Interleukin-2 muteins for expansion of regulatory t cells

Abstract

To provide IL-2 that selectively expands and activates regulatory T cells and is amenable to large scale production with optimum properties for human treatment.SOLUTION: The invention relates to an IL-2 mutein with reduced possibility to elicit immune response, and an IL-2 mutein-Fc fusion protein, where the Fc is a variant human IgG1 Fc molecule having no or very low effector function, and being highly stable though aglycosylated at N297. The invention also provides an IL-2 mutein comprising a linker peptide which linker peptide is glycosylated when expressed in a mammalian cell, as well as an IL-2 mutein-Fc fusion protein.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 61 / 784,669, filed March 14, 2013, which is incorporated herein by reference. Sequence Listing Reference

[0002] This application is filed in electronic format via EFS-Web with a Sequence Listing, which is provided as a text file entitled A-1826-WO-PCT_ST25.txt, created on February 25, 2014, and is 40,849 bytes in size. The information contained in the electronic Sequence Listing is incorporated herein by reference in its entirety. [Background technology]

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

[0004] T cells require expression of CD25 to respond to low concentrations of IL-2, which are typically present in tissues. T cells expressing CD25 include both FOXP3+ inhibitory T cells (Treg cells), which are essential for suppressing autoimmune inflammation, and FOXP3- T cells that are activated to express CD25. FOXP3-CD25+ T effector cells (Teff) can be either CD4+ or CD8+ cells, both of which can contribute to inflammation, autoimmunity, organ transplant rejection, or graft-versus-host disease. STAT5 signaling stimulated by IL-2 is crucial for normal T-reg cell growth and survival, as well as high FOXP3 expression.

[0005] In co-filed application WO2010 / 085495, we describe the use of IL-2 muteins to selectively expand or stimulate Treg cells. When administered to a subject, the effect on Treg cells is useful for treating inflammatory and autoimmune diseases. Although the IL-2 muteins described herein are useful for expanding Treg cells over Teff cells in vivo, it was desirable to generate IL-2 muteins with optimal attributes for human therapy. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2010 / 085495 Summary of the Invention

[0007] Described herein are IL-2 muteins that are amenable to high-yield manufacturability and have optimal pharmacological activity. In efforts to generate exemplary IL-2 mutein-based human therapeutics, numerous unexpected and unpredictable observations were made. The IL-2 muteins described herein are the result of those efforts.

[0008] The IL-2 muteins described herein have minimal alterations to IL-2, thereby reducing the likelihood of eliciting an immune response against the IL-2 mutein and / or endogenous IL-2, while still maintaining the selective proliferation and activation of Tregs. Additionally, in certain embodiments, the IL-2 mutein is fused to a molecule, e.g., an antibody Fc, that increases serum half-life upon administration to a subject. IL-2 muteins have a short serum half-life (3-5 hours for subcutaneous injection). Exemplary IL-2 mutein Fc fusions described herein have a half-life in humans of at least 1 day, at least 3 days, at least 5 days, at least 10 days, at least 15 days, at least 20 days, or at least 25 days. This effect on the pharmacokinetics of the IL-2 mutein allows for less or less frequent dosing of the IL-2 mutein therapeutic.

[0009] Additionally, when creating large pharmaceutical molecules, one must consider the ability to produce large quantities of the large molecule while minimizing molecular aggregation and maximizing molecular stability. The IL-2 mutein Fc fusion molecules exhibit such attributes.

[0010] Furthermore, in certain embodiments, the IL-2 mutein Fc fusion protein contains an IgG1 Fc region. When it is desired to abolish the effector functions of IgG1 (e.g., ADCC activity), it has been found that mutation of asparagine at position 297 to glycine (N297G; EU numbering scheme) provides significantly improved purification efficiency and physical properties compared to other mutations resulting in aglycosylated IgG1 Fc. In a preferred embodiment, a cysteine ​​is introduced into the Fc to allow disulfide bonds and thereby increase the stability of the aglycosylated Fc-containing molecule. The utility of the aglycosylated Fc goes beyond IL-2 mutein Fc fusions. Thus, provided herein are Fc-containing molecules, Fc fusions, and antibodies that include an N297G substitution and, optionally, the substitution of one or more additional residues for the cysteine.

[0011] Another embodiment of the invention includes a glycosylated peptide linker. Preferred linker peptides capable of N-glycosylation include GGNGT (SEQ ID NO: 6) or YGNGT (SEQ ID NO: 7).

