of folding into biologically active molecules. Host cells may be eukaryotes or prokaryotes.

[0111] Mammalian cell lines available as expression hosts are well known in this art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC). Recombinant polypeptides of the present invention can be formed using any cell line used in expression systems known in this art. Generally, host cells are transformed with a recombinant expression vector containing DNA encoding a desired IL-2 mutant protein, an IL-2 mutant protein Fc fusion, or an anti-IL-2 antibody. Host cells that may be used include prokaryotes, yeasts, 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 their derivatives, 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 lines, and the African green monkey kidney cell line CVI (ATCC) as described in McMahan et al., 1991, EMBO J.10:2821. Examples include CVI / EBNA cell lines derived from CCL70), human embryonic kidney cells, e.g., 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, when polypeptide use is desired in various signal transduction assays or reporter assays, mammalian cell lines, e.g., HepG2 / 3B, KB, NIH3T3, or S49 may be used for polypeptide expression.

[0112] In addition to the above, polypeptides can be produced in lower eukaryotes such as yeast, or in prokaryotes such as bacteria. Suitable yeasts include budding yeast (Saccharomyces cerevisiae), fission yeast (Schizosaccharomyces pombe), strains of the genus Kluyveromyces, the genus Candida, or any yeast strain capable of expressing heterologous polypeptides. Suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous polypeptides. If polypeptides are formed within yeast or bacteria, it may be desirable to modify the polypeptides produced within the yeast or bacteria, for example, by phosphorylation or glycosylation at appropriate sites, to obtain functional polypeptides. Such covalent bonding can be achieved using known chemical or enzymatic methods.

[0113] Furthermore, polypeptides can be generated by using an insect expression system, in which the isolated nucleic acids of the present invention are operably ligated to a suitable regulatory sequence in one or more insect expression vectors. Materials and methods for baculovirus / insect cell expression systems are commercially available, for example, in kit form from Invitrogen, San Diego, Calif., USA (MaxBac® kits), 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 generate polypeptides using RNA derived from the nucleic acid constructs disclosed herein. Cloning and expression vectors suitable for use in bacteria, fungi, 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 present invention, preferably operably linked to at least one expression regulatory sequence, is a "recombinant host cell."

[0114] In a particular embodiment, 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,D2 0A,M104V;H16E,V91H;V91H,D20A,M104V;H16E,V91H,M104T;H16E,V91H, E61Q,M104T;V91K,E61Q,H16E;V91K,H16R,M104L;H16E,V91H,E16Q;V91 K,E61Q,H16R;D20W,V91K,E61Q;V91H,H16R;V91K,H16R;D20W,V91K,E61Q ,M104T;V91K,D20A;V91H,D20A,E16Q;V91K,D20A,M104L;V91H,D20A;V9 1K,E61Q,D20A;V91H,M104T;V91H,M104V;V91K,E61Q;V91K,N88K,E61Q,M 104T;V91K,N88K,E61Q;V91H,E61Q;V91K,N88K;D20A,H16E,M104T;D20A ,M104T;H16E,N88K;D20A,M104V;D20A,M104L;H16E,M104T;H16E,M104V; N88K,M104V;N88K,E61Q;D20A,E61Q;H16R,D20A;D20W,E61Q;H16E,E61Q ;H16E,M104L;N88K,M104T;D20A,H16E;D20A,H16E,E16Q;D20A,H16R,E16 Q;V91K,D20W;V91A,H16A;V91A,H16D;V91A,H16E;V91A,H16S;V91E,H16A ;V91E,H16D;V91E,H16E;V91E,H16S;V91K,H16A;V91K,H16D;V91K,H16S;and / or comprising isolated nucleic acids encoding a human IL-2 mutant protein containing the V91S,H16E substitution and an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in SEQ ID NO: 1.

[0115] Also included are isolated nucleic acids encoding one 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 encoded within a single open reading frame, with a linker optionally encoded between the Fc region and the IL-2 mutant protein.

