Interleukin-2 muteins and their uses

Abstract

The present invention provides, inter alia, compositions and methods for preventing and treating autoimmune diseases. The present invention is based, in part, on the surprising discovery that human interleukin-2 muteins activate the proliferation of regulatory T cells. In one aspect, the present invention provides compositions and methods for expanding regulatory T cells. In another aspect, human interleukin-2 (IL-2) muteins are described that comprise an amino acid sequence at least 90% identical to the wild-type IL-2 protein, and that have at least one amino acid substitution selected from the group consisting of T111H, T37Y, E15T, M23L, P34F, E68F, and E62A.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of and priority to U.S. Application No. 63 / 073,208, filed September 1, 2020, and U.S. Application No. 63 / 231,471, filed August 10, 2021, the contents of which are incorporated herein. BACKGROUND OF THE INVENTION

[0002] Autoimmune diseases occur when the immune system is unable to distinguish between self and non-self tissue, thereby attacking and destroying the body's cells and tissues.

[0003] One of the functions of regulatory T cells (Tregs) is to suppress pathological activation of the immune system and prevent autoimmune diseases. Regulatory T cells (Tregs) are CD4+CD25+ T cells that suppress the activity of other immune cells and play an important role in maintaining tolerance to self-antigens, regulating responses to foreign antigens, and controlling the immune system. The number or function of Tregs has been confirmed to be reduced in several autoimmune and inflammatory diseases, including type 1 diabetes (T1D), systemic lupus erythematosus (SLE), and graft-versus-host disease (GVHD).

[0004] Interleukin-2 (IL-2) is a potent stimulator of the immune system, activating T cells, B cells, and monocytes, in addition to stimulating the development of regulatory T cells (Tregs). Therapeutic administration of IL-2 can result in undesirable toxicities, for example, due to nonspecific activation of NK cells.

[0005] T cells must express CD25 to respond to the low concentrations of IL-2 normally present in tissues. CD25-expressing T cells include both FOXP3+CD4+ regulatory T cells (Treg cells), which are essential for suppressing autoimmune inflammation. On the other hand, FOXP3- T effector cells express CD25 upon activation and become either CD4+ or CD8+, potentially contributing to inflammation, autoimmunity, organ transplant rejection, or graft-versus-host disease. IL-2-stimulated STAT5 signaling is thought to be important for the growth and survival of normal Treg cells and for the high expression of FOXP3. Summary of the Invention

[0006] There is a need for therapeutic methods that selectively stimulate the production and / or activity of Treg cells for immune system regulation. The present invention provides, inter alia, compositions and methods for expanding regulatory T cells (Tregs). The present invention provides, inter alia, human interleukin-2 (IL-2) muteins and IgG Fc fusion proteins thereof that activate the proliferation of regulatory T cells. The present invention provides, inter alia, compositions and methods for preventing and treating autoimmune diseases.

[0007] One approach to treating autoimmune diseases is the transplantation of ex vivo expanded autologous Treg cells. Although successful in animal models and early human clinical trials, this approach is technically complex and invasive, requiring personalized treatment with the patient's own T cells.

[0008] Proleukin (Prometheus Laboratories, San Diego), a recombinant IL-2, is approved for the treatment of metastatic melanoma and metastatic renal carcinoma, but is highly toxic and associated with severe side effects. Clinical treatment with low-dose IL-2 has been used in chronic GVHD and HCV-associated autoimmune vasculitis, and has demonstrated an increase in Treg levels. However, even clinical trials of low-dose IL-2 have raised safety and tolerability concerns. Therefore, there is a need for a safe and tolerable therapeutic agent for treating humans that specifically targets and activates Treg cells to prevent and / or treat autoimmune diseases.

[0009] IL-2 receptors are widely expressed on many types of immune cells, including T cells, NK cells, eosinophils, and monocytes, resulting in the pleiotropic effects and high systemic toxicity resulting from IL-2 administration. IL-2 receptors exist in three forms: α (alpha) (also known as IL-2Ra, CD25, or Tac antigen), β (beta) (also known as IL-2Rb or Cd122), and γ (gamma).

[0010] When administered to human patients, IL-2 has a short half-life of 85 minutes after intravenous administration and 3.3 hours after subcutaneous administration (Kirchner, GI et al., 1998, Br J. Clin. Pharmacol. 46:5-10). High doses are generally considered necessary because in vitro studies have shown that at least 5-6 hours of exposure to IL-2 is required to stimulate T cell proliferation.

[0011] In one aspect, the present invention identifies improved methods for treating autoimmune diseases using IL-2 variants that are selective for Treg cells over other types of immune cells.

[0012] In one embodiment, an IL-2 mutein is fused to the Fc region of an IgG to increase the half-life of circulating IL-2.

[0013] Although IL-2 targets many types of immune cells, Treg cells respond to lower concentrations of IL-2 than many other cell types because they express high levels of the high-affinity receptor IL2Rαβγ, which is composed of receptors for IL2Rα (CD25), IL2Rβ (CD122), and IL2Rγ (CD132). Treg development is responsive to IL-2. Treg cells (CD4+ cells) express IL2Rα (also known as CD25), whereas other non-Treg T cells, which are CD8+, express IL2Rβ (CD122).

[0014] The present invention is based, in part, on the surprising discovery that exemplary IL-2 muteins preferentially expand or stimulate Treg cells. The present invention provides human interleukin-2 muteins comprising at least one amino acid substitution selected from the group consisting of T111H, T37Y, E15T, M23L, P34F, E68F, and E62A relative to wild-type IL-2 (SEQ ID NO: 1), which can selectively activate the proliferation of regulatory T cells.

[0015] In one aspect, the present invention provides a human interleukin-2 (IL-2) mutein comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1, wherein the IL-2 mutein has at least one amino acid substitution selected from the group consisting of T111H, T37Y, E15T, M23L, P34F, E68F, and E62A. Thus, in some embodiments, the IL-2 mutein has at least one amino acid substitution characterized by a T111H substitution. In some embodiments, the IL-2 mutein has at least one amino acid substitution characterized by a T37Y substitution. In some embodiments, the IL-2 mutein has at least one amino acid substitution characterized by an E15T substitution. In some embodiments, the IL-2 mutein has at least one amino acid substitution characterized by an M23L substitution. In some embodiments, the IL-2 mutein has at least one amino acid substitution characterized by a P34F substitution. In some embodiments, the IL-2 mutein has at least one amino acid substitution characterized by an E68F substitution. In some embodiments, the IL-2 mutein has at least one amino acid substitution characterized by an E62A substitution. In some embodiments, the IL-2 mutein has a combination of amino acid substitutions including one or more of the following amino acid substitutions: T111H, T37Y, E15T, M23L, P34F, E68F, and E62A.

[0016] In some embodiments, the human interleukin-2 (IL-2) mutein further comprises an amino acid substitution of C125A.

[0017] In one embodiment, the present invention provides a nucleotide sequence encoding the amino acid sequence of a human interleukin-2 (IL-2) mutein.

[0018] In some embodiments, the present invention provides a medicament comprising a human interleukin-2 (IL-2) mutein or a salt thereof, hi some embodiments, the IL-2 mutein is fused to an IgG Fc fusion partner.

[0019] In some embodiments, the medicament is a Treg activator.

[0020] In some embodiments, the medicament is an agent for preventing or treating an autoimmune disease.

[0021] In some embodiments, the IL-2 mutein may comprise one or more compounds to increase the serum half-life of the IL-2 mutein when administered to a patient. Such half-life extenders include soluble polymers (e.g., polyethylene glycol (PEG)), low-density and high-density lipoproteins, antibody Fc (monomer or dimer), transthyretin (TTR), and TGF-β latency-associated peptide (LAP). IL-2 variants containing combinations of serum half-life extenders, such as PEGylated TTR (from U.S. Patent Application Publication No. 2003 / 0195154), are also contemplated.

[0022] In some embodiments, the present invention provides a method for expanding regulatory T cells (Treg cells) in a mammal, the method comprising administering to the mammal an effective amount of a human interleukin-2 (IL-2) mutein or a salt thereof.

[0023] In some embodiments, the present invention provides a method for preventing or treating an autoimmune disease in a mammal, comprising administering to the mammal an effective amount of a human interleukin-2 (IL-2) mutein or a salt thereof.

[0024] In some embodiments, the present invention provides human interleukin-2 (IL-2) muteins for use in methods of treating autoimmune diseases. A variety of autoimmune diseases are recognized in the art, including, for example, diseases associated with an enhanced inflammatory response, such as inflammatory skin diseases, including psoriasis and dermatitis (e.g., atopic dermatitis); responses associated with inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); dermatitis; allergic conditions such as eczema and asthma; rheumatoid arthritis; systemic lupus erythematosus (SLE) (including, but not limited to, lupus nephritis, cutaneous lupus); diabetes mellitus (e.g., type 1 diabetes or insulin-dependent diabetes mellitus); multiple sclerosis and juvenile-onset diabetes.

[0025] In some embodiments, the present invention provides use of a human interleukin-2 (IL-2) mutein or a salt thereof for producing an agent for preventing or treating an autoimmune disease.

[0026] In some embodiments, the present invention provides a method for expanding regulatory T cells (Tregs), comprising contacting a population of T cells with an effective amount of a human interleukin-2 (IL-2) mutein described herein.

[0027] Any aspect or embodiment described herein may be combined with any other aspect or embodiment disclosed herein. While the present disclosure has been described in conjunction with its detailed description, the foregoing description is intended to illustrate, but not to limit, the scope of the disclosure, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0028] All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are incorporated by reference. All other publications, documents, manuscripts, and scientific literature cited herein are incorporated by reference.

[0029] Other features and advantages of the present invention will become apparent from the following detailed description, drawings, and claims. It should be understood, however, that the detailed description, drawings, and claims, while describing embodiments of the present invention, are given by way of example only and not by way of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art. [Brief explanation of the drawings]