[0012] In another aspect, the present invention provides a human interleukin-2 (IL-2) mutein comprising a V91K substitution and 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 selectively stimulates regulatory T cells. In one embodiment, the human IL-2 mutein comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1. In another embodiment, the mutein comprises the amino acid sequence set forth in SEQ ID NO: 1. In another embodiment, position 125 is an alanine. In another embodiment, position 125 is a cysteine.

[0013] In another embodiment, the invention provides an Fc fusion protein comprising an Fc and a human IL-2 mutein. In one embodiment, the Fc is a human IgG1 Fc. In another embodiment, the human IgG1 Fc comprises one or more mutations that alter the effector function of the Fc. In another embodiment, the human IgG1 comprises a substitution at N297. In another embodiment, the substitution at N297 is N297G. In another embodiment, the Fc fusion protein comprises a substitution or deletion of the C-terminal lysine of the human IgG Fc. In another embodiment, the C-terminal lysine of the human IgG Fc is deleted. In another embodiment, a linker connects the Fc and human IL-2 mutein portions of the protein. In another embodiment, the linker is GGGGS, GGNGT, or YGNGT. In another embodiment, the linker is GGGGS. In another embodiment, the IL-2 mutein further comprises an amino acid addition, substitution, or deletion that alters the glycosylation of the Fc fusion protein when expressed in a mammalian cell. In another embodiment, the IL-2 mutein comprises a T3 substitution. In another embodiment, the IL-2 mutein comprises a T3N or T3A substitution. In another embodiment, the IL-2 mutein comprises a T3N substitution. In another embodiment, the IL-2 mutein further comprises an S5 mutation. In another embodiment, the IL-2 mutein further comprises an S5T mutation. In another embodiment, the Fc fusion protein comprises an Fc dimer. In another embodiment, the Fc fusion protein comprises two IL-2 muteins. In another embodiment, the Fc fusion protein comprises a single IL-2 mutein.

[0014] In another aspect, the invention provides an isolated nucleic acid encoding a human IL-2 mutein or an Fc portion of an antibody and a human IL-2 mutein. In one embodiment, the Fc portion of the antibody and the human IL-2 mutein are encoded in a single open reading frame. In another embodiment, the Fc is a human IgG1 Fc. In another embodiment, the human IgG1 Fc comprises one or more mutations that alter the effector function of the Fc. In another embodiment, the human IgG1 comprises a substitution at N297. In another embodiment, the substitution at N297 is N297G. In another embodiment, the human IgG1 Fc comprises a substitution or deletion of the C-terminal lysine. In another embodiment, the C-terminal lysine of the human IgG Fc is deleted. In another embodiment, the nucleic acid further encodes a linker connecting the Fc portion of the antibody and the human IL-2 mutein. In another embodiment, the linker is GGGGS, GGNGT, or YGNGT. In another embodiment, the linker is GGGGS. In another embodiment, the IL-2 mutein further comprises an amino acid addition, substitution, or deletion that alters the glycosylation of the portion comprising the IL-2 mutein when expressed in a mammalian cell. In another embodiment, the IL-2 mutein comprises a T3 substitution. In another embodiment, the IL-2 mutein comprises a T3N or T3A substitution. In another embodiment, the IL-2 mutein comprises a T3N substitution. In another embodiment, the IL-2 mutein further comprises an S5 mutation. In another embodiment, the IL-2 mutein further comprises an S5T mutation.

[0015] In another aspect, the invention provides an expression vector comprising the aforementioned isolated nucleic acid operably linked to a promoter.

[0016] In another aspect, the present invention provides a host cell comprising the isolated nucleic acid described above. In one embodiment, the isolated nucleic acid is operably linked to a promoter. In another embodiment, the host cell is a prokaryotic cell. In another embodiment, the host cell is E. coli. In another embodiment, the host cell is a eukaryotic cell. In another embodiment, the host cell is a mammalian cell. In another embodiment, the host cell is a Chinese Hamster Ovary (CHO) cell line.

[0017] In another aspect, the invention provides a method for producing a human IL-2 mutein comprising culturing said host cell under conditions in which the promoter is expressed and recovering the human IL-2 mutein from said culture. In one embodiment, the method comprises culturing said host cell under conditions in which the promoter is expressed and recovering the Fc fusion protein from said culture.

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

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

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

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

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

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

[0024] In another aspect, the present invention provides a method of treating a patient suffering from an inflammatory or autoimmune disease, the method comprising administering to the subject a therapeutically effective amount of an IL-2 mutein as described above.