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

[0117] In another embodiment, provided herein are host cells comprising isolated nucleic acids encoding the IL-2 mutant protein, the IL-2 mutant protein Fc fusion protein, or an anti-IL-2 antibody. The host cells may be prokaryotic cells such as Escherichia coli (E. coli) or eukaryotic cells such as mammalian cells. In certain embodiments, the host cells are Chinese hamster ovary (CHO) cell lines.

[0118] In another embodiment, a method for forming a human IL-2 mutant protein is provided herein. The method comprises culturing host cells under conditions in which a promoter operably linked to the human IL-2 mutant protein is expressed. The human IL-2 mutant protein is then harvested from the culture. The IL-2 mutant protein may be harvested from the culture medium and / or host cell lysates.

[0119] In another embodiment, a method for forming a human IL-2 mutant protein Fc fusion protein is provided herein. The method comprises culturing host cells under conditions in which a promoter operably linked to the human IL-2 mutant protein Fc fusion protein is expressed. The human IL-2 mutant protein Fc fusion protein is then harvested from the culture. The human IL-2 mutant protein Fc fusion protein may be harvested from the culture medium and / or host cell lysates.

[0120] In another embodiment, a method for forming an anti-IL-2 antibody is provided herein. The method comprises culturing host cells under conditions in which promoters operably linked to the heavy and light chains of the anti-IL-2 antibody are expressed. The anti-IL-2 antibody is then harvested from the culture. The anti-IL-2 antibody may be harvested from the culture medium and / or host cell lysates.

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

[0122] Preferably, the formulation material is non-toxic to the recipient at the dosage and concentration used. In certain embodiments, a pharmaceutical composition is provided comprising a therapeutically effective amount of an IL-2 mutant protein-containing therapeutic molecule, 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 the composition's properties, such as pH, osmolality, viscosity, transparency, color, isotonicity, odor, sterility, stability, dissolution rate or release rate, absorbency, or permeability. In such embodiments, suitable formulation materials include: amino acids (glycine, glutamine, asparagine, arginine, proline, or lysine, etc.); antimicrobial agents; antioxidants (ascorbic acid, sodium sulfite, or sodium bisulfite, etc.); buffers (boric acid, bicarbonate, tris-HCl, citric acid, phosphoric acid, or other organic acids, etc.); fillers (mannitol or glycine, etc.); chelating agents (ethylenediaminetetraacetic acid (EDTA), etc.); complexing agents (caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin, etc.); fillers; monosaccharides, disaccharides, and other carbohydrates (glucose, mannose, or dextrin, etc.); proteins (serum albumin, gelatin, or immunoglobulin, etc.); colorants, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (polyvinylpyrrolidone, etc.); low molecular weight polypeptides; and salt-forming pairs. Ions (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 Pluronic acid, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, Triton, tromethamine, lecithin, cholesterol, tyroxapol, etc.); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol, etc.); delivery vehicles; diluents; excipients; and / or pharmaceutical adjuvants. See REMINGTON'S PHARMACEUTICAL SCIENCES, 18” Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company.

[0124] In certain embodiments, the optimal pharmaceutical composition will be determined by those skilled in the art, for example, depending on the intended route of administration, the form of delivery, and the desired dose. See, for example, Remington's Pharmaceutical Sciences cited above. In certain embodiments, such a composition may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the antigen-binding protein of the present invention. In certain embodiments, the main vehicle or carrier in the pharmaceutical composition may actually be either aqueous or non-aqueous. For example, suitable vehicles or carriers may be water for injection, saline, or artificial cerebrospinal fluid, but other materials common in compositions for parenteral administration may be supplemented. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, the pharmaceutical composition comprises Tris buffer at approximately pH 7.0–8.5, or acetate buffer at approximately pH 4.0–5.5, and may further comprise sorbitol or a suitable substitute 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 aqueous solution by mixing a selected composition having a desired purity with an optional formulation agent (REMINGTON'S PHARMACEUTICAL SCIENCES, as mentioned above). Furthermore, 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. Alternatively, the compositions may be selected for inhalation or for delivery 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 the administration site in an acceptable concentration. In certain embodiments, a buffer is used to maintain the composition at a physiological pH or slightly lower, typically within a pH range of about 5 to about 8.