[0030] The following drawings are for illustrative purposes only and are not limiting. [Figure 1A] FIG. 1A is a graph showing the binding affinity of WT IL-2 to IL-2Rα (CD25) in comparison with two exemplary IL-2 muteins. [Figure 1B] FIG. 1B shows that IL-2 mutein K77A has similar binding affinity to IL-2Rα as WT IL-2. [Figure 1C] FIG. 1C shows that the E96A mutant does not bind to IL-2Rα. [Figure 2A-D] Figure 2A shows a graph of median fluorescence intensity to quantify pSTAT5 induction in CD25+ CD4 T cells. Figure 2B shows a graph of pSTAT5 levels in CD8 T cells. Figure 2C shows a graph of pSTAT5 levels in CD25- CD4 T cells. Figure 2D shows a graph of pSTAT5 levels in CD25- NK cells. [Figure 2E] FIG. 2E shows a series of graphs and an associated table showing pSTAT5a induction with IL-2 muteins M23L, T111H, E68F, E15T, P34F, T37Y compared to wild-type IL-2. [Figure 3] Figure 3 is a graph showing a comparison of the dose-dependent increase in Treg cell numbers (foxp3+% of CD4+ T cells) upon treatment with E96-HLE compared to WT mIL-2-HLE, F906-hIL2, F906-E62A, and IL-2-S4B6 antibodies. [Figure 4A]FIG. 4A shows the representative Treg populations measured by flow cytometry in vehicle-treated cells graphed in FIG. [Figure 4B] FIG. 4B shows exemplary Treg populations in WT mIL-2 HLE cells. [Figure 4C] FIG. 4C shows an exemplary dose-dependent expansion of Treg populations by E96-HLE. [Figure 4D] FIG. 4D shows an exemplary dose-dependent expansion of Treg populations by E96-HLE. [Figure 4E] FIG. 4E shows an exemplary dose-dependent expansion of Treg populations by E96-HLE. [Figure 4F] FIG. 4F shows representative control flow cytometry results with IL2+SB46 antibody. [Figure 5A] FIG. 5A is a graph showing a comparison of the dose-dependent increase in the percentage of foxp3+ cells as a subset of CD3+ cells in mice treated with E96-HLE versus WT mIL-2-HLE. [Figure 5B] FIG. 5B is a graph showing a comparison of the dose-dependent expansion of splenocytes, including CD3+ cells, in E96-HLE versus WT mIL-2-HLE treated mice. [Figure 6A] FIG. 6A is a schematic diagram showing the experimental design for testing the effects of E62A-HLE and E96A-HLE in WT mice after one round of administration. [Figure 6B] FIG. 6B shows the percentage of mean body weight in mice administered with IL-2 muteins E62A-HLE and E96A-HLE relative to WT mice. [Figure 6C] FIG. 6C is a graph showing the total number of splenocytes scored when mice were treated with E62A-HLE or E96A-HLE relative to WT mIL-2-HLE or WT hIL2-HLE at low and high doses. [Figure 7A] FIG. 7A is a schematic diagram showing the experimental design for testing the effects of E62A-HLE and E96A-HLE in WT mice after two rounds of administration. [Figure 7B] FIG. 7B is a graph showing the percentage of mean body weight in mice administered two rounds of the IL-2 muteins E62A-HLE and E96A-HLE relative to WT mice. [Figure 7C] FIG. 7C is a graph showing the total number of splenocytes scored when mice were treated with E62A-HLE or E96A-HLE relative to WT mIL-2-HLE or WT hIL2-HLE at low and high doses. [Figure 8A-F] Figures 8A and 8D are graphs showing the percentage of Treg cells as a percentage of CD4+ cells after treatment of mice with WT or IL-2 E62A-HLE or E96A-HLE muteins. Figures 8B and 8E are graphs showing the percentage of CD8+ cells as a percentage of CD3+ cells after treatment of mice with WT or IL-2 E62A-HLE or E96A-HLE muteins. Figures 8C and 8F are graphs showing the CD8:Treg ratio after treatment of mice with E62A-HLE or E96A-HLE compared to WT IL-2. Figures 8A-8C relate to one round of administration of IL-2 muteins or WT IL-2. Figures 8D-8F relate to two rounds of administration of IL-2 muteins or WT IL-2. [Figure 9A] FIG. 9A is a graph of the percentage of body weight monitored through 30 weeks upon administration of IL-2 muteins in two dosing regimens. [Figure 9B] FIG. 9B is a graph of blood glucose levels measured in mice treated with IL-2 muteins in two dosing regimens. [Figure 9C] FIG. 9C is a graph showing the incidence of diabetes in populations of mice treated with IL-2 muteins relative to vehicle controls. [Figure 10A-B] Figure 10A is a graph of the percentage of CD45+ cells in PBMCs and splenocytes in NK cells after an IL-2 mutein treatment regimen versus a vehicle control, and Figure 10B is a graph of the percentage of CD45+ cells in PBMCs and splenocytes in B cells after an IL-2 mutein treatment regimen versus a vehicle control. [Figure 10C-D] Figure 10C is a graph of the percentage of CD45+ cells among CD4+ T cells after an IL-2 mutein treatment regimen versus a vehicle control, and Figure 10D is a graph of the percentage of CD45+ cells among CD8+ T cells after an IL-2 mutein treatment regimen versus a vehicle control. [Figure 11] Figure 11A is a graph of CD45+ Tregs in PBMCs and splenocytes after an IL-2 mutein treatment regimen versus a vehicle control. Figure 11B is a graph of CD45+ GITR (glucocorticoid-induced tumor necrosis factor) expressing Tregs in PBMCs and splenocytes after an IL-2 mutein treatment regimen versus a vehicle control. [Figure 12] 12A and 12B are graphs of the percentage of immune cells in the peripheral blood of mice 96 hours after the IL-2 mutein treatment regimen versus vehicle control. [Figure 13] 13A and 13B are graphs of the percentage of immune cells in lymph nodes 96 hours after the IL-2 mutein treatment regimen versus vehicle control. [Figure 14] 14A and 14B are graphs of the percentage of immune cells in the spleen 96 hours after the IL-2 mutein treatment regimen versus vehicle control. [Figure 15] 15A and 15B are graphs of the percentage of immune cells in the tumor after 96 hours of IL-2 mutein treatment regimen versus vehicle control. [Figure 16A] FIG. 16A is a graph of the binding affinity between WT human IL-2 and the human CD25 (IL-2Rα) receptor. [Figure 16B] FIG. 16B is a graph of the binding affinity between human IL-2 muteins and the human CD25 (IL-2Rα) receptor. [Figure 17A] FIG. 17A is a graph of the binding affinity between WT IL-2 and the CD122 (IL-2Rβ) receptor. [Figure 17B] FIG. 17B is a graph of the binding affinity between IL-2 muteins and the CD25 (IL-2Rβ) receptor. [Figure 18A] FIG. 18A is a graph of the binding affinity between hCTLA4 and IL-2 WT fusion protein. [Figure 18B] FIG. 18B is a graph of the binding affinity between hCTLA4 and the IL-2 mutein, E62A. [Figure 19A] FIG. 19A is a schematic diagram showing the experimental design for testing the effects of M23L, T111H, and WT hIL-2 in cynomolgus monkeys after multiple rounds of administration. [Figure 19B] Figure 19B is a graph showing changes in the number of CD4, memory CD4, naive CD4, CD4 Treg, memory Treg, naive Treg, CD8, NK, and NKT lymphocyte cells in vehicle-treated, WT hIL-2-treated, and IL-2 mutein, M23L-treated cynomolgus monkeys on days 0, 1, 4, 7, 8, 11, and 14 of treatment. [Figure 19C] Figure 19C is a graph showing changes in the number of CD4, memory CD4, naive CD4, CD4 Treg, memory Treg, naive Treg, CD8, NK, and NKT lymphocyte cells in vehicle-treated, WT hIL-2-treated, and IL-2 mutein, T111H-treated cynomolgus monkeys on days 0, 1, 4, 7, 8, 11, and 14 of treatment. [Figure 20] Figure 20A shows a graph of blood glucose measurements in mice treated with PBS, E62A-HLE, and M23L-HLE, and Figure 20B shows a graph of the incidence of hyperglycemia over time in mice treated with E62A-HLE, M23L-HLE, and WT hIL-2-HLE. DETAILED DESCRIPTION OF THE INVENTION

[0031] [Definition] In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions for these and other terms are set forth throughout the specification.

[0032] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0033] Unless otherwise stated or clear from context, as used herein, the term "or" is understood to be inclusive, covering both "or" and "and."

[0034] As used herein, the terms "for example" and "i.e." are not intended to be limiting and are used merely as an illustration, and should not be construed to refer only to the items explicitly listed herein.

[0035] Terms such as "or more," "at least," "greater than," e.g., "at least one," mean at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 5 8, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, 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, at least 100, at least 101, at least 102, at least 103, at least 104, at least 105, at least 106, at least 107, at least 108, at least 109, at least 110, at least 111, at least 112, at least 113, at least 114, at least 115, at least 116, at least 117, at least 118,at least 119, at least 120, at least 121, at least 122, at least 123, at least 124, at least 125, at least 126, at least 127, at least 128, at least 129, at least 130, at least 131, at least 132, at least 133, at least 134, at least 135, at least 136, at least 137, at least 138, at least 139, at least 140, at least 141, at least 142, at least 143, It is understood to include, but not be limited to, at least 144, at least 145, at least 146, at least 147, at least 148, at least 149, or 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, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, or even higher values, including any higher numbers or fractions therebetween.

[0036] Conversely, the term "less than or equal to" includes each value less than the specified value. For example, "less than or equal to 100 nucleotides" means 100 nucleotides, 99 nucleotides, 98 nucleotides, 97 nucleotides, 96 nucleotides, 95 nucleotides, 94 nucleotides, 93 nucleotides, 92 nucleotides, 91 nucleotides, 90 nucleotides, 89 nucleotides, 88 nucleotides, 87 nucleotides, 86 nucleotides, 85 nucleotides, 84 nucleotides, 83 nucleotides, 82 nucleotides, 81 nucleotides, 80 nucleotides, 79 nucleotides, 78 nucleotides, 77 nucleotides, 76 nucleotides, 75 nucleotides, 74 nucleotides, 73 nucleotides, 72 nucleotides, 71 nucleotides, 70 nucleotides, 69 nucleotides, 68 nucleotides, 67 nucleotides, 66 nucleotides, 65 nucleotides, 64 nucleotides, 63 nucleotides, 62 nucleotides, 61 nucleotides, 60 nucleotides, 59 nucleotides, 58 nucleotides, 57 nucleotides, 56 nucleotides, 55 nucleotides, 54 nucleotides, 53 nucleotides, 52 nucleotides, 5 Includes 1 nucleotide, 50 nucleotides, 49 nucleotides, 48 ​​nucleotides, 47 nucleotides, 46 nucleotides, 45 nucleotides, 44 nucleotides, 43 nucleotides, 42 nucleotides, 41 nucleotides, 40 nucleotides, 39 nucleotides, 38 nucleotides, 37 nucleotides, 36 nucleotides, 35 nucleotides, 34 nucleotides, 33 nucleotides, 32 nucleotides, 31 nucleotides, 30 nucleotides, 29 nucleotides, 28 nucleotides, 27 nucleotides, 26 nucleotides, 25 nucleotides, 24 nucleotides, 23 nucleotides, 22 nucleotides, 21 nucleotides, 20 nucleotides, 19 nucleotides, 18 nucleotides, 17 nucleotides, 16 nucleotides, 15 nucleotides, 14 nucleotides, 13 nucleotides, 12 nucleotides, 11 nucleotides, 10 nucleotides, 9 nucleotides, 8 nucleotides, 7 nucleotides, 6 nucleotides, 5 nucleotides, 4 nucleotides, 3 nucleotides, 2 nucleotides, 1 nucleotide, and 0 nucleotides. Any smaller number or fractional number therebetween is also included.

[0037] Terms such as "plurality," "at least two," "two or more," "at least a second," and the like mean at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, at least 69, at least 70, at least 71, at least 72, at least 73, at least 74, at least 75, at least 76, at least 77, at least 78, at least 79, at least 80, at least 81, at least 82, at least 83, at least 84, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 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, at least 100, at least 101, at least 102, at least 103, at least 104, at least 105, at least 106, at least 107, at least 108, at least 109, at least 110, at least 111, at least 112, at least 113, at least 114, at least 115, at least 116, at least 117, at least 118, at least 119,It is understood to include, but is not limited to, at least 120, at least 121, at least 122, at least 123, at least 124, at least 125, at least 126, at least 127, at least 128, at least 129, at least 130, at least 131, at least 132, at least 133, at least 134, at least 135, at least 136, at least 137, at least 138, at least 139, at least 140, at least 141, at least 142, at least 143, at least 144, at least 145, at least 146, at least 147, at least 148, at least 149, or 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, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, or more. Any larger numbers or fractions between them are also included.

[0038] Throughout the specification, the word "comprising" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of the specified element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps.

[0039] Unless otherwise specified or clear from the context, as used herein, the term "about" is understood to mean within normal tolerances in the art, e.g., within two standard deviations of the mean. "About" can be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% of the specified value. Unless otherwise clear from the context, all numerical values ​​provided herein reflect normal variations that would be understood by one of ordinary skill in the art.

[0040] As used herein, a fusion protein generally refers to a fusion polypeptide molecule comprising an immunoglobulin molecule and an IL-2 molecule, wherein the components of the fusion protein are linked to each other by peptide bonds, either directly or via a peptide linker. For clarity, the individual peptide chains of the immunoglobulin components of the fusion protein may be non-covalently linked, for example, by disulfide bonds.

[0041] Fusion means that the components are linked by peptide bonds, either directly or via one or more peptide linkers.

[0042] Specific binding means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an immunoglobulin to bind to a particular antigen can be measured through enzyme-linked immunosorbent assay (ELISA) or other techniques well known to those skilled in the art, such as surface plasmon resonance (SPR) technology (analyzed on a BIAcore instrument) and conventional binding assays. In one embodiment, the extent of binding of the immunoglobulin to an unrelated protein is less than about 10% of the binding of the immunoglobulin to the antigen, as measured, for example, by SPR. In certain embodiments, immunoglobulins that bind to an antigen have a binding affinity of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 It has a dissociation constant (D) of 1 M.

[0043] Affinity or binding affinity refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, binding affinity refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (koff and kon, respectively). Thus, equivalent affinities can involve different rate constants as long as the ratio of the rate constants remains unchanged. Affinity can be measured by common methods known in the art, including those described herein. A specific method for measuring affinity is surface plasmon resonance (SPR).

[0044] Decreased binding, e.g., decreased binding to an Fc receptor or IL-2 receptor, refers to a decrease in affinity for the respective interaction, as measured, e.g., by SPR. For clarity, the term also includes a decrease in affinity to zero (or below the detection limit of the analytical method), i.e., a complete abolition of the interaction. Conversely, increased binding refers to an increase in binding affinity for the respective interaction.

[0045] Fc domain or Fc region is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. The IgG Fc region comprises the IgG CH2 and IgG CH3 domains. The CH2 domain of a human IgG Fc region typically extends from about amino acid residue 231 to about amino acid residue 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" comprises the stretch of residues from the C-terminus of the Fc region to the CH2 domain (i.e., from about amino acid residue 341 to about amino acid residue 447 of IgG). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain with an introduced "protuberance" ("knob") in one chain and a corresponding introduced "depression" ("hole") in the other chain). Such variant CH3 domains can be used to promote heterodimerization of two non-identical immunoglobulin heavy chains, as described herein. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine of the Fc region (Lys447) may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0046] Effector functions refer to biological activities attributable to the Fc region of an immunoglobulin and vary depending on the immunoglobulin isotype. Examples of immunoglobulin effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0047] An activating Fc receptor is an Fc receptor that, after engagement with the Fc region of an immunoglobulin, triggers signaling events that stimulate receptor-bearing cells to exert effector function. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). A specific activating Fc receptor is human FcγRIIIa (see UniProt Accession No. P08637 (Version 141)).