[0025] In another aspect, the present invention provides a method for treating a patient suffering from inflammatory disease or autoimmune disease, comprising administering to the subject a therapeutically effective amount of the above-mentioned Fc fusion protein. In one embodiment, the administration results in the alleviation of at least one symptom of the disease. In another embodiment, the ratio of regulatory T cells (Treg) to non-regulatory T cells in the peripheral blood of the subject increases after administration. In another embodiment, the ratio of regulatory T cells (Treg) to non-regulatory T cells in the peripheral blood of the subject remains essentially the same after administration. In another embodiment, the inflammatory disease or autoimmune disease is lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type I diabetes, multiple sclerosis, spontaneous abortion, atopic disease, or inflammatory bowel disease.

[0026] In another aspect, the present invention provides an Fc region of a human IgG1 antibody, the Fc region comprising an N297G mutation, the Fc region of the human IgG1 having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 3. In one embodiment, the Fc region of the human IgG1 has at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 3. In another embodiment, the Fc region of the human IgG1 comprises the amino acid sequence set forth in SEQ ID NO: 3. In another embodiment, the Fc region of the human IgG1 further comprises one or more mutations to stabilize the polypeptide. In another embodiment, one or more amino acids set forth in SEQ ID NO: 3 are substituted with cysteine. In another embodiment, V259, A287, R292, V302, L306, V323, or I332 of the amino acid sequence set forth in SEQ ID NO: 3 are substituted with cysteine. In another embodiment, the Fc region comprises A287C and L306C substitutions in the amino acid sequence set forth in SEQ ID NO: 3. In another embodiment, the Fc region comprises a V259C and a L306C substitution in the amino acid sequence set forth in SEQ ID NO: 3. In another embodiment, the Fc region comprises a R292C and a V302C substitution in the amino acid sequence set forth in SEQ ID NO: 3. In another embodiment, the Fc region comprises a V323C and a I332C substitution in the amino acid sequence set forth in SEQ ID NO: 3.

[0027] In another embodiment, the present invention provides an antibody comprising the above-described Fc region.

[0028] In another embodiment, the present invention provides an Fc fusion protein comprising an Fc region as described above.

[0029] In another aspect, the present invention provides a polypeptide comprising a linker, wherein the linker is GGNGT or YGNGT. In one embodiment, the linker comprises N-glycosylation. In another embodiment, the linker is inserted into or replaces a loop in the polypeptide structure.

[0030] In another embodiment, the invention provides a method of making an aglycosylated IgG1 Fc-containing molecule, the method comprising expressing a nucleic acid encoding an aforementioned polypeptide in a mammalian cell culture and recovering the aglycosylated IgG1 Fc-containing molecule from the culture.

[0031] In another aspect, the invention provides a method of making an IgG1 Fc-containing molecule that is aglycosylated upon expression in a mammalian cell, the method comprising the step of mutating the codon for N297 in the Fc region to a glycine codon.

[0032] In another aspect, the present invention provides an Fc fusion protein consisting of the sequence of SEQ ID NO: 18 or SEQ ID NO: 20. In one embodiment, the present invention provides a nucleic acid encoding the Fc fusion. In another embodiment, the present invention provides a cell comprising the nucleic acid. In another embodiment, the present invention provides a method of making an Fc fusion protein, comprising incubating a cell under conditions that allow the cell to express the Fc fusion protein. In another embodiment, the present invention provides a method of treating an inflammatory or autoimmune condition in a subject, comprising administering to the subject an effective amount of an Fc fusion protein. In another embodiment, the inflammatory or autoimmune condition is graft-versus-host disease.

[0033] In another aspect, the present invention provides a method for monitoring a subject's response to treatment with the human interleukin-2 (IL-2) mutein or the Fc fusion protein, comprising detecting a change in the subject, the change being an increase in body temperature, an increase in CRP in the peripheral blood of the subject, a decrease in platelets in the peripheral blood of the subject, a decrease in neutrophils in the peripheral blood of the subject, or a decrease in albumin in the peripheral blood of the subject, and after the change is detected, the treatment is stopped, suspended, reduced in frequency, or reduced in dosage.In one embodiment, the change comprises an increase in body temperature of at least 0.5°C, an increase in CRP in the peripheral blood of the subject of at least 0.2 mg / mL, a decrease in platelets in the peripheral blood of the subject of at least 0.8-fold, a decrease in neutrophils in the peripheral blood of the subject of at least 0.8-fold, or a decrease in albumin in the peripheral blood of the subject of at least 0.4-fold. [Brief description of the drawings]