[0126] When parenteral administration is intended, the therapeutic composition used in the present invention may be provided in the form of a pyrogenically-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 appropriately retained. In certain embodiments, the preparation may involve formulation of the desired molecule with an agent capable of achieving controlled or sustained release of the product that 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 increasing 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, including those comprising formulations containing an IL-2 mutant protein composition or an anti-IL-2 antibody composition in a sustained-release or controlled-release formulation, will become apparent to those skilled in the art. Furthermore, techniques for formulating various other sustained or controlled delivery means, e.g., liposome carriers, biodegradable microparticles, or porous beads, and depot injections, are known to those skilled in the art. See, for example, International Application PCT / US93 / 00829 (incorporated by reference), which describes the controlled release of porous polymer microparticles for delivering pharmaceutical compositions. The sustained-release preparation may comprise a semipermeable polymer matrix in the form of a molded article, e.g., a film or microcapsules. The sustained-release matrix may include polyester, hydrogel, polylactide (disclosed in U.S. Patent No. 3,773,919 and European Patent Application Publication No. 058481, which are incorporated by reference, respectively), copolymer 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, op. cit.), or poly-D(-)-3-hydroxybutyric acid (European Patent Application Publication No. 133,988). Furthermore, the sustained-release composition may include liposomes that can be prepared by any of several methods known in the art. See, for example, Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-3692; European Patent Application Publication No. 036,676; European Patent Application Publication No. 088,046; and European Patent Application Publication No. 143,949, incorporated by reference.

[0128] Pharmaceutical compositions used 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 freeze-dried, sterilization using this method may be performed either before or after freeze-drying and reconstitution. Compositions for parenteral administration can be stored in freeze-dried form or in solution. Parenteral compositions are generally placed in containers with a sterile access port, such as intravenous solution bags or vials with a stopper that can be penetrated by a subcutaneous needle.

[0129] Aspects of the present invention include self-buffering IL-2 mutant protein preparations or anti-IL-2 antibody preparations, which can be used as pharmaceutical compositions as described in International Publication No. 06138181(A2) (PCT / US2006 / 022599), which is incorporated herein by reference in whole.

[0130] As discussed above, certain embodiments provide a pharmaceutical Il-2 mutant protein Fc fusion protein comprising an IL-2 mutant protein composition or an anti-IL-2 antibody composition, in particular an IL-2 mutant protein or anti-IL-2 antibody composition, plus one or more excipients, such as those described exemplary in this section and elsewhere in this specification. Excipients can be used in the present invention for a wide range of purposes, including the processes of the present invention for modifying the physical, chemical, or biological properties of a formulation, such as viscosity adjustment, and / or improving efficacy and / or stabilizing such a formulation, as well as processes against degradation and spoilage caused by stress during, for example, manufacturing, transportation, storage, preparation before use, administration, and thereafter.

[0131] Various explanations are possible for protein stabilization and useful formulation materials and methods therein, 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 these in whole is incorporated herein by reference), and in particular, various explanations are possible with respect to protein pharmaceutical products and processes for veterinary and / or human medical uses, especially in the parts relating to excipients and processes for self-buffering protein formulations according to the present invention.

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

[0133] As is well known, ions can stabilize native proteins by binding to charged residues on the protein surface and shielding charged and polar groups within the protein, thereby reducing the intensity of their electrostatic, attractive, and repulsive interactions. Furthermore, ions can stabilize denatured proteins, particularly by binding to denatured peptide bonds (--CONH). Additionally, ionic interactions with charged and polar groups within proteins can reduce intermolecular electrostatic interactions, thereby preventing or mitigating protein aggregation and insolubilization.