[0048] Interleukin-2 or IL-2, as used herein, unless otherwise specified, refers to any native IL-2 of any vertebrate origin, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses unprocessed IL-2 as well as any form of IL-2 produced by intracellular processing. The term also encompasses naturally occurring variants of IL-2, such as splice variants or allelic variants. The amino acid sequence of an exemplary human IL-2 is set forth in SEQ ID NO: 1. Unprocessed human IL-2 further comprises a 20-amino acid signal peptide at the N-terminus, which is not present in the mature IL-2 molecule.

[0049] Native IL-2, also referred to as wild-type IL-2 or wild-type IL-2, refers to naturally occurring IL-2. The sequence of a native human IL-2 molecule is set forth in SEQ ID NO: 1. For purposes of the present invention, the term wild-type also encompasses forms of IL-2 that contain one or more amino acid mutations that do not alter IL-2 receptor binding compared to naturally occurring native IL-2, such as, for example, a substitution of alanine for cysteine ​​at the position corresponding to residue 125 of human IL-2. In some embodiments, for purposes of the present invention, wild-type IL-2 contains the amino acid substitution C125A (see SEQ ID NO: 3).

[0050] CD25 or IL-2 receptor alpha, as used herein, unless otherwise specified, refers to any native CD25 of any vertebrate origin, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed CD25 as well as any form of CD25 that results from processing within the cell. The term also encompasses naturally occurring variants of CD25, such as splice variants or allelic variants. In a specific embodiment, the CD25 is human CD25.

[0051] As used herein, the high-affinity IL-2 receptor refers to a heterotrimeric form of the IL-2 receptor consisting of a receptor gamma subunit (also known as the common cytokine receptor gamma subunit, γc, or CD132), a receptor beta subunit (also known as CD122 or p70), and a receptor alpha subunit (also known as CD25 or p55). The term intermediate-affinity IL-2 receptor or IL-2 receptor βγ, in contrast, refers to an IL-2 receptor that does not contain an alpha subunit, but contains only the gamma and beta subunits (for a review, see, e.g., Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008)).

[0052] Regulatory T cells, or Treg cells, are a special type of CD4+ T cell that can suppress the responses of other T cells (effector T cells). Treg cells are characterized by expression of CD4, the α subunit of the IL-2 receptor (CD25), and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)), and play an important role in inducing and maintaining peripheral self-tolerance to antigens, including those expressed by tumors.

[0053] CD4+ T cells refer to CD4+ T cells other than regulatory T cells. Conventional CD4+ memory T cells are characterized by expressing CD4 and CD3 but not FOXP3. Conventional CD4+ memory T cells are a subset of conventional CD4+ T cells further characterized by not expressing CD45RA, in contrast to conventional CD4+ naive T cells, which express CD45RA.

[0054] Selective activation of Treg cells essentially refers to activation of Treg cells without concomitant activation of other T cell subsets (e.g., CD4+ T helper cells, CD8+ cytotoxic T cells, NK T cells) or natural killer (NK) cells. Methods for identifying and distinguishing these cell types are described in the Examples. Activation can include induction of IL-2 receptor signaling (e.g., as measured by detection of phosphorylated STAT5a), induction of proliferation (e.g., as measured by detection of Ki-67), and / or upregulation of expression of activation markers (e.g., CD25, etc.).

[0055] The term peptide linker refers to a peptide comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides include, for example, (G4S)n, (SG4)n, or G4(SG4) ” Including a peptide linker, "n" is generally a number from 1 to 10, typically 2 to 4.

[0056] The term modified refers to any manipulation of the peptide backbone (eg, amino acid sequence) of a polypeptide or post-translational modification (eg, glycosylation).

[0057] A knob-into-hole modification refers to a modification in the interface between two immunoglobulin heavy chains in the CH3 domains in which: i) in the CH3 domain of one heavy chain, an amino acid residue is substituted with an amino acid residue having a larger side chain volume to create a protuberance ("knob") in the interface at the CH3 domain of one heavy chain that can be positioned in a depression ("hole") in the interface at the CH3 domain of the other heavy chain; and ii) in the CH3 domain of the other heavy chain, an amino acid residue is substituted with an amino acid residue having a smaller side chain volume to create a depression ("hole") in the interface at the second CH3 domain that can be positioned in the protuberance ("knob") in the interface at the first CH3 domain. In one embodiment, a "knob-into-hole modification" comprises the amino acid substitution T366W and, optionally, the amino acid substitution S354C in one of the antibody heavy chains, and the amino acid substitutions T366S, L368A, Y407V, and, optionally, Y349C in the other antibody heavy chain. Knob-into-hole technology has been described, for example, in U.S. Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding depression ("hole") at the interface of a second polypeptide, such that the protuberance can be positioned within the depression to promote heterodimer formation and prevent homodimer formation. The protuberance is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensatory depression of identical or similar size to the protuberance is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). The introduction of two cysteine ​​residues at positions S354 and Y349, respectively, allows the formation of disulfide bridges between the two antibody heavy chains in the Fc region, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0058] An amino acid substitution refers to the substitution of one amino acid for another amino acid in a polypeptide. In one embodiment, an amino acid is substituted with another amino acid that has similar structural and / or chemical properties, e.g., a conservative amino acid substitution.

[0059] Conservative amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Non-conservative substitutions involve exchanging a member of one of these classes for another. Amino acid substitutions may also replace one amino acid with another amino acid having different structural and / or chemical properties, such as substituting an amino acid from one group (e.g., polar) with another amino acid from a different group (e.g., basic). Amino acid substitutions can be made using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. It is contemplated that methods other than genetic engineering, such as chemical modification, that alter the side chain group of an amino acid may also be useful. Various designations may be used herein to represent the same amino acid substitution. For example, a proline to glycine substitution at position 329 of an immunoglobulin heavy chain may be represented as 329G, G329, G329, P329G, or Pro329Gly.

[0060] The percent (%) amino acid sequence identity with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to maximize the percent sequence identity; any conservative substitutions are not considered part of the sequence identity. Alignment to determine percent amino acid sequence identity can be performed in a variety of ways within the skill of the art, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve optimal alignment over the full length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are determined using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code, together with user documentation, has been filed with the U.S. Copyright Office (Washington, DC, 20559) and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California), or may be compiled from the source code. The ALIGN-2 program must be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.In the context of using ALIGN-2 to compare amino acid sequences, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (alternatively, it can be expressed as a given amino acid sequence A having or containing a particular % amino acid sequence identity to a given amino acid sequence B) is calculated as follows: 100 × fraction X / Y, where X is the number of amino acid residues scored as perfect matches by the sequence alignment program ALIGN-2 in an alignment of A and B by this program, and Y is the total number of amino acid residues in B. It is understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless otherwise specifically specified, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program. As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can also contain post-synthetic modification(s), such as conjugation to a label.

[0061] A nucleic acid or polypeptide having a nucleotide sequence that is, for example, at least 95% "identical" to a reference nucleotide sequence of the present invention means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain no more than five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, no more than 5% of the nucleotides in the reference sequence may be deleted or substituted with alternative nucleotides, or no more than 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These changes to the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence, or anywhere between these terminal positions, and may be interspersed individually between residues in the reference sequence or in one or more contiguous groups within the reference sequence. In practice, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence of the present invention can be conventionally determined using known computer programs such as those discussed above for polypeptides (e.g., ALIGN-2).

[0062] As used herein, a vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which the vector is introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors." The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. Host cells are any type of cell line that can be used to produce the fusion proteins of the invention. Host cells include, to name just a few, cultured cells, e.g., cultured mammalian cells such as CHO cells, BH cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, but also cells contained within transgenic animals, transgenic plants, or cultured plant or animal tissue.

[0063] An effective amount of an agent refers to the amount necessary to effect a physiological change in the cells or tissue to which it is administered.

[0064] A therapeutically effective amount of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. For example, a therapeutically effective amount of an agent eliminates, reduces, delays, minimizes, or prevents the adverse effects of a disease.

[0065] The individual or subject is a mammal. Mammals include, but are not limited to, livestock animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

[0066] A pharmaceutical composition refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered.

[0067] A pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical composition, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0068] Treatment (and grammatical variations such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of disease in the individual being treated, and may be performed prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological consequence of the disease, prevention of metastasis, reduction in the rate of disease progression, remission or palliation of the disease state, and remission or improvement of prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or slow the progression of disease.

[0069] Autoimmune diseases refer to non-malignant diseases or disorders that arise from and affect an individual's own tissues. Examples of autoimmune diseases or disorders include inflammatory responses such as inflammatory skin diseases, including psoriasis and dermatitis (e.g., atopic dermatitis); responses associated with inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); dermatitis; allergic conditions such as eczema and asthma; rheumatoid arthritis; systemic lupus erythematosus (SLE) (including, but not limited to, lupus nephritis and cutaneous lupus); diabetes (e.g., type 1 diabetes or insulin-dependent diabetes mellitus); multiple sclerosis and juvenile-onset diabetes. Additional examples of autoimmune diseases include, for example, multiple sclerosis (MS), lupus, ankylosing spondylitis, arthritis, colitis, type 1 diabetes, Crohn's disease, heart disease, graft-versus-host disease, complications from immune responses during pregnancy, allergies, rejection of cell or solid organ transplants, amyotrophic lateral sclerosis (ALS), and myasthenia gravis.

[0070] Substantially refers to a qualitative state exhibiting the entire or nearly entire extent or degree of a feature or characteristic of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection, or achieve or avoid absolute results. Thus, the term substantially is used herein to capture the potential lack of perfection inherent in many biological and chemical phenomena.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs and as commonly used in the art to which this application belongs, and such art is incorporated by reference in its entirety. In case of conflict, the present specification, including definitions, will control.

[0072] [Description of the embodiment] The present invention provides, inter alia, compositions and methods for preventing and treating autoimmune diseases. The present invention provides compositions and methods for expanding regulatory T cells. In one aspect, the present invention uses human interleukin-2 muteins in methods for activating the proliferation of regulatory T cells.

[0073] Various aspects of the present invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section may be applicable to any aspect of the present invention. In this application, the use of "or" means "and / or" unless otherwise specified.

[0074] IL-2 mutein Described herein are various IL-2 muteins that can be used to enhance the presence and / or activity of Treg cells. The IL-2 muteins described herein can be used to treat autoimmune diseases.

[0075] IL-2 variants (also referred to herein as "IL-2 muteins") comprise an amino acid sequence at least 70%, at least 75%, at least 80%, at least 85%, 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 wild-type IL-2. IL-2 variants further comprise a sequence of amino acids at least 70%, at least 75%, at least 80%, at least 85%, 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 a functional fragment of wild-type IL-2. As used herein, "wild-type IL-2" is intended to mean a polypeptide having the amino acid sequence of SEQ ID NO: 1 (see Table 1).

[0076] Variants may contain one or more substitutions, deletions, or insertions within the amino acid sequence of wild-type IL-2. Residues are designated herein by the single-letter code for the amino acid followed by the amino acid position in IL-2. Substitutions are designated herein by the single-letter code for the amino acid followed by the amino acid position in IL-2 followed by the single-letter code for the replacing amino acid.

[0077] In one aspect, the present invention provides human interleukin-2 muteins comprising at least one amino acid substitution relative to wild-type IL-2 selected from the group consisting of T111H, T37Y, E15T, M23L, P34F, E68F, and E62A, which can selectively activate the proliferation of regulatory T cells. Thus, in some embodiments, the IL-2 mutein has at least one amino acid substitution characterized by a T111H substitution. In some embodiments, the IL-2 mutein has at least one amino acid substitution characterized by a T37Y substitution. In some embodiments, the IL-2 mutein has at least one amino acid substitution characterized by an E15T substitution. In some embodiments, the IL-2 mutein has at least one amino acid substitution characterized by an M23L substitution. In some embodiments, the IL-2 mutein has at least one amino acid substitution characterized by a P34F substitution. In some embodiments, the IL-2 mutein has at least one amino acid substitution characterized by an E68F substitution. In some embodiments, the IL-2 mutein has at least one amino acid substitution characterized by an E62A substitution.