[0034] [Figure 1] In short-term stimulation assays, homodimerization by fusion to the C-terminus of IgG-Fc does not alter the activity of an IL-2 mutein with low potency and high affinity for CD25. [Figure 2A] IL-2 muteins with the indicated mutations fused to one C-terminus of the Fc-heterodimer were tested for their ability to stimulate STAT5 phosphorylation in T cells. These muteins also contained three mutations (V69A, N71R, Q74P) that confer high affinity to CD25. Their activity was compared to three forms of IL-2 without Fc fusion (open shapes): WT IL-2, HaWT (high affinity for CD25) (N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P), and HaD (high affinity for CD25 and low signaling activity) (N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P, N88D). Phospho-STAT5 responses of gated FOXP3+CD4+ and FOXP3-CD4+ T cells are shown. [Figure 2B]IL-2 muteins with the indicated mutations fused to one C-terminus of the Fc-heterodimer were tested for their ability to stimulate STAT5 phosphorylation in T cells. These muteins also contained three mutations (V69A, N71R, Q74P) that confer high affinity to CD25. Their activity was compared to three forms of IL-2 without Fc fusion (open shapes): WT IL-2, HaWT (high affinity for CD25) (N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P), and HaD (high affinity for CD25 and low signaling activity) (N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P, N88D). Phospho-STAT5 responses of gated FOXP3+CD4+ and FOXP3-CD4+ T cells are shown. [Diagram 3] Proliferation of T cell subsets in response to titration of IL-2 muteins fused to Fc-heterodimers. The activity of the fusion proteins was compared to three forms of IL-2 without Fc fusion (open figures): WT IL-2, HaWT (high affinity for CD25) (N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P), and HaD (high affinity for CD25 and low signaling activity) (N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P, N88D). [Figure 4] NK cell proliferation in response to titration of IL-2 muteins fused to Fc-heterodimers. The activity of the fusion proteins was compared to three forms of IL-2 without Fc fusion (open figures): WT IL-2, HaWT (high affinity for CD25) (N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P), and HaD (high affinity for CD25 and low signaling activity) (N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P, N88D). [Diagram 5]Proliferation of T cell subsets in response to titration of IL-2 muteins fused to the Fc-homodimer N297G. The activity of the Fc muteins was compared to WT IL-2 (open circles) and Fc.WT (closed circles). Mutations conferring high affinity to CD25 (HaMut1) were V69A and Q74P. [Figure 6] NK cell proliferation in response to titration of IL-2 muteins fused to Fc-homodimer N297G. The activity of Fc.muteins was compared to WT IL-2 (open circles) and Fc.WT (closed circles). [Figure 7A] An Fc.IL-2 mutein that does not contain the mutation that confers high affinity for CD25 promoted Treg expansion and upregulation of FOXP3 in humanized mice. [Figure 7B] An Fc.IL-2 mutein that does not contain the mutation that confers high affinity for CD25 promoted Treg expansion and upregulation of FOXP3 in humanized mice. [Figure 8] Low weekly doses (0.5 μg per animal) of Fc.IL-2 muteins promoted Treg expansion and FOXP3 upregulation in humanized mice, with better activity observed with Fc.V91K compared to Fc.N88D and Fc.WT. [Figure 9A] Fc.V91K and Fc.N88D remain on the surface of activated T cells via their association with CD25. [Figure 9B] Duration of IL-2R signaling with Fc.V91K and Fc.N88D compared to Fc.WT. [Figure 10A] Comparison of 2-week and 4-week dosing intervals and IV and SC administration routes of Fc.V91K in cynomolgus monkeys. [Figure 10B] Comparison of 2-week and 4-week dosing intervals and IV and SC administration routes of Fc.V91K in cynomolgus monkeys. [Figure 11A] Kinetics of cellular responses, body temperature, and serum CRP in cynomolgus monkeys treated with different dosing regimens of PROLEUKIN®, Fc.V91K, and Fc.N88D. [Figure 11B]Kinetics of cellular responses, body temperature, and serum CRP in cynomolgus monkeys treated with different dosing regimens of PROLEUKIN®, Fc.V91K, and Fc.N88D. [Figure 11C] Kinetics of cellular responses, body temperature, and serum CRP in cynomolgus monkeys treated with different dosing regimens of PROLEUKIN®, Fc.V91K, and Fc.N88D. [Figure 11D] Kinetics of cellular responses, body temperature, and serum CRP in cynomolgus monkeys treated with different dosing regimens of PROLEUKIN®, Fc.V91K, and Fc.N88D. [Figure 11E] Kinetics of cellular responses, body temperature, and serum CRP in cynomolgus monkeys treated with different dosing regimens of PROLEUKIN®, Fc.V91K, and Fc.N88D. [Figure 11F] Kinetics of cellular responses, body temperature, and serum CRP in cynomolgus monkeys treated with different dosing regimens of PROLEUKIN®, Fc.V91K, and Fc.N88D. [Figure 12A] Effect of increasing doses of PROLEUKIN®, Fc.V91K, or Fc.N88D on levels of Treg cells, NK cells, CD4+FOXP3- T cells, and CD8+FOXP3- T cells in cynomolgus monkeys. Each data point represents the average peak response of four animals. [Figure 12B] Effect of increasing doses of PROLEUKIN®, Fc.V91K, or Fc.N88D on levels of Treg cells and eosinophils in cynomolgus monkeys. Each data point represents the average peak response of four animals. [Figure 12C] Effect of increasing doses of PROLEUKIN®, Fc.V91K, or Fc.N88D on Treg cell and CRP levels, and body temperature in cynomolgus monkeys. Each data point represents the average peak response of four animals. [Figure 12D]Effect of increasing doses of PROLEUKIN®, Fc.V91K, or Fc.N88D on Treg cell, platelet, neutrophil, and albumin levels in cynomolgus monkeys. Each data point represents the average peak response of 4 animals. The right Y-axis is inverted to show the fold change in platelets, neutrophils, or albumin compared to pre-dose samples. [Figure 13] Kinetics of anti-drug antibody (ADA) development in cynomolgus monkeys treated with Fc.V91K. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All references cited within the body of this specification are expressly incorporated herein by reference in their entirety.