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

[0135] Free amino acids can be used as bulking agents, stabilizers, and antioxidants, and for other standard uses, in IL-2 mutant protein formulations or anti-IL-2 antibody formulations according to various embodiments of the present invention. Lysine, proline, serine, and alanine can be used to stabilize proteins in the formulation. Glycine is useful in lyophilization to ensure accurate cake structure and properties. Arginine may 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 this specification, polyethylene glycol (PEG) and related substances. Polyols are cosmotropic. Polyols are useful stabilizers in both liquid and lyophilized formulations to protect proteins from physical and chemical degradation processes. Polyols are also useful for adjusting the tonicity of formulations.

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

[0138] Embodiments of IL-2 mutant protein formulations and / or anti-IL-2 antibody formulations further include surfactants. Protein molecules may be susceptible to adsorption to surfaces and denaturation and resulting aggregation at gas-liquid interfaces, solid-liquid interfaces, and liquid-liquid interfaces. These effects are generally inversely proportional to the protein concentration. These harmful interactions are generally inversely proportional to the protein concentration and are typically exacerbated by physical agitation, for example, that occurs 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] Furthermore, surfactants are generally used to control the three-dimensional stability of proteins. In this respect, the use of surfactants is protein-specific, as any given surfactant typically stabilizes some proteins and destabilizes others.

[0141] Polysorbates are susceptible to oxidative degradation and, if supplied, often contain sufficient peroxides to cause oxidation of protein side chains, particularly methionine. Consequently, polysorbates should be used cautiously and, if 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 formulation or anti-IL-2 antibody formulation further include one or more antioxidants. The deleterious oxidation of proteins in pharmaceutical formulations 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 formulations according to the invention are preferably water-soluble and maintain their activity throughout the product's shelf life. In this regard, EDTA is a preferred antioxidant according to the invention.

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

[0144] The formulations according to the invention may contain metal ions, which are protein cofactors and essential for forming protein coordination complexes, such as zinc, which is essential for forming certain insulin suspensions. Metal ions can also inhibit some of the processes that degrade proteins. However, metal ions can also catalyze the 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 destabilized by Mg+2 Mn +2 and Zn +2 Cu +2 and Fe +2 It can be destabilized by, and its aggregation is Al +3 It can be amplified by ions.

[0146] Embodiments of IL-2 mutant protein formulations or anti-IL-2 antibody formulations further include one or more preservatives. Preservatives are essential when developing multi-dose parenteral formulations involving two or more extractions from the same container. Their main function is to inhibit microbial growth throughout the entire shelf life or use period of the drug product and to ensure the sterility of the product. Commonly used preservatives include benzyl alcohol, phenol, and m-cresol. While preservatives have a long history of use in low-molecular-weight parenteral drugs, the development of protein formulations containing preservatives can be challenging. Preservatives almost always have an destabilizing effect (aggregation) on proteins, which is a major factor limiting their use in multi-dose protein formulations. To date, most protein drugs have only been formulated for single use. However, the possibility of multi-dose formulations offers the added advantage of increased patient convenience and marketability. A good example is human growth hormone (hGH), where the development of a preserved formulation led to the commercialization of more convenient multi-dose injectable pens. At least four such pen devices containing hGH-preserved formulations are currently available on the market. Norditropin (liquid, Novo Nordisk), Nutropin AQ (liquid, Genentech), and Genotropin (lyophilized - dual-chamber cartridge, Pharmacia & Upjohn) contain phenol, 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, such as the IL-2 mutant protein or Fc-fusion of an IL-2 mutant protein described herein, is formulated in 10 mM KPi and 161 mM L-arginine at pH 7.6.

[0148] Several aspects need to be considered during the formulation and development of preservative-type formulations. The effective preservative concentration in the drug product must be optimized. This requires testing a given preservative in a dosage form within a concentration range that imparts antimicrobial efficacy without compromising protein stability.