[0078] In one embodiment, the IL-2 mutations are V91I, V91L, V91W, E95Q, E95S, E95N, L12Y, L19V, D84E, L19F, E95D, I92Y, E95T, I92V, L12V, I92W, D84T, D84S, M23L, I92F, M23I, H16Y, E15D, L12I, E15S, L12M, D20N, H16R, E15T, D20T, N88S, S87T, V91F, V91M, H16K, L19M, L19I, T111W, F42R, T111F, D109H, P34Q , P34W, D109W, T111N, T41Y, Y45W, L72W, L72F, E68Q, P34F, P65R, P65E, P65Q, E61W, T111H, F42M, T37Y, K43W, T111M, E68F, T111Y, N71W, L72R, E68W, K35T, E106W, K48V, P34Y, D109K, T111Q, E68R, K48S, K48H, P65N, E68Y, D109R, M104H, T41H, M104I, K48I, or S87E. Thus, in some embodiments, the IL-2 mutein comprises a V91I amino acid substitution. In some embodiments, the IL-2 mutein comprises a V91L substitution. In some embodiments, the IL-2 mutein comprises a V91W substitution. In some embodiments, the IL-2 mutein comprises an E95Q substitution. In some embodiments, the IL-2 mutein comprises an E95N substitution. In some embodiments, the IL-2 mutein comprises an L12Y substitution. In some embodiments, the IL-2 mutein comprises an L19V substitution. In some embodiments, the IL-2 mutein comprises a D84E substitution. In some embodiments, the IL-2 mutein comprises an L19F substitution. In some embodiments, the IL-2 mutein comprises an E95D substitution. In some embodiments, the IL-2 mutein comprises an I92Y substitution. In some embodiments, the IL-2 mutein comprises an E95T substitution. In some embodiments, the IL-2 mutein comprises an I92V substitution. In some embodiments, the IL-2 mutein comprises an L12V substitution. In some embodiments, the IL-2 mutein comprises a I92W substitution. In some embodiments, the IL-2 mutein comprises a D84T substitution. In some embodiments, the IL-2 mutein comprises a D84S substitution.In some embodiments, the IL-2 mutein comprises an M23L substitution. In some embodiments, the IL-2 mutein comprises an I92F substitution. In some embodiments, the IL-2 mutein comprises an M23I substitution. In some embodiments, the IL-2 mutein comprises an H16Y substitution. In some embodiments, the IL-2 mutein comprises an E15D substitution. In some embodiments, the IL-2 mutein comprises an L12I substitution. In some embodiments, the IL-2 mutein comprises an E15S substitution. In some embodiments, the IL-2 mutein comprises an L12M substitution. In some embodiments, the IL-2 mutein comprises a D20N substitution. In some embodiments, the IL-2 mutein comprises an H16R substitution. In some embodiments, the IL-2 mutein comprises an E15T substitution. In some embodiments, the IL-2 mutein comprises a D20T substitution. In some embodiments, the IL-2 mutein comprises an N88S substitution. In some embodiments, the IL-2 mutein comprises a S87T substitution. In some embodiments, the IL-2 mutein comprises a V91F substitution. In some embodiments, the IL-2 mutein comprises a V91M substitution. In some embodiments, the IL-2 mutein comprises a H16K substitution. In some embodiments, the IL-2 mutein comprises a L19M substitution. An L19I substitution. In some embodiments, the IL-2 mutein comprises a T111W substitution. In some embodiments, the IL-2 mutein comprises a F42R substitution. In some embodiments, the IL-2 mutein comprises a T111F substitution. In some embodiments, the IL-2 mutein comprises a D109H substitution. In some embodiments, the IL-2 mutein comprises a P34Q substitution. In some embodiments, the IL-2 mutein comprises a P34W substitution. In some embodiments, the IL-2 mutein comprises a D109W substitution. In some embodiments, the IL-2 mutein comprises a T111N substitution. In some embodiments, the IL-2 mutein comprises a T41Y substitution. In some embodiments, the IL-2 mutein comprises a Y45W substitution. In some embodiments, the IL-2 mutein comprises a L72W substitution. In some embodiments, the IL-2 mutein comprises a L72F substitution. In some embodiments, the IL-2 mutein comprises an E68Q substitution.In some embodiments, the IL-2 mutein comprises a P34F substitution. In some embodiments, the IL-2 mutein comprises a P65R substitution. In some embodiments, the IL-2 mutein comprises a P65E substitution. In some embodiments, the IL-2 mutein comprises a P65Q substitution. In some embodiments, the IL-2 mutein comprises an E61W substitution. In some embodiments, the IL-2 mutein comprises a T111H substitution. In some embodiments, the IL-2 mutein comprises an F42M substitution. In some embodiments, the IL-2 mutein comprises a T37Y substitution. In some embodiments, the IL-2 mutein comprises a K43W substitution. In some embodiments, the IL-2 mutein comprises a T111M substitution. In some embodiments, the IL-2 mutein comprises an E68F substitution. In some embodiments, the IL-2 mutein comprises a T111Y substitution. In some embodiments, the IL-2 mutein comprises an N71W substitution. In some embodiments, the IL-2 mutein comprises an L72R substitution. In some embodiments, the IL-2 mutein comprises an E68W substitution. In some embodiments, the IL-2 mutein comprises a K35T substitution. In some embodiments, the IL-2 mutein comprises an E106W substitution. In some embodiments, the IL-2 mutein comprises a K48V substitution. In some embodiments, the IL-2 mutein comprises a P34Y substitution. In some embodiments, the IL-2 mutein comprises a D109K substitution. In some embodiments, the IL-2 mutein comprises a T111Q substitution. In some embodiments, the IL-2 mutein comprises an E68R substitution. In some embodiments, the IL-2 mutein comprises a K48S substitution. In some embodiments, the IL-2 mutein comprises a K48H substitution. In some embodiments, the IL-2 mutein comprises a P65N substitution. In some embodiments, the IL-2 mutein comprises an E68Y substitution. In some embodiments, the IL-2 mutein comprises a D109R substitution. In some embodiments, the IL-2 mutein comprises a M104H substitution. In some embodiments, the IL-2 mutein comprises a T41H substitution. In some embodiments, the IL-2 mutein comprises a M104I substitution. In some embodiments, the IL-2 mutein comprises a K48I substitution.In some embodiments, the IL-2 mutein comprises a S87E substitution. In some embodiments, the IL-2 mutein comprises a V91I, V91L, V91W, E95Q, E95S, E95N, L12Y, L19V, D84E, L19F, E95D, I92Y, E95T, I92V, L12V, I92W, D84T, D84S, M23L, I92F, M23I, H16Y, E15D, L12I, E15S, L12M, D20N, H16R, E15T, D20T, N88S, S87T, V91F, V91M, H16K, L19M, L19I, T111W, F42R, T111F, D109H, P34 and more substitutions selected from Q, P34W, D109W, T111N, T41Y, Y45W, L72W, L72F, E68Q, P34F, P65R, P65E, P65Q, E61W, T111H, F42M, T37Y, K43W, T111M, E68F, T111Y, N71W, L72R, E68W, K35T, E106W, K48V, P34Y, D109K, T111Q, E68R, K48S, K48H, P65N, E68Y, D109R, M104H, T41H, M104I, K48I, and S87E.

[0079] In one aspect, the present invention provides immunosuppressive IL-2 variants that have a higher affinity for IL-2Rα than wild-type IL-2. For example, in some embodiments, IL-2 muteins with a higher affinity for IL-2Rα include IL-2 muteins with amino acid substitutions selected from T111H, T37Y, P34F, and E68F. In some embodiments, the IL-2 variants described herein have a cytotoxicity of about 4.35×10 -6 (M)~Approx. 7.62×10 -11 (M). Thus, in some embodiments, the IL-2 variants described herein have a binding affinity of about 4×10 -6 (M), approx. 5 x 10 -6 (M), approx. 6×10 -6 (M), approx. 7 x 10 -6 (M), or approximately 8 x 10 -6 It has a binding affinity of (M).

[0080] In some embodiments, IL-2 variants contain one or more mutations at positions in the IL-2 sequence that contact IL-2Rα or that alter the orientation of other positions that contact IL-2Rα, resulting in higher affinity for IL-2Rα. The mutations can be within or near regions known to be in close proximity to IL-2Rα, as predicted based on the published crystal structure. Specific IL-2 muteins that have been designed and tested for functional properties in in vitro binding and in vivo assays are described herein.

[0081] In another aspect, the present invention provides immunosuppressive IL-2 variants that have lower affinity for IL-2Rβ than wild-type IL-2. For example, in some embodiments, the IL-2 mutein with lower affinity for IL-2Rβ comprises an IL-2 mutein with an amino acid substitution selected from E15T and M23L. In some embodiments, the IL-2 variant has a cytotoxicity of 1.6×10 -6 (M) has an affinity for IL-2Rβ greater than that of

[0082] In yet another aspect, the present invention provides immunosuppressive IL-2 muteins having lower affinity for IL-2Rα, hi one embodiment, the IL-2 mutein having lower affinity for IL-2Rα comprises an E62A substitution.

[0083] In one embodiment, the IL-2 mutein has a higher binding affinity for IL-2Rβ than WT IL-2.

[0084] Immunosuppressive IL-2 variants also include variants that exhibit altered signaling through specific pathways activated by wild-type IL-2 via the IL-2R, resulting in preferential proliferation, survival, and activation of T-regs. Molecules known to be phosphorylated upon IL-2R activation include STAT5, p38, ERK, SYK, LCK, AKT, and mTOR. Compared to wild-type IL-2, immunosuppressive IL-2 variants may exhibit reduced PI3K signaling in FOXP3 T cells, as measured by reduced phosphorylation of AKT and / or mTOR compared to wild-type IL-2. Such variants may contain mutations at positions that contact IL-2Rβ or IL-2Rγ or alter the orientation of other positions that contact IL-2Rβ or IL-2Rγ.

[0085] In certain embodiments, the IL-2 variant comprises a combination of mutations that increase or decrease binding to IL-2Rα or IL-2Rβ, or both. In preferred embodiments, the IL-2 variant stimulates STAT5 phosphorylation in FOXP3-positive regulatory T cells, but has a reduced ability to induce STAT5 and AKT phosphorylation in FOXP3-negative T cells compared to wild-type IL-2.

[0086] In some embodiments, the IL-2 variant may further comprise one or more mutations compared to the wild-type IL-2 sequence that do not affect affinity for IL-2Rβ or IL-2Rγ, provided that the IL-2 variant enhances FOXP3 expression over other T cells that do not express FOXP3. + provided that it promotes preferential proliferation, survival, activation, or function of T-regs. In a preferred embodiment, such mutations are conservative mutations.

[0087] In some embodiments, as used herein, an IL-2 mutein suitable for the present invention includes any wild-type and modified IL-2 variant (e.g., IL-2 proteins with amino acid mutations, deletions, insertions, and / or fusion proteins) that retain substantial IL-2 biological activity. Typically, recombinant IL-2 proteins are produced using recombinant techniques. However, IL-2 proteins (wild-type or modified) purified from natural sources or chemically synthesized can also be used in accordance with the present invention.

[0088] In some embodiments, a suitable recombinant IL-2 mutein has an in vivo half-life of about 1 minute, about 2 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, or about 24 hours, or longer. In some embodiments, a suitable recombinant IL-2 mutein or recombinant IL-2 fusion protein has an in vivo half-life of about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, or about 60 hours, or longer. In some embodiments, the recombinant IL-2 mutein has an in vivo half-life of 0.5 to 24 hours, 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, or 1 to 5 days.

[0089] In some embodiments, genetically engineered recombinant IL-2 variants are provided herein. In some embodiments, the genetically engineered recombinant variants are fused to IgG Fc. In some embodiments, the genetically engineered recombinant IL-2 variants are fused to human IgG1 Fc.

[0090] As will be appreciated by those skilled in the art, any such heavy chain CDR sequence, if present in an antibody or binding molecule of any format disclosed herein or otherwise known in the art, can be readily combined with IL-2 by, for example, molecular biology techniques, with any other antibody sequence or domain provided herein or otherwise known in the art, including any framework region, CDR, or constant domain, or portion thereof, disclosed herein or otherwise known in the art.

[0091] autoimmune disease Autoimmune diseases, disorders, or conditions are amenable to treatment or may be prevented by administration of an IL-2 mutein that promotes Treg proliferation and / or activity in a subject, hi some embodiments, one or more of the IL-2 muteins described herein are used to treat autoimmune diseases, disorders, or conditions.

[0092] Such diseases, disorders, and conditions that may be reduced in incidence and / or severity include inflammation, autoimmune diseases, paraneoplastic autoimmune diseases, cartilage inflammation, fibrotic diseases and / or bone degradation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, and the like. Arthritis, juvenile Rather syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Rather syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), Dermatomyositis, psoriatic arthritis, scleroderma, systemic lupus erythematosus, vasculitis, myositis, polymyositis, dermatomyositis, osteoarthritis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, scleroderma, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, These include, but are not limited to, arteriosclerosis, lupus, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, Guillain-Barré disease, type 1 diabetes, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD, transplant rejection, etc. In certain embodiments, a pharmaceutical composition comprising a therapeutically effective amount of a T-reg-selective IL-2 variant is provided.

[0093] In some embodiments, the disease is selected from the group consisting of MS, lupus, ankylosing spondylitis, arthritis, colitis, type 1 diabetes, reducing the severity of inflammatory diseases, Crohn's disease, heart disease, reducing complications due to the immune response during pregnancy, reducing complications due to graft-versus-host disease (GVHD), reducing the severity of allergies, reducing rejection of HSC or allogeneic solid organ transplants, depression, ALS, and / or myasthenia gravis.