[0036] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, tissue culture and transformation, protein purification, and the like. Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The following procedures and techniques may generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manuel, 3 rded., Cold Spring Harbor Laboratory Press, cold Spring Harbor, NY, incorporated herein by reference for any purpose. Unless specific definitions are provided, the nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0037] IL-2 The IL-2 muteins described herein are variants of wild-type human IL-2. As used herein, "wild-type human IL-2," "wild-type IL-2," or "WT IL-2" refers to a polypeptide having the following amino acid sequence:

[0038] [ka] X is C, S, V, or A (SEQ ID NO:2).

[0039] Mutants may contain one or more substitutions, deletions, or insertions in the wild-type IL-2 amino acid sequence. Residues are represented herein by the one-letter amino acid code followed by the position of the IL-2 amino acid: for example, K35 is the lysine residue at position 35 of SEQ ID NO: 2. Substitutions are represented herein by the one-letter amino acid code followed by the position of the IL-2 amino acid followed by the replacing one-letter amino acid code: for example, K35A is the lysine residue at position 35 of SEQ ID NO: 2 with an alanine residue.

[0040] IL-2 muteins Provided herein is a human IL-2 mutein that selectively stimulates regulatory T (Treg) cells. As used herein, "selectively stimulates regulatory T cells" means that the mutein promotes the proliferation, survival, activation, and / or function of CD3+FoxP3+ T cells over CD3+FoxP3- T cells. The ability to selectively stimulate Tregs can be measured by flow cytometry of peripheral blood leukocytes, where an increase in the percentage of FOXP3+CD4+T cells among total CD4+T cells, an increase in the percentage of FOXP3+CD8+T cells among total CD8+T cells, an increase in the percentage of FOXP3+T cells relative to NK cells, and / or a greater increase in the expression level of CD25 on the surface of FOXP3+T cells relative to an increase in CD25 expression on other T cells is observed. Selective expansion of Treg cells can also be detected as increased expression of demethylated FOXP3 promoter DNA (i.e., Treg-specific demethylated region, or TSDR) relative to the demethylated CD3 gene in DNA extracted from whole blood, as detected by sequencing polymerase chain reaction (PCR) products from bisulfite-treated genomic DNA (J. Sehouli, et al. 2011. Epigenetics 6:2,236-246).

[0041] IL-2 muteins that selectively stimulate Treg cells increase the ratio of CD3+FoxP3+ T cells to CD3+FoxP3- T cells in a subject or peripheral blood sample by at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%.