[0149] In another embodiment, the present invention provides an IL-2 mutant protein, or an Fc fusion of an IL-2 mutant protein, in a lyophilized formulation. The lyophilized product is lyophilized without preservatives and can be reconstituted at use with a preservative-containing diluent. This reduces the time the preservative is in contact with the protein, significantly minimizing the associated stability risks. In the case of liquid formulations, the effectiveness and stability of the preservative should be maintained throughout the entire product shelf life (approximately 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 are generally designed for specific routes and methods of administration, specific dosages and frequencies of administration, specific treatments for specific diseases, and, in particular, within the scope of bioavailability and persistence. Thus, formulations may be designed in accordance with the present invention for delivery by any suitable route, including but not limited to oral, transaural, transocular, transrectal, and transvaginal routes, and for delivery by parenteral routes, including intravenous and intra-arterial injections, intramuscular injections, and subcutaneous injections.

[0151] Once a pharmaceutical composition is formulated, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. Such formulations can be stored in an immediate-use form or in a form that is reconstituted before administration (e.g., lyophilized form). The present invention also provides kits for generating single-dose administration units. Each kit of the present invention may 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 rio-syringes).

[0152] The therapeutically effective dose of the IL-2 mutant protein pharmaceutical composition to be used will depend, for example, on the context and purpose of the treatment. Those skilled in the art will understand that the appropriate dose level for the 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 used, the route of administration, and the patient's size (body weight, body surface, or organ size) and / or condition (age and health status). In certain embodiments, the clinician may potentiate the dose and modify the route of administration to obtain the optimal therapeutic effect. Typical doses may range from about 0.1 μg / kg to over about 1 mg / kg, depending on the factors mentioned above. In certain embodiments, doses may range from 0.5 μg / kg to over 100 μg / kg, and possibly from 2.5 μg / kg to over 50 μg / kg.

[0153] A therapeutically effective amount of 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-asymptomatic periods, or prevention of disability or impairment caused by the suffering of the disease.

[0154] Pharmaceutical compositions may be administered using medical devices. Examples of medical devices for administering pharmaceutical compositions are described in U.S. Patent Nos. 4,475,196; 4,439,196; 4,447,224; 4,447,233; 4,486,194; 4,487,603; 4,596,556; 4,790,824; 4,941,880; 5,064,413; 5,312,335; 5,312,335; 5,383,851; and 5,399,163 (all incorporated herein by reference).

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

[0156] Methods for treating autoimmune disorders or inflammatory disorders In certain embodiments, the IL-2 mutant protein or anti-IL-2 antibody of the present invention is used to treat autoimmune disorders or inflammatory disorders. In preferred embodiments, an IL-2 mutant protein Fc fusion protein is used.

[0157] Disorders particularly suitable for treatment with the IL-2 mutant protein or anti-IL-2 antibody disclosed herein include inflammation, autoimmune diseases, atopic diseases, paraneoplastic autoimmune diseases, chondritis, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, small-joint juvenile rheumatoid arthritis, polyjoint juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathy-related arthritis, juvenile reactive arthritis, and juvenile rheumatoid arthritis. Juvenile Reiter syndrome, SEA syndrome (seronegative, enthesopathy, arthritis syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, small-joint rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, intestinal arthritis, reactive arthritis, Reiter syndrome, SEA syndrome (seronegative, enthesopathy, arthritis syndrome), dermatomyositis, psoriatic arthritis, Scleroderma, vasculitis, myelitis, polymyositis, dermatomyositis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjögren's syndrome, psoriasis, psoriasis vulgaris, guttate psoriasis, reverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, inflammation Examples of conditions that may be present include, but are not limited to, symptomatic bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, sinusitis, sinusitis with polyps, eosinophilic esophagitis, eosinophilic bronchitis, Guillain-Barré disease, type 1 diabetes mellitus, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD, transplant rejection, renal impairment, hepatitis C-induced vasculitis, and spontaneous abortion.