[0094] The term "treatment" encompasses the alleviation or prevention of at least one symptom or other aspect of a disorder, or the reduction in the severity of a disease, etc. A T-reg-selective IL-2 variant need not bring about a complete cure or eliminate all symptoms or signs of a disease to constitute a viable therapeutic agent. As recognized in the relevant art, a drug used as a therapeutic agent can reduce the severity of a given disease state, but need not eliminate all signs of the disease to be considered a useful therapeutic agent. Similarly, a treatment administered prophylactically need not completely prevent the onset of the condition to constitute a viable prophylactic agent. Simply reducing the impact of a disease (e.g., by reducing the number or severity of its symptoms, by enhancing the effectiveness of another treatment, or by producing another beneficial effect) or reducing the likelihood of the disease developing or worsening in a subject is sufficient. One embodiment of the present invention relates to a method comprising administering a T-reg-selective IL-2 variant to a patient in an amount and for a time sufficient to prevent or treat, i.e., induce a sustained improvement beyond baseline, in an indicator reflective of the severity of a particular disorder.

[0095] Regulatory T cells (Treg) in the suppression of autoimmune inflammation Regulatory T cells (Treg) are FOXP3+CD4+ cells that play an important role in maintaining self-tolerance and normal immune homeostasis, as well as suppressing autoimmune inflammation. Current immunosuppressive therapies generally target individual pro-inflammatory pathways and are therefore often only partially effective or can only be applied to specific diseases. The present invention provides a method for suppressing autoimmune diseases, which involves selectively increasing the production and activation of natural suppressor cells.

[0096] Described herein are therapeutic agents that selectively promote the proliferation, survival, activation, and / or function of T-reg cells. By "selectively promote," we mean that the therapeutic agent promotes activity in T-reg cells but has limited or no ability to promote activity in non-regulatory T cells.

[0097] In certain embodiments, the agent is an IL-2 variant, specifically an IL-2 variant that promotes these activities of T-reg cell growth / survival but has a reduced ability to promote proliferation, survival, activation, and / or function of non-regulatory T cells (FOXP3 CD25 − ) and NK cells compared to wild-type IL-2, thereby minimizing side effects.

[0098] In certain embodiments, such IL-2 variants function through a combination of increased affinity for the IL-2R subunit IL-2Rα (CD25) and decreased affinity for the signaling subunits IL-2Rβ and / or IL-2Rγ. While IL-2 and its variants have been used in the art as immunostimulatory agents, e.g., in methods for treating cancer or infectious diseases, the IL-2 variants described herein are particularly useful as immunosuppressive agents, e.g., in methods for treating inflammatory disorders.

[0099] IL-2 fusion protein In some embodiments, a suitable IL-2 mutein described herein may be fused to another peptide. For example, a recombinant IL-2 mutein may be a fusion protein of the IL-2 domain with another domain or moiety that can enhance the therapeutic effect of IL-2, for example, by enhancing or increasing the stability, potency, and / or delivery of the IL-2 protein, or by reducing or eliminating immunogenicity or clearance. Suitable domains or moieties for IL-2 fusion proteins include, but are not limited to, an Fc domain, an XTEN domain, or a human albumin fusion. In other embodiments, suitable domains or moieties for IL-2 fusion proteins include the VH domain of an antibody.

[0100] Fc domain In some embodiments, a suitable recombinant IL-2 protein comprises an Fc domain or portion thereof that binds to the FcRn receptor. By way of non-limiting example, a suitable Fc domain may be derived from an immunoglobulin subclass, such as IgG. In some embodiments, a suitable Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, a suitable Fc domain is derived from IgM, IgA, IgD, or IgE. Particularly suitable Fc domains include those derived from human or humanized antibodies. In some embodiments, a suitable Fc domain is a modified Fc portion, such as a modified human Fc portion.

[0101] In some embodiments, a suitable Fc domain comprises an amino acid sequence as provided in Table 1.

[0102] [Table 1]

[0103] In some embodiments, a suitable Fc domain comprises an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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 more homologous or identical to an Fc domain sequence disclosed in Table 1.

[0104] It is contemplated that improved binding of the Fc domain to the FcRn receptor results in an extended serum half-life of the recombinant protein. Accordingly, in some embodiments, a suitable Fc domain comprises one or more amino acid mutations that improve binding to FcRn. A variety of mutations in Fc domains that have the effect of improving binding to FcRn are known in the art and can be adapted to the practice of the present invention. In some embodiments, a suitable Fc domain comprises one or more mutations at one or more positions corresponding to Thr250, Met252, Ser254, Thr256, Thr307, Glu380, Met428, His433, and / or Asn434 of human IgG1 according to EU numbering.

[0105] In some embodiments, a suitable Fc domain comprises one or more mutations at one or more positions corresponding to L234, L235, H433, and N434 of human IgG1 according to EU numbering.

[0106] The Fc portion of the recombinant fusion protein can lead to targeting of cells expressing Fc receptors, resulting in pro-inflammatory effects. Some mutations in the Fc domain reduce binding of the recombinant protein to Fc gamma receptors, thereby inhibiting effector function. In one embodiment, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). For example, a suitable Fc domain can contain the mutations L234A (Leu234Ala) and / or L235A (Leu235Ala) (EU numbering). In some embodiments, the L234A and L235A mutations are also referred to as LALA mutations. As a non-limiting example, a suitable Fc domain can contain the mutations L234A and L235A (EU numbering).

[0107] In some embodiments, a suitable Fc domain may contain the mutations H433K (His433Lys) and / or N434F (Asn434Phe) (EU numbering). As a non-limiting example, a suitable Fc domain may contain the mutations H433K and N434F (EU numbering). In some embodiments, the H433K and N434F mutations are also referred to as NHance mutations.

[0108] In some embodiments, a suitable Fc domain may contain the mutations L234A (Leu234Ala), L235A (Leu235Ala), H433K (His433Lys), and / or N434F (Asn434Phe) (EU numbering). As a non-limiting example, a suitable Fc domain may contain the mutations L234A, L235A, H433K, and N434F (EU numbering). Additional amino acid substitutions that can be included in an Fc domain include those described in, for example, U.S. Patent Nos. 6,277,375, 8,012,476, and 8,163,881, which are incorporated herein by reference.

[0109] Exemplary IL-2 Fusion Proteins The present invention provides IL-2 muteins that preferentially expand Tregs over, for example, Teff or NK cells. The IL-2 muteins provided herein may be modified to include or be fused to a molecule that extends the serum half-life of the mutein without increasing the risk of increasing the likelihood or intensity of side effects or adverse events in patients by extending such half-life. Subcutaneous administration of such serum half-life extended muteins results in a lower maximum systemic exposure (C max ) provides prolonged target coverage. The increased serum half-life allows for smaller and less frequent dosing regimens of the muteins.

[0110] The IL-2 variant may contain one or more compounds to increase the serum half-life of the IL-2 variant when administered to a patient. Such half-life extenders include water-soluble polymers (e.g., polyethylene glycol (PEG)), low-density and high-density lipoproteins, antibody Fc (monomer or dimer), transthyretin (TTR), and TGF-β latency-associated peptide (LAP). IL-2 variants containing combinations of serum half-life extenders, such as PEGylated TTR (U.S. Patent Application Publication No. 2003 / 0195154), are also contemplated.

[0111] The serum half-life of the IL-2 muteins provided herein can be extended by essentially any method known in the art. Such methods include modifying the sequence of the IL-2 mutein to include a peptide that binds to the neonatal Fey receptor or to a protein with an extended serum half-life, such as IgG or human serum albumin. In other embodiments, the IL-2 mutein is fused to a polypeptide that confers an extended half-life to the fusion molecule. Such polypeptides include polypeptides that bind to IgG Fc or other polypeptides that bind to the neonatal Fey receptor, human serum albumin, or proteins with an extended serum half-life.

[0112] In some embodiments, the IL-2 mutein is fused to an IgG Fc molecule. The IL-2 mutein may be fused to the N-terminus or C-terminus of the IgG Fc region.

[0113] One embodiment of the present invention relates to a dimer comprising two Fc fusion polypeptides created by fusing an IL-2 mutein to the Fc region of an antibody. The dimer can be produced, for example, by inserting a gene fusion encoding the fusion protein into an appropriate expression vector, expressing the gene fusion in a host cell transformed with the recombinant expression vector, and allowing the expressed fusion protein to assemble in a manner similar to an antibody molecule, forming interchain bonds between the Fc regions to obtain a dimer.

[0114] As used herein, the term "Fc polypeptide" or "Fc region" includes native and mutein forms of polypeptides derived from the Fc region of an antibody and may be part of either an IL-2 mutein fusion protein or an anti-IL-2 antibody of the present invention. Truncated forms of such polypeptides containing the hinge region that promotes dimerization are also included. In certain embodiments, the Fc region comprises the CH2 and CH3 domains of an antibody. In addition to having a long serum half-life, fusion proteins containing the Fc portion (and oligomers formed therefrom) have the advantage of being easily purified by affinity chromatography using Protein A or Protein G columns. Preferred Fc regions are derived from human IgG, including IgG1, IgG2, IgG3, and IgG4. Specific residues within the Fc are identified herein by position. All Fc positions are based on the EU numbering scheme.

[0115] One function of the Fc portion of an antibody is to signal the immune system when the antibody binds to its target. This is considered an "effector function." Signaling can lead to antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). ADCC and ADCP are mediated through binding of Fc to Fc receptors on the surface of cells of the immune system. CDC is mediated through binding of Fc to proteins of the complement system, such as Clq.

[0116] IgG subclasses vary in their ability to mediate effector function. For example, IgG1 is better at mediating ADCC and CDC than IgG2 and IgG4. 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 mutein Fc fusion proteins with engineered Fc to enhance effector function (U.S. Pat. No. 7,317,091 and Strohl, Curr. Opin. Biotech., 20:685-691, 2009; both of which are incorporated by reference in their entirety).

[0117] Manufacturing method The IL-2 variants described herein can be produced using any suitable method known in the art. Such methods include, for example, constructing a DNA sequence encoding the IL-2 variant and expressing that sequence in a suitable transformed host. This method produces recombinant variants of the invention. However, variants can also be produced by chemical synthesis or a combination of chemical synthesis and recombinant DNA technology. Methods for batch or perfusion production are known in the art. Freshey, RI (ed), 3 “Animal Cell Culture: A Practical Approach,” 2nd ed., 1992, IRL Press. See Mammalian Cell Culture, "Curr Opin Biotechnol 8:148-153 (1997); Konstantinov, KB, Tsai, Y., Moles, D., Matanguihan, R., "Control of long-term perfusion Chinese hamster ovary cell culture by glucose auxostat.," Biotechnol Prag 12:100-109 (1996).

[0118] In some embodiments for generating the IL-2 variants described herein, a DNA sequence encoding wild-type IL-2 is isolated or synthesized, and then one or more codons are altered by site-directed mutagenesis to construct the DNA sequence. See, e.g., Mark et al., "Site-specific Mutagenesis of the Human Fibroblast Interferon Gene," Proc. Natl. Acad. Sci. USA 81, pp. 5662-66 (1984); and U.S. Pat. No. 4,588,585, which are incorporated herein by reference. Various mutations and methods for making them are known in the art and include, for example, amino acid and / or nucleic acid deletions, insertions, substitutions, and / or fusions.

[0119] Another method for constructing a DNA sequence encoding an IL-2 variant is chemical synthesis. This includes, for example, direct peptide synthesis by chemical means of a protein sequence encoding an IL-2 variant exhibiting the properties described herein. This method allows for the incorporation of both natural and unnatural amino acids. Alternatively, genes encoding the desired IL-2 variant can be synthesized by chemical means using an oligonucleotide synthesizer. In some embodiments, such oligonucleotides are designed based on the amino acid sequence of the desired IL-2 variant, with codons selected that are favorable in the host cell in which the recombinant variant will be produced. In this regard, it is well recognized that the genetic code is degenerate, and an amino acid may be encoded by more than one codon. For example, Phe (F) is encoded by two codons, TTC or TTT, Tyr (Y) is encoded by TAC or TAT, and His (H) is encoded by CAC or CAT. Trp (W) is encoded by a single codon, TGG. Thus, it will be understood that for any given DNA sequence encoding a particular IL-2 variant, there are many degenerate DNA sequences that also encode that IL-2 variant.

[0120] The DNA sequence encoding the IL-2 variant, whether prepared by site-directed mutagenesis, chemical synthesis, or other methods, may or may not include a DNA sequence encoding a signal sequence. In some embodiments, such a signal sequence, if present, is one that is recognized by the cell selected for expression of the IL-2 variant. It may be prokaryotic, eukaryotic, or a combination of the two. It may also be the signal sequence of native IL-2. The inclusion of a signal sequence depends on whether it is desired that the IL-2 variant be secreted from the recombinant cell in which it is produced. In some embodiments, if the selected cell is prokaryotic, the DNA sequence does not encode a signal sequence. In some embodiments, if the selected cell is eukaryotic, a signal sequence is encoded and may have a wild-type IL-2 signal sequence.

[0121] Standard methods can be applied to synthesize genes encoding IL-2 variants. For example, a reverse-translated gene can be constructed using the complete amino acid sequence. DNA oligomers containing nucleotide sequences encoding IL-2 variants can also be synthesized. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides can contain 5' or 3' overhangs for complementary assembly.