[0158] In preferred embodiments, the autoimmune or inflammatory disorders include lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type 1 diabetes, multiple sclerosis, spontaneous abortion, atopic diseases, and inflammatory bowel disease.

[0159] In another embodiment, a patient suffering from or at risk of developing an autoimmune or inflammatory disorder is treated with an IL-2 mutant protein or an anti-IL-2 antibody (e.g., an IL-2 mutant protein disclosed herein, e.g., an IL-2 mutant protein Fc fusion as disclosed herein, or possibly as part of an Fc fusion molecule of the type described herein, or another IL-2 mutant protein or wild-type IL-2 known in the art) and the patient's response to the treatment is monitored. The monitored patient response may be any detectable or measurable response of the patient to the treatment, or any combination of such responses. For example, the response may be a change in the patient's physiological state, e.g., body temperature or fever, appetite, sweating, headache, nausea, fatigue, hunger, thirst, cognitive function, etc. Otherwise, the response may be, for example, a change in the cell type or amount of gene products (e.g., proteins, peptides, or nucleic acids) in a peripheral blood sample taken from the patient. In one embodiment, the patient's treatment regimen is modified if the patient has a detectable or measurable response to the treatment, or if such response exceeds a certain threshold. This change may involve a decrease or increase in the frequency of medication, a decrease or increase in the amount of IL-2 mutant protein or anti-IL-2 antibody administered per single dose, a “pause” of medication (i.e., a temporary interruption of treatment for a specific period, or until the physician administering the treatment decides that the treatment should be continued, or until the monitored patient’s response indicates that the treatment should or can be resumed), or termination of treatment. In one embodiment, the response is a change in the patient’s body temperature or CRP level. For example, the response may be an increase in the patient’s body temperature, an increase in CRP levels in a peripheral blood sample, or both. In a particular embodiment, the patient’s treatment is reduced, temporarily suspended, or terminated if the patient’s body temperature rises 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 treatment.In another specific embodiment, the patient's treatment is reduced, suspended, 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. Other patient responses that may be monitored and used when deciding whether to modify, reduce, suspend, 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 infection, or detection thereof), thrombocytopenia, thromboangiopathy, injection site reactions, vasculitis (e.g., hepatitis C virus vasculitis), or inflammatory signs or diseases. Further patient responses that may be monitored and used when deciding whether to modify, reduce, increase, suspend, or terminate the treatment include NK cells, Treg cells, and FOXP3. - CD4T cells, FOXP3 + An increase in the number of CD4 T cells, FOXP3-CD8 T cells, or eosinophils may be observed. Increases in these cell types can be detected, for example, as an increase in the number of such cells per unit of peripheral blood (e.g., expressed as an increase in cells per milliliter of blood), or as an increase in the percentage of such cell types compared to other cell types in the blood sample. Another patient response that may be monitored is CD25 in the patient's peripheral blood sample. + This involves an increase in the amount of cell surface-bound IL-2 mutant protein or anti-IL-2 antibody on the cell.

[0160] Methods for increasing Treg cells IL-2 mutant proteins, anti-IL-2 antibodies, or IL-2 mutant protein Fc fusion proteins may be used to proliferate Treg cells in a subject or sample. Provided herein is a method for increasing the ratio of Tregs to unregulated T cells. This method involves contacting a population of T cells with an effective amount of human IL-2 mutant protein, anti-IL-2 antibody, or IL-2 mutant protein Fc fusion. The ratio can be measured by determining the ratio of CD3+FOXP3+ cells to CD3+FOXP3- cells in the T cell population. The typical frequency of Tregs in human blood is 5-10% of total CD4+CD3+ T cells. However, in the diseases listed above, this percentage may be lower or higher. In preferred embodiments, the percentage of Tregs increases by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%. The maximum Treg increase ratio may vary depending on the specific disease; however, the maximum Treg frequency that can be obtained by IL-2 mutant protein treatment is 50% or 60% of total CD4+CD3+ T cells. In certain embodiments, when an IL-2 mutant protein, anti-IL-2 antibody, or IL-2 mutant protein Fc fusion protein is administered to a subject, the ratio of regulatory T cells (Tregs) to unregulatory T cells in the subject's peripheral blood increases.