[0122] Once assembled (by synthesis, site-directed mutagenesis, or other methods), the DNA sequence encoding the IL-2 variant is inserted into an expression vector and operably linked to expression control sequences suitable for expression of the IL-2 variant in the desired transformed host. Proper assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and expression of a biologically active polypeptide in a suitable host. As is known in the art, to obtain high expression levels of a transfected gene in a host, the gene is operably linked to transcriptional and translational expression control sequences that function in the selected expression host. The choice of expression control sequences and expression vector depends on the choice of host. A wide variety of expression host / vector combinations can be used.

[0123] Any suitable host may be used to produce an IL-2 variant, including bacterial, fungal (including yeast), plant, insect, mammalian, or other suitable animal cells or cell lines, as well as transgenic animals or plants. These hosts include well-known eukaryotic and prokaryotic hosts such as E. coli, Pseudomonas, Bacillus, and Streptomyces strains, fungi, yeast, insect cells such as Spodoptera frugiperda (Sf9), Chinese hamster ovary cells (CHO) and mouse cells such as NS / 0, African green monkey cells such as COS1, COS7, BSC1, BSC40, and BNT10, and animal cells such as human cells, as well as plant cells in tissue culture. In some embodiments, for expression in animal cells, CHO cells and COS7 cells in culture may be used, as well as the CHO cell lines CHO(DHFR-) or HKB strains.

[0124] It should be understood that not all vectors and expression control sequences function equally well for expressing the DNA sequences described herein. Also, not all hosts function equally well in the same expression system. However, one of ordinary skill in the art can select from among these vectors, expression control sequences, and hosts without undue experimentation. For example, when selecting a vector, the host cell is considered, since the vector must replicate within the host cell. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, may also be considered. For example, in some embodiments, vectors for use in the present invention include those capable of amplifying the copy number of DNA encoding an IL-2 variant. Such amplifiable vectors are well known in the art. For example, vectors that can be amplified by DHFR amplification (see, e.g., Kaufman, U.S. Pat. No. 4,470,461; Kaufman and Sharp, "Construction of a Modular Dihydrafolate Reductase cDNA Gene: Analysis of Signals Utilized for Efficient Expression," Mol. Cell. Biol., 2, pp. 1304-19 (1982)) or glutamine synthetase ("GS") amplification (see, e.g., U.S. Pat. No. 5,122,464 and European Patent Application Publication No. 338,841).

[0125] IL-2 variants may be glycosylated or non-glycosylated, depending on the host organism used to produce the variant. In some embodiments, when a bacterium is selected as the host, the IL-2 variant produced will be non-glycosylated. In some embodiments, eukaryotic cells glycosylate the IL-2 variant. IL-2 variants produced by transformed hosts can be purified according to any suitable method. Various methods for purifying IL-2 are known. See, for example, Current Protocols in Protein Science, Vol. 2, Eds: John E. Coligan, Ben M. Dunn, Hidde L. Ploehg, David W. Speicher, Paul T. Wingfield, Unit 6.5 (Copyright 1997, John Wiley and Sons, Inc.).

[0126] Pharmaceutical Compositions and Administration The present invention further provides pharmaceutical compositions comprising a therapeutically active ingredient of the present invention (e.g., a recombinant IL-2 mutein protein, a recombinant IL-2 mutein fusion protein, or a recombinant IL-2 mutein-Fc fusion protein) together with one or more pharmaceutically acceptable carriers or excipients. Such pharmaceutical compositions may optionally contain one or more additional therapeutically active substances.

[0127] Although the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for ethical administration to humans, one of ordinary skill in the art will understand that such compositions are generally suitable for administration to any animal. Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to a variety of animals are well understood, and a veterinary pharmacologist of ordinary skill can design and / or implement such modifications with no more than routine experimentation, if any.

[0128] The formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such preparatory methods include the step of bringing the active ingredient into association with a diluent or other excipient or carrier, and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into a desired single-dose or multi-dose unit.

[0129] Pharmaceutical compositions according to the invention may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject, and / or a convenient fraction of such a dosage, such as, for example, one-half or one-third of such a dosage.

[0130] The relative amounts of active ingredient, pharmaceutically acceptable excipient or carrier, and / or any additional ingredients in a pharmaceutical composition of the present invention will vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition is administered. By way of example, the composition may contain from 0.1% to 100% (w / w) active ingredient.

[0131] Pharmaceutical formulations may further comprise pharmaceutically acceptable excipients or carriers, which as used herein include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surface active agents, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, appropriate for the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21 stEdition, A.R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for preparing them. Except insofar as any conventional excipient vehicle or carrier is incompatible with the substance or its derivatives, for example, by producing some undesirable biological effect or otherwise interacting adversely with any other component(s) of the pharmaceutical composition, its use is contemplated within the scope of the present invention.

[0132] In some embodiments, the pharmaceutically acceptable excipient or carrier has a purity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In some embodiments, the excipient or carrier is approved for human and veterinary use. In some embodiments, the excipient or carrier is approved by the U.S. Food and Drug Administration. In some embodiments, the excipient or carrier is pharmaceutical grade. In some embodiments, the excipient or carrier conforms to the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0133] Pharmaceutically acceptable excipients or carriers used in preparing pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifying agents, disintegrating agents, binders, preservatives, buffers, lubricants, and / or oils. Optionally, such excipients or carriers may be included in the pharmaceutical formulation. Excipients or carriers such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and / or perfuming agents may also be present in the composition, according to the discretion of the formulator.

[0134] Suitable pharmaceutically acceptable excipients or carriers include, but are not limited to, water, salt solutions (e.g., NaCl), saline, buffered saline, alcohol, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin; carbohydrates such as lactose, amylose, or starch; sugars such as mannitol, sucrose, or others; dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, flavor oils, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc., and combinations thereof. Pharmaceutical preparations may, if necessary, be mixed with auxiliary substances (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring, and / or aromatic substances) that do not adversely react with the active compound or interfere with its activity. In a preferred embodiment, a water-soluble carrier suitable for intravenous administration is used.

[0135] Suitable pharmaceutical compositions or medicaments may contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if necessary.The compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, sustained-release formulations, or powders.The compositions may also be formulated as suppositories using conventional binders and carriers such as triglycerides.Oral formulations may contain standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0136] A pharmaceutical composition or medicament can be formulated according to routine procedures as a pharmaceutical composition adapted for administration to humans. For example, in some embodiments, compositions for intravenous administration are typically sterile isotonic aqueous buffer solutions. Where necessary, the compositions may also include a solubilizing agent and a local anesthetic to ease pain at the injection site. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet, indicating the quantity of active agent. When the composition is administered by infusion, it may be dispensed using an infusion bottle containing pharmaceutical-grade sterile water, saline, or dextrose / water. When the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients can be mixed prior to administration.

[0137] The recombinant IL-2 mutein proteins or recombinant IL-2 mutein-Fc fusion proteins described herein can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with free carboxyl groups, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0138] General considerations in the formulation and / or manufacture of pharmaceuticals are found, for example, in Remington: The Science and Practice of Pharmacy 21 st ed., Lippincott Williams & Wilkins, 2005 (hereby incorporated by reference).

[0139] Administration route The recombinant IL-2 mutein protein or recombinant IL-2 mutein-Fc fusion protein described herein (or a composition or medicament containing the recombinant IL-2 mutein protein described herein) can be administered by any suitable route. In some embodiments, the recombinant IL-2 mutein protein, recombinant IL-2 mutein-Fc fusion protein, or pharmaceutical composition containing the same is administered systemically. Systemic administration may be intravenous, intradermal, inhalation, transdermal (topical), intraocular, intramuscular, subcutaneous, intramuscular, oral, and / or transmucosal administration. In some embodiments, the recombinant IL-2 mutein protein, recombinant IL-2 mutein-Fc fusion protein, or pharmaceutical composition containing the same is administered subcutaneously. As used herein, the term "subcutaneous tissue" is defined as the layer of loose, irregular connective tissue immediately beneath the skin. For example, subcutaneous administration may be performed by injecting the composition into areas including, but not limited to, the thigh, abdomen, buttocks, or scapular region. In some embodiments, the recombinant IL-2 mutein protein, recombinant IL-2 mutein-Fc fusion protein, or pharmaceutical composition containing same is administered intravenously. In some embodiments, the recombinant IL-2 mutein protein, recombinant IL-2 mutein-Fc fusion protein, or pharmaceutical composition containing same is administered orally. In some embodiments, the recombinant IL-2 mutein protein, recombinant IL-2 mutein-Fc fusion protein, or pharmaceutical composition containing same is administered intramuscularly. In some embodiments, more than one route may be used simultaneously.

[0140] In some embodiments, administration results in only a localized effect in an individual, while in other embodiments, administration results in an effect throughout multiple parts of an individual, e.g., a systemic effect. Typically, administration results in systemic delivery of the recombinant IL-2 mutein protein or recombinant IL-2 mutein-Fc fusion protein. In some embodiments, the recombinant IL-2 mutein protein or recombinant IL-2 mutein-Fc fusion protein is delivered to one or more target tissues, including, but not limited to, the heart, brain, spinal cord, striated muscle (e.g., skeletal muscle), smooth muscle, kidney, liver, lung, and / or spleen.

[0141] Dosage Forms and Administration Regimen In some embodiments, the compositions are administered in a therapeutically effective amount and / or according to a dosing regimen that correlates with a particular desired outcome (eg, treatment or reduced risk of an autoimmune disease).

[0142] The specific dose or amount administered in accordance with the present invention may vary depending, for example, on the nature and / or extent of the desired outcome, characteristics of the route and / or timing of administration, and / or one or more characteristics (e.g., body weight, age, personal history, genetic characteristics, lifestyle parameters, etc., or a combination thereof). Such doses or amounts can be determined by one of ordinary skill in the art. In some embodiments, an appropriate dose or amount is determined in accordance with standard clinical techniques. Alternatively, or in addition, in some embodiments, an appropriate dose or amount is determined through the use of one or more in vitro or in vivo assays to help identify desirable or optimal dosage ranges or amounts to be administered.

[0143] In various embodiments, the recombinant IL-2 mutein protein is administered in a therapeutically effective amount. Generally, a therapeutically effective amount is sufficient to achieve a meaningful benefit in the subject (e.g., treatment, modulation, cure, prevention, and / or amelioration of the underlying disease or condition).

[0144] In some embodiments, provided compositions are provided as pharmaceutical formulations, hi some embodiments, the pharmaceutical formulations are or comprise unit doses for administration according to a dosing regimen that correlates with achieving a reduced incidence or risk of autoimmune disease.

[0145] In some embodiments, a formulation comprising a recombinant IL-2 mutein protein or a recombinant IL-2 mutein-Fc fusion protein described herein is administered as a single dose. In some embodiments, a formulation comprising a recombinant IL-2 mutein protein or a recombinant IL-2 mutein-Fc fusion protein described herein is administered at regular intervals. As used herein, administration at an "interval" indicates that a therapeutically effective amount is administered periodically (as distinguished from a one-time administration). This interval can be determined by standard clinical techniques. In some embodiments, a formulation comprising a recombinant IL-2 mutein protein or a recombinant IL-2 mutein-Fc fusion protein described herein is administered bimonthly, monthly, twice monthly, once every three weeks, every other week, weekly, twice weekly, three times weekly, daily, twice daily, or every six hours. The administration interval for an individual need not be constant and may vary over time depending on the individual's needs.

[0146] As used herein, the term "bimonthly" means administration once every two months (i.e., once every two months); the term "monthly" means administration once per month; the term "once every three weeks" means administration once every three weeks (i.e., once every three weeks); the term "biweekly" means administration once every two weeks (i.e., once every two weeks); the term "weekly" means administration once per week; and the term "daily" means administration once per day.

[0147] In some embodiments, a formulation comprising a recombinant IL-2 mutein protein or a recombinant IL-2 mutein-Fc fusion protein described herein is administered at regular intervals indefinitely, hi some embodiments, a formulation comprising a recombinant IL-2 mutein protein or a recombinant IL-2 mutein-Fc fusion protein described herein is administered at regular intervals for a defined period of time.

[0148] As described herein, the term "therapeutically effective amount" is determined primarily based on the total amount of the therapeutic agent contained in the pharmaceutical composition of the present invention. The therapeutically effective amount is generally administered in a dosage regimen that may include multiple unit doses. For any particular composition, the therapeutically effective amount (and / or the appropriate unit dose within an effective dosage regimen) may vary depending, for example, on the route of administration or combination with other pharmaceutical agents.

[0149] In some embodiments, the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of one or more IL-2 muteins described herein, together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant.

[0150] Combination Therapy: In further embodiments, the IL-2 muteins described herein are administered in combination with other agents useful for treating the condition from which the patient is afflicted. Examples of such agents include both proteinaceous and non-proteinaceous drugs. When multiple therapeutic agents are co-administered, dosages may be adjusted accordingly, as recognized in the relevant art. "Co-administration" and combination therapy are not limited to simultaneous administration, but also include therapeutic regimens in which a T-reg-selective IL-2 variant is administered at least once during a course of treatment that includes administering at least one other therapeutic agent to the patient.