[0161] Furthermore, IL-2 mutant proteins, anti-IL-2 antibodies, and IL-2 mutant protein Fc fusion proteins preferentially promote the proliferation of Tregs over other cell types, making them useful for increasing the ratio of regulatory T cells (Tregs) to natural killer (NK) cells in the target peripheral blood. This ratio can be measured by determining the ratio of CD3+FOXP3+ cells to CD19- and CD3- CD16+ and / or CD56+ lymphocytes.

[0162] IL-2 mutant proteins, anti-IL-2 antibodies, or IL-2 mutant protein Fc fusion proteins are thought to have therapeutic effects on diseases or disorders in patients without significantly increasing the ratio of Tregs to unregulated T cells or NK cells in the patient's peripheral blood. These therapeutic effects 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 actual and hypothetical examples are provided for the purpose of illustrating specific embodiments or features of the present invention and are not intended to limit its scope.

[0164] Example 1: pSTAT5 signaling of IL-2 mutant protein We investigated IL-2 mutant proteins in relation to relative pSTAT5 activation. An activity screen was designed to identify mutant proteins that enhance the Treg:Teff window, i.e., those that retain high levels of activity in Treg cells while showing significant attenuation in Teff cells. Since activated Teff cells express high levels of CD25, and the number of such cells increases in patients with autoimmune and inflammatory diseases, the inventors used CD25+-gating on Teff cells to mimic a more realistic differentiation of CD25 expression in patients. The activity of IL-2 mutant proteins was evaluated by the intracellular phosphorus-STAT5 response measured by a FACS-based assay. In short, previously frozen human PBMCs were thawed and allowed to stand in complete medium for 0.5–2 hours. Cells were suspended 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). Cells were stimulated with IL-2 mutant protein by 10× dose titration ranging from 1nM to 200nM in a final volume of 10 μl for 30 minutes. The level of STAT5 phosphorylation was measured using the BD phosflow buffer kit. Briefly, stimulation was stopped by adding 1 ml of BD lyse / fix phosflow buffer. Cells were fixed at 37°C for 10-15 minutes, permeabilized on ice with 1× BD phosflow perm buffer, and then stained for CD3, CD4, CD25, FOXP3, CD8, and pSTAT5. The results from two PBMC donors are shown in Table 2 below.

[0165] [Table 2]

[0166] [Table 3]

[0167] Example 2 - Stability of IL-2 mutant protein The DSC Tm measurement molecular evaluation assay was tested for the selection of stable molecules with good potential for easy manufacturing, along with their stability at 40°C for 10 days as measured by SEC chromatography. The MP% results after 10 days are shown in Figure 1.

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

[0169] Example 4 - In vivo activity of human IL-2 mutant protein in mice C57Bl6 mice were administered a single dose of PBS (vehicle control), wild-type IL-2-Fc, V91K D20A M104V, H16R, or D20W on day 0. Splenocytes were collected on day 4 and analyzed for their effects on Treg cells, CD8 T cells, and NK cells. Three doses (1 μg, 5 μg, or 25 μg per mouse) were evaluated, except for D20W, which was administered at 25 μg. The percentage of Treg cells, defined as CD4+CD25+FoxP3+ cells, in total gated viable cells is shown in (A), and the calculated total number of Treg cells (B), CD8 T cells (C), and NK cells (D) is shown. As shown in Figure 3, wild-type IL-2, H16R, and V91K D20A M104V induced significant dose-dependent in vivo proliferation of Treg cells. Surprisingly, the two mutant proteins, H16R and V91K D20A M104V, exhibited robust activity on Treg cells to a degree similar to, or even greater 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 significant attenuation of activity as measured by in vitro pSTAT5 readout, the attenuated mutant proteins retain the ability to induce a robust Treg response in vivo while simultaneously inducing minimal CD8 T and NK cell responses. Therefore, the attenuation disproportionately affects Treg:non-Treg in vivo selectivity.