[0151] In certain embodiments, the IL-2 mutein is administered in combination with an inhibitor of the PI3-K / AKT / mTOR pathway, such as rapamycin (rapamune, sirolimus). Combining an inhibitor of this pathway with IL-2 is advantageous for the enrichment of T-regs.

[0152] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The documents cited herein are not admitted to be prior art to the claimed invention. Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting. [Example]

[0153] Example 1. Design and construction of IL-2 muteins This example describes the design and construction of exemplary IL-2 muteins.

[0154] Standard recombinant DNA techniques were used to manipulate DNA (Sambrook et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) to design constructs containing the nucleotide sequences of the light and heavy chains of heavy immunoglobulin (Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No. 91-3242). Double-stranded DNA was sequenced to confirm the nucleotide sequence of the constructs.

[0155] Exemplary nucleotide residues in the WT IL-2 protein (SEQ ID NO: 1) were mutated to generate exemplary IL-2 muteins (Table 2), which were then characterized for binding affinity to IL-2Rα, IL-2Rβ, CD25, or CD122. Exemplary IL-2 muteins contain at least one amino acid substitution relative to the wild-type IL-2 protein (SEQ ID NO: 1) selected from the group consisting of T111H, T37Y, E15T, M23L, P34F, E68F, and E62A.

[0156] Furthermore, IgG-fused IL-2 muteins were constructed. To prevent intermolecular disulfide bond formation, a C125A mutation was introduced into all IL-2 muteins constructed. Therefore, in some embodiments of the present invention, the IL-2 muteins contain a C125A mutation. To minimize avidity effects and heterodimerization, knob-in-hole mutations were introduced into the IgG Fc region, and all IL-2 muteins were fused only to IgGs with knob mutations.

[0157] This example demonstrates the design and construction of exemplary IL-2 muteins (Table 2).

[0158] [Table 2]

[0159] Example 2. Generation of IgG-fused IL-2 muteins This example describes the production of an exemplary IgG-fused IL-2 mutein protein.

[0160] First, gene fragments were generated by synthetic gene synthesis and / or PCR from suitable templates and subcloned into standard mammalian expression vectors. To generate IgG IL-2 mutein fusion proteins, mammalian expression vectors for the IgG light chain, the IgG heavy chain with the hole mutation, and the IgG heavy chain with the knob mutation fused to the IL-2 mutein were co-transfected into exponentially growing Expi293 cells using ExpiFectamine transfection reagent.

[0161] The fusion protein was then purified from the supernatant by one-step affinity purification using Protein A beads equilibrated in PBS. After loading the supernatant, the column was first washed with PBS. The fusion protein was eluted with 0.1 M glycine-HCl / 0.3 M NaCl (pH 3.0). Fractions were neutralized with 1 M Tris-HCl pH 8.0 (1:10) and buffer-exchanged into PBS by dialysis. The protein concentration of the purified protein sample was determined by measuring the optical density (OD) at 280 nm using the molar extinction coefficient calculated based on the amino acid sequence. This resulted in the production of a purified IgG-fused IL-2 mutein.

[0162] Example 3. Binding affinity of IL-2 mutein fusion proteins to the IL-2 receptor In this example, the binding affinity of an exemplary IL-2 mutein fusion protein to the IL-2 receptor was measured. The affinity of the fusion protein to the IL-2 receptor was determined by biolayer interferometry (BLI) using recombinant IL-2Rα and IL-2Rβ on Octet Red 96e (Forte Bio) for the human IL-2Rα and IL-2Rβ receptors under the following conditions: the ligand contained biotinylated human IL-2Rα or IL-2Rβ immobilized on an SA chip, and the analyte contained a single dose of IgG-fused IL-2 mutein in PBS buffer at 25°C. Association time: 120 s, dissociation time: 600 s, fitting: 1:1 Langmuir binding model. Affinity was determined based on the kinetic rate constants k and k (Table 3).

[0163] [Table 3-1]

[0164] [Table 3-2]

[0165] For example, the K77A IL-2 mutein exhibited binding affinity to IL-2Rα similar to that of WT IL-2 (Figures 1A and 1B), whereas the murine E96A mutant IL-2 exhibited reduced binding to IL-2Rα (CD25) (Figure 1C).

[0166] This example demonstrates the binding affinity of exemplary IL-2 muteins to the IL-2 receptor.

[0167] Example 4. Induction of pSTAT5a in human peripheral blood cell subsets This example demonstrates the functional effect of exemplary IL-2 muteins on STAT5a phosphorylation in human CD4+ and CD8+ T cells.

[0168] Binding of IL-2 to the IL-2 receptor on the cell surface induces phosphorylation of STAT5a, activating several signaling pathways and resulting in the transcription of target genes that contribute to various functions associated with the IL-2 / IL-2R pathway. Therefore, to determine the integrated signaling response to IL-2 mediated by various combinations of high- and intermediate-affinity receptors, we measured pSTAT5a levels in individual cells by flow cytometry.

[0169] The effect of a single dose of IgG-fused IL-2 mutein (10 pM) on the induction of STAT5a phosphorylation in human CD4+ and CD8+ T cells was evaluated. Briefly, frozen PBMCs from healthy adult humans were incubated at 37°C for 2 hours. IgG-fused IL-2 mutein (10 pM) was added to 100 μL of PBMCs at 0.5 million cells / well and incubated at 37°C. After 2 hours, PBMCs were fixed and permeabilized with prewarmed lysis / fixation buffer for 10 minutes at 37°C, washed twice with PBS containing 0.2% BSA, and then permeabilized with pre-chilled methanol at -20°C for 20 minutes on ice. After extensively washing the cells four times with PBS containing 0.2% BSA, FACS staining was performed using a panel of fluorescent antibodies to distinguish between CD4+ and CD8+ T cells and the phosphorylation status of pSTAT5a in these cells. The antibodies used were anti-CD4-Alexa Fluor® 700 (clone RPA-T4), CD3-PerCP / Cy5.5 (UCHT1), CD8-Brilliant Violet 605 (RPA-T8), and pSTAT5a-Alexa Fluor® 488 (pY694) (Becton Dickinson). Samples were acquired using an LSR Fortessa cell analyzer (Becton Dickinson), and data were analyzed using FlowJo software (FlowJo, LLC). Intracellular pSTAT5a levels were quantified in the two cell subsets, and results are shown for CD4+ and CD8+ cells (Table 4).

[0170] [Table 4-1]

[0171] [Table 4-2]

[0172] Based on the results of measuring affinity for the IL-2 receptor and induction of pSTAT5a in human PBMCs, six clones (M23L, E15T, P34F, T111H, T37Y, and E68F) were selected for further analysis.

[0173] Dose-response analysis of selected muteins in the pSTAT5a assay was performed. Briefly, frozen PBMCs from healthy adult humans were incubated at 37°C for 2 hours. IgG-fused IL-2 muteins were added to 100 μL of PBMCs at various concentrations (10 nM to 0.01 pM) at 0.5 million cells per well and incubated at 37°C. After 2 hours, PBMCs were fixed, and pSTAT5a levels were measured as described above.

[0174] The results showed that all six muteins tested exhibited pSTAT5a induction activity in CD4+ cells that was equal to or greater than that induced by wild-type IL-2.

[0175] Furthermore, the exemplary E62A mutation inhibited the activity of human IL-2 mutants to induce pSTAT5 in CD25+ CD4 T cells, but not in CD8 T cells (Figures 2A and 2B, Table 5). The E62A mutation did not inhibit the activity of human IL-2 mutants to induce pSTAT5 in CD25- CD4 T cells and NK cells (Figures 2C and 2D). F906 is the VH domain of an anti-CTLA-4 antibody. Wild-type human IL-2 or a human IL-2 mutein linked to F906 via a peptide linker is specifically delivered to Tregs.

[0176] [Table 5]

[0177] FIG. 2E shows a series of graphs depicting pSTAT5a induction with IL-2 muteins M23L, T111H, E68F, E15T, P34F, and T37Y compared to wild-type IL-2, along with a table of associated EC50 and Emax values.

[0178] This example demonstrates exemplary pSTAT5a induction by IL-2 muteins.

[0179] Example 5. Effect of E96A-HLE in WT mice after multiple administrations This example demonstrates the in vivo effects of the E96A-HLE mutein in mice after multiple administrations.

[0180] WT mIL-2-HLE (wild-type mouse IL-2 half-life extension, w ild- t type m ouse IL-2- h alf- l ife e To understand the biological activity of E96A-HLE compared to that of E96A-HLE (extended), mice were treated with these cytokines and examined for changes in the development of immune cell subsets, including CD8 and CD4 T cells, including Tregs.

[0181] Each biologic was diluted in 1x PBS. A volume of 100 μL was administered intraperitoneally to mice for each condition, based on an approximate dose of 20 g per mouse. Non-tumor-bearing female C57BL / 6 mice, 6-8 weeks old, were treated daily for 5 days, followed by a 2-day break and then another 4-day treatment. 24 hours after the final dose, mice were euthanized, and their spleens were removed for PD analysis. Three mice per group were tested.

[0182] Spleens were mashed through a 70 μm cell strainer in an RP10 tube. Cells were subjected to RBC lysis with RBC lysis buffer, and viable splenocytes were counted using a ViCell (Beckman Coulter) microscope. Spleens were stained with live / dead e780, anti-CD3, anti-CD4, anti-CD8, anti-CD25, and anti-foxp3 antibodies according to the manufacturer's instructions. Spleen cells were washed and incubated with antibodies in staining buffer, and intracellular staining was performed using the foxp3 staining buffer set. Cells were analyzed using an LSRFortessa FACS machine (BD), and data were analyzed using FloJo software (TreeStar).

[0183] Lymphocytes were gated based on FSC / SSC and doublets were excluded. Naive T cells were identified as CD3+CD4+foxp3+ (Figure 4).

[0184] The results showed that treatment of mice with WT mIL-2-HLE resulted in a small expansion of CD4+foxp3+ T cells, whereas treatment with E96A-HLE resulted in a greater and dose-dependent expansion of CD4+foxp3+ T cells (Figures 3 and 4A-F).

[0185] Treatment of mice with WT mIL-2-HLE expanded foxp3+ cells as a subset of CD3+ cells, whereas treatment of mice with E96A-HLE expanded the proportion of foxp3+ cells as a subset of CD3+ cells in a dose-dependent manner to a greater extent (Fig. 5A).

[0186] Treatment of mice with E96A-HLE resulted in a dose-dependent expansion of splenocytes, including CD3+ cells (Fig. 5B).

[0187] The results demonstrated the in vivo activity of IL-2-HLE and a dose-dependent increase in Tregs by administration of E96A-HLE.

[0188] Example 6. Effects of E62A-HLE and E96A-HLE in WT mice after a single administration This example demonstrates the in vivo effects of E62A-HLE and E96A-HLE compared to WT hIL2-HLE and WT mIL2-HLE after a single dose.

[0189] WT hIL2-HLE (wild-type human IL-2 half-life extension, w ild- t type h uman IL-2- h alf- l ife eTo understand the biological activity of E62A-HLE and E96A-HLE compared to WT mIL-2-HLE (extended) and WT mIL-2-HLE, mice were treated with these cytokines. CD8 and CD4 T cells, including Tregs, were examined for changes in the development of immune cell subsets. Each biologic was diluted in 1x PBS. Mice were intraperitoneally administered a volume of 100 μL based on an approximate dose of 20 g per mouse (Figure 6A).

[0190] Four 6- to 8-week-old non-tumor-bearing female C57BL / 6 mice per group were treated with a single low-dose (0.45 mg / kg / mouse) or high-dose (2 mg / kg / mouse) of mouse or human wild-type IL2 or IL2 mutein. After 7 days, the mice were euthanized. Wet lung weights were measured, cardiac puncture was performed for plasma cytokine analysis, and spleens were removed for PD analysis.

[0191] The mean weight change rate was measured by thermogravimetry (TG) (FIG. 6B).

[0192] Spleens were mashed through a 70 μm cell strainer in an RP10 tube. Cells were subjected to RBC lysis using RBC lysis buffer, and viable splenocytes were counted using a ViCell™ system. Spleens were stained with live / dead e780, anti-CD3, anti-CD4, anti-CD8, anti-CD25, and anti-foxp3, all according to the manufacturer's instructions. Spleen cells were washed and incubated with antibodies in staining buffer, followed by intracellular staining using the foxp3 staining buffer set. Cells were analyzed using an LSR Fortessa FACS machine, and data were analyzed using FloJo software. Lymphocytes were gated based on FSC / SSC, and doublets were excluded. Naive T cells were identified as CD3+CD4+foxp3+.

[0193] Treatment of mice with WT mIL-2-HLE or WT hIL2-HLE increased the number of splenocytes, whereas treatment with E62A-HLE or E96A-HLE resulted in a greater dose-dependent expansion of splenocytes (Figure 6C). Treatment of mice with E96A-HLE or E62A-HLE resulted in a dose-dependent expansion of CD4+foxp3+ T cells. Treatment of mice with WT hIL2-HLE or WT mIL-2-HLE reduced the CD8:Treg ratio, whereas treatment with E62A-HLE or E96A-HLE preferentially expanded Tregs, resulting in a greater decrease in the CD8:Treg ratio (Figures 8A-8C).

[0194] Example 7. Effects of E62A-HLE and E96A-HLE in WT mice after two doses This example shows the in vivo effects of E62A-HLE and E96A-HLE compared to WT hIL2-HLE and WT mIL2-HLE after two doses.

[0195] To understand the biological activity of E62A-HLE and E96A-HLE compared to WT hIL2-HLE and WT mIL-2-HLE, mice were treated with these cytokines. CD8 and CD4 T cells, including Tregs, were examined for changes in the development of immune cell subsets. Each biologic was diluted in 1x PBS. Mice were intraperitoneally administered a volume of 100 μL based on an approximate dose of 20 g per mouse (Figure 7A).

[0196] Four 6- to 8-week-old non-tumor-bearing female C57BL / 6 mice per group were treated with a single low dose of 0.45 mg / kg / mouse or a high dose of 2 mg / kg / mouse on day 0, then treated again 7 days later and euthanized 4 days later. Wet lung weights were measured, cardiac puncture was performed for plasma cytokine analysis, and spleens were removed for PD analysis.

[0197] Administration of high doses of WT or mutant IL-2 resulted in a transient decrease in body weight (Fig. 7B).

[0198] Spleens were mashed through a 70 μm cell strainer in an RP10 tube. Cells were subjected to RBC lysis using RBC lysis buffer, and viable splenocytes were counted using a ViCell™ system. Spleens were stained with live / dead e780, anti-CD3, anti-CD4, anti-CD8, anti-CD25, and anti-foxp3, all according to the manufacturer's instructions. Spleen cells were washed and incubated with antibodies in staining buffer, followed by intracellular staining using the foxp3 staining buffer set. Cells were analyzed using an LSRFortessa FACS machine, and data were analyzed using FloJo software (TreeStar). Lymphocytes were gated based on FSC / SSC, and doublets were excluded. Naive T cells were identified as CD3+CD4+foxp3+. Although treating mice twice with WT mIL-2-HLE or WT hIL2-HLE increased the number of splenocytes, treating mice twice with E62A-HLE resulted in a greater dose-dependent expansion of splenocytes (Fig. 7C).

[0199] Treatment of mice with E96A-HLE or E62A-HLE resulted in a greater dose-dependent expansion of CD4+foxp3+ T cells. Treatment of mice with WT hIL2-HLE or WT mIL-2-HLE increased the CD8:Treg ratio, whereas treatment of mice with E62A-HLE or E96A-HLE preferentially expanded Tregs, resulting in a greater decrease in the CD8:Treg ratio (Figures 8D–8F).

[0200] Example 8. Administration of E62A-HLE protected mice from autoimmune hyperglycemia This example demonstrates that administration of E62A-HLE results in increased Treg expansion in vivo as well as increased protection of treated mice from autoimmune hyperglycemia, an exemplary autoimmune pathology.

[0201] Seven-week-old mice were assigned to treatment groups based on non-fasting blood glucose levels and administered 100 μL / mouse by intraperitoneal (IP) injection according to Table 6. Body weight, clinical findings, and non-fasting blood glucose levels were recorded weekly throughout the study. All animals with non-fasting blood glucose levels above 250 mg / dL for two consecutive days were humanely euthanized.

[0202] [Table 6]

[0203] At 10 weeks of age, three mice from treatment groups 1 and 2 were humanely euthanized and splenocytes were assessed by flow cytometry using a standard panel of T and NK cells.

[0204] At 30 weeks of age, the remaining mice in the study were humanely euthanized and tissues were collected for flow cytometry using standard T cell and NK cell panels.

[0205] Neither administration regimen of E62A-HLE resulted in a loss of body weight (Fig. 9A).

[0206] Blood glucose levels were measured (Figure 9B). Results showed that, in contrast to control mice, 60% of which developed hyperglycemia by 20 weeks of age (n = 10), none of the E62A-HLE-treated mice (n = 19) developed hyperglycemia by 30 weeks of age, suggesting that Treg expansion protects mice from autoimmune hyperglycemia (Figure 9C).

[0207] Tregs were expanded in splenocytes from mice treated with E62A-HLE (Figures 11A and 11B). NK cells (Figure 10A) and CD8+ T cells (Figure 10D) were also expanded in E62A-treated mice. B cells were expanded in splenocytes from E62A-treated mice (Figure 10B), but CD4+ T cells were not expanded (Figure 10C).

[0208] Example 9. PD after a single dose of E62A-HLE at multiple time points and in multiple tissues This example shows PD after a single dose of E62A-HLE at multiple time points and in multiple tissues.

[0209] To understand the biological activity of E62A-HLE compared to WT hIL-2-HLE (referred to as Cyto5(2124-T60) in the figure), mice were treated with these cytokines. CD8 and CD4 T cells, including Tregs, were examined for changes in the development of immune cell subsets. Female C57BL / 6 mice were inoculated with 0.1 mL of B16F10 tumor cells (8 × 10 4 Mice were inoculated subcutaneously with E62A-HLE (cell suspension). A single dose of E62A-HLE was administered intraperitoneally. After E62A-HLE treatment, mice were euthanized at 96, 168, and 240 hours post-injection, and whole blood, lymph nodes, spleens, and tumors were collected for analysis. Flow antibodies included anti-CD45, anti-CD8, anti-CD4, anti-ICOS, live / dead, anti-hCTLA-4, Ki67, anti-NKp46, anti-CD19, anti-Ly6G, anti-CD25, anti-CD62L, anti-foxp3, anti-CD44, and anti-TCRb, all stained according to the manufacturer's instructions. The antibodies were washed and stained with staining buffer, followed by intracellular staining using the foxp3 staining buffer set. Cells were analyzed using an LSRFortessa FACS machine (BD), and data were analyzed using FloJo software (TreeStar). Lymphocytes were gated based on FSC / SSC and doublets were excluded. Naive T cells were identified as CD3+CD4+foxp3+.

[0210] In the blood, a single dose of E62A-HLE (represented as Cyto6(2124-T61) in the figure) expanded CD4+foxp3+ as a percentage of hematopoietic CD45+ cells at 96 hours post-injection. In the blood, a single dose of E62A-HLE expanded CD4+foxp3+ as a percentage of TCRb+ cells at 96 hours post-injection. In the blood, a single dose of E62A-HLE expanded CD4+foxp3+ as a percentage of hematopoietic CD4+ cells at 96 hours post-injection (Figures 12A and 12B).

[0211] In lymph nodes, a single dose of E62A-HLE expanded CD4+foxp3+ as a percentage of hematopoietic CD45+ cells at 96 hours post-injection. In lymph nodes, a single dose of E62A-HLE expanded CD4+foxp3+ as a percentage of TCRb+ cells at 96 hours post-injection. In lymph nodes, a single dose of E62A-HLE expanded CD4+foxp3+ as a percentage of hematopoietic CD4+ cells at 96 hours post-injection. In lymph nodes, a single dose of E62A-HLE expanded Ki67+ Tregs at 96 hours post-injection (Figures 13A and 13B).

[0212] In the spleen, a single dose of E62A-HLE expanded CD4+foxp3+ as a percentage of hematopoietic CD45+ cells at 96 hours post-injection. In the spleen, a single dose of E62A-HLE expanded CD4+foxp3+ as a percentage of TCRb+ cells at 96 hours post-injection. In the spleen, a single dose of E62A-HLE expanded CD4+foxp3+ as a percentage of hematopoietic CD4+ cells at 96 hours post-injection. In the spleen, a single dose of E62A-HLE expanded Ki67+ Tregs at 96 hours post-injection. In the spleen, a single dose of E62A-HLE expanded CD44+CD62L-neg Tregs at 96 hours post-injection (Figures 14A and 14B).

[0213] In tumors, a single dose of E62A-HLE expanded CD4+foxp3+ as a percentage of hematopoietic CD45+ cells at 96 hours post-injection. In tumors, a single dose of E62A-HLE expanded CD4+foxp3+ as a percentage of TCRb+ cells at 96 hours post-injection. In tumors, a single dose of E62A-HLE expanded CD4+foxp3+ as a percentage of hematopoietic CD4+ cells at 96 hours post-injection (Figures 15A and 15B).

[0214] Binding assays showed that WT IL-2 bound to IL-2Rα (CD25) (Fig. 16A), but this ability was lost by the E62A IL-2 mutein (Fig. 16B). Binding assays also showed that not only WT IL-2 but also the E62A IL-2Rα mutein bound to IL-2Rβ CD122 (Fig. 17A and 17B).

[0215] The results of the binding assay showed that not only WT IL-2 but also the E62A IL-2 mutant fusion protein bound to hCTLA-4 (FIGS. 18A and 18B).

[0216] Example 10. Effect of M23L and T111H in cynomolgus monkeys after multiple administrations This example demonstrates the in vivo efficacy of exemplary M23L and T111H muteins compared to WT hIL-2 after multiple administrations in monkeys.

[0217] To understand the biological activity of M23L, T111H, and WT hIL-2, monkeys were treated with these cytokines. CD8 and CD4 T cells, including Tregs, were examined for changes in the development of immune cell subsets. Each biologic was diluted in 1x PBS. For each biologic, 800 pmol / kg of cytokine was administered subcutaneously to monkeys (n=6 per group) on days 0, 2, 4, 7, 9, and 11 (Figure 19A).

[0218] Four monkeys per group received multiple treatments, and blood samples were collected on days 0, 1, 4, 7, 8, 11, and 14. Blood cells were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD25, anti-CD45RA, anti-CD56, anti-CD16, and anti-Foxp3, all according to the manufacturer's instructions. Lymphocytes were washed and incubated with antibodies in staining buffer, and intracellular staining was performed using the foxp3 staining buffer set. Cells were analyzed on an LSRFortessa FACS machine, and data were analyzed using FloJo software. Lymphocytes were gated based on FSC / SSC to exclude doublets. Lymphocytes were gated to identify CD4 T cells, memory CD4, naive CD4, CD4 Tregs, memory Tregs, naive Tregs, CD8 T, NK, and NKT cells.

[0219] The results showed that treatment of monkeys with WT hIL-2 increased the number of memory CD4 cells and CD4 Treg cells. Treatment of monkeys with the M23L mutein, designated mutant #19 in the graph, increased the number of CD4 Treg cells, but not the number of memory CD4 cells (Figure 19B). Treatment of monkeys with T111H, designated mutant #57 in the graph, increased the number of memory CD4 cells, but not the number of CD4 Treg cells (Figure 19C).

[0220] Example 11. Effect of M23L-HLE on autoimmune hyperglycemia in mice This example demonstrates that administration of M23L-HLE results in increased protection of treated mice from autoimmune hyperglycemia, an exemplary autoimmune pathology.

[0221] At 7 weeks of age, mice were assigned to treatment groups based on non-fasting blood glucose levels and administered 100 μL / mouse by intraperitoneal (IP) injection according to Table 7. Body weight, clinical findings, and non-fasting blood glucose levels were recorded weekly throughout the study. All animals with non-fasting blood glucose levels above 250 mg / dL for two consecutive days were humanely euthanized.

[0222] [Table 7]

[0223] Blood glucose levels were measured (Fig. 20A). Results showed that, in contrast to 75% of PBS-treated mice (n = 16), none of the E62A-HLE-treated mice (n = 16) developed hyperglycemia by 20 weeks of age, and only 30% of the M23L-HLE-treated mice and 18% of the IL-2-HLE-treated mice developed hyperglycemia by 20 weeks of age, suggesting that M23L-HLE protects mice from autoimmune hyperglycemia (Fig. 20B).

[0224] [Equivalent] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as defined in the following claims.

Claims

1. A human interleukin-2 (IL-2) mutein having one or two amino acid substitutions in the amino acid sequence set forth in SEQ ID NO: 1, (1) an IL-2 mutein having any one amino acid substitution selected from the group consisting of T111H, T37Y, E15T, M23L, P34F, E68F, and E62A; or (2) An IL-2 mutein having (i) any one amino acid substitution selected from the group consisting of T111H, T37Y, E15T, M23L, P34F, E68F, and E62A, and (ii) C125A.

2. A nucleotide encoding the amino acid sequence of the human interleukin-2 (IL-2) mutein of claim 1.

3. A pharmaceutical comprising the human interleukin-2 (IL-2) mutein or a salt thereof according to claim 1.

4. The pharmaceutical agent according to claim 3, which is a Treg activator.

5. The pharmaceutical composition according to claim 3, which is an agent for preventing or treating an autoimmune disease.

6. 10. The human interleukin-2 (IL-2) mutein of claim 1 for use in a method for treating an autoimmune disease.

7. Use of the human interleukin-2 (IL-2) mutein or a salt thereof according to claim 1 for producing an agent for preventing or treating an autoimmune disease.

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