[0170] Example 5 - pSTAT5 signaling of IL-2 mutant protein We investigated IL-2 mutant proteins in relation to relative pSTAT5 activation. An activity screen was designed to identify mutant proteins that enhance the Treg:Teff window, i.e., those that retain high levels of activity in Treg cells while showing significant attenuation in Teff cells. Since activated Teff cells express high levels of CD25, and the number of such cells increases in patients with autoimmune and inflammatory diseases, the inventors used CD25+-gating on Teff cells to mimic a more realistic differentiation of CD25 expression in patients. The activity of IL-2 mutant proteins was evaluated by the intracellular phosphorus-STAT5 response measured by a FACS-based assay. In short, previously frozen human PBMCs were thawed and allowed to stand in complete medium for 0.5–2 hours. Cells were suspended 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). Cells were stimulated with IL-2 mutant protein by 10× dose titration 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, stimulation was stopped by adding 1 ml of BD lyse / fix phosflow buffer. Cells were fixed at 37°C for 10-15 minutes, permeabilized on ice with 1× BD phosflow perm buffer, and then stained for CD3, CD4, CD25, FOXP3, CD8, and pSTAT5. The results from two PBMC donors are shown in Table 3 below.

[0171] [Table 4]

Claims

1. A human interleukin-2 (IL-2) mutant protein having an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 2, and which includes the V91K and H16E mutations compared to SEQ ID NO: 2, and which preferentially stimulates regulatory T cells compared to other T cells.

2. Furthermore, the human IL-2 mutant protein according to claim 1, which preferentially stimulates regulatory T cells compared to NK cells.

3. The human IL-2 mutant protein according to claim 1, wherein the amino acid sequence of the human IL-2 mutant protein includes alanine at position 125 of the numbering sequence number 2.

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

5. An Fc fusion protein comprising Fc and the human IL-2 mutant protein described in any one of claims 1 to 4.

6. The Fc fusion protein according to claim 5, wherein the Fc is human IgG1 Fc.

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

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

9. The Fc fusion protein according to claim 5, wherein a linker connects the Fc portion of the Fc fusion protein to the human IL-2 mutant protein portion.

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

11. The Fc fusion protein according to claim 5, wherein the human IL-2 mutant protein, when expressed in mammalian cells, further comprises the addition, substitution, or deletion of amino acids that alter the glycosylation of the Fc fusion protein.

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

13. The Fc fusion protein according to claim 12, comprising two human IL-2 mutant proteins.

14. The Fc fusion protein according to claim 12, comprising a single human IL-2 mutant protein.

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

16. An isolated nucleic acid encoding the Fc fusion protein according to claim 5.

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

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

19. A pharmaceutical composition comprising a human IL-2 mutant protein 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 target.

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

21. A pharmaceutical composition comprising the human IL-2 mutant protein according to any one of claims 1 to 4, for use in treating subjects suffering from inflammatory diseases or autoimmune diseases.

22. A pharmaceutical composition comprising the Fc fusion protein according to claim 5, for use in treating subjects suffering from inflammatory diseases or autoimmune diseases.

23. The pharmaceutical composition according to claim 21 or 22, wherein the inflammatory disease or autoimmune disease is lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type 1 diabetes, type 2 diabetes, multiple sclerosis, rheumatoid arthritis, alopecia areata, atherosclerosis, psoriasis, organ graft rejection, Sjögren's syndrome, Behçet's disease, spontaneous abortion, atopic disease, asthma, or inflammatory bowel disease.

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