[0042] Preferred IL-2 muteins include, but are not limited to, IL-2 muteins comprising a V91K or N88D substitution in the amino acid sequence set forth in SEQ ID NO:2. An exemplary IL-2 mutein is set forth in SEQ ID NO:1. Particularly preferred is the amino acid sequence set forth in SEQ ID NO:1 comprising a C125A substitution. Although it may be advantageous to reduce the number of additional mutations relative to the wild-type IL-2 sequence, the present invention includes IL-2 muteins having truncations or further insertions or substitutions in addition to the V91K or N88D substitution, provided that the muteins maintain the activity of selectively stimulating Tregs. Thus, embodiments include IL-2 muteins that selectively stimulate Treg cells and comprise an amino acid sequence having V91K or N88D that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:2. In particularly preferred embodiments, such IL-2 muteins comprise an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:2.

[0043] For amino acid sequences, sequence identity and / or sequence similarity are determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. USA 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis), the Best Fit sequence program described by Devereux et al., 1984, Nucl. Acid Res. 12:387-395, preferably using default settings, or by inspection. Preferably, percent identity is calculated by FastDB based on the following parameters: mismatch penalty = 1; gap penalty = 1; gap size penalty = 0.33; and joining penalty = 30 ("Current Methods in Sequence Comparison and Analysis" Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp127-149 (1988), Alan R. Liss, Inc.).

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

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

[0046] A further useful algorithm is Gapped BLAST, reported by Altschul et al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST uses the BLOSUM-62 substitution score; a threshold T parameter set to 9; a two-hit method for initiating ungapped extensions, imposing a gap length k of 10+k cost; and X set to 16. u , and X was set to 40 in the database search stage and 67 in the output stage of the algorithm. g The gapped alignment is started with a score corresponding to approximately 22 bits.

[0047] The site or region where the amino acid sequence mutation is introduced may be predetermined, but the mutation itself need not be predetermined. For example, to optimize the performance of a mutation at a given site, random mutagenesis can be performed at the target codon or region, and the expressed IL-2 muteins can be screened for the optimal combination of desired activity. Techniques for making substitution mutations at a given site in DNA with a known sequence are well known, such as, for example, M13 primer mutagenesis and PCR mutagenesis. Screening of mutants can be performed, for example, using the assays described herein.

[0048] Amino acid substitutions are typically of single residues; insertions are usually on the order of about 1 to about 20 amino acid residues, although considerably larger insertions can be tolerated. Deletions range from about 1 to about 20 amino acid residues, although in some cases the deletions may be much larger.

[0049] Substitution, deletion, insertion, or any combination thereof may be used to arrive at the final derivative or variant. Generally, these changes are made to a few amino acids to minimize the change in the molecule, particularly the immunogenicity and specificity of the antigen-binding protein. However, larger changes may be tolerated in certain circumstances. Conservative substitutions are generally made according to the following chart shown as Table 1.

[0050] [Table 1] Substantial alterations in function or immunological identity are made by selecting substitutions that are less conservative than those shown in Table 1. For example, substitutions may be made that more significantly affect the structure of the polypeptide backbone, e.g., alpha-helical or beta-sheet structure, the charge or hydrophobicity of the target site molecule, or the bulk of the side chains in the region of the change. The substitutions that are generally predicted to result in the most significant changes in the properties of a polypeptide are those in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted with (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine ​​or proline is substituted with (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted with (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted with (or by) a residue having no side chain, e.g., glycine.

[0051] Mutants typically exhibit the same qualitative biological activity and induce the same immune response as naturally occurring analogs, but mutants are also selected to modify the characteristics of IL-2 muteins, if necessary. Alternatively, mutants may be designed to change the biological activity of IL-2 muteins. For example, glycosylation sites may be altered or removed as described herein.

[0052] IL-2 muteins with extended serum half-lives The IL-2 muteins provided herein are expected to have a different safety profile when administered to patients than wild-type IL-2 or PROLEUKIN® (aldesleukin; Novartis, Basel, Switzerland) because they selectively expand Tregs over, for example, Teff or NK cells. Side effects associated with wild-type IL-2 or PROLEUKIN® include flu-like symptoms, chills / chills, joint pain, fever, rash, pruritus, injection site reactions, hypotension, diarrhea, nausea, anxiety, confusion, and depression. The IL-2 muteins provided herein can be altered to contain or be fused to a molecule that extends the serum half-life of the mutein without increasing the risk that such half-life extension will increase the likelihood or intensity of side effects or adverse events in the patient. Subcutaneous administration of such serum half-life extended muteins can result in lower maximum systemic exposure (C max ) may allow for long-term target coverage. The increased serum half-life may allow for fewer or less frequent dosing regimens of the mutein.

[0053] The serum half-life of the IL-2 muteins provided herein may be extended by essentially any method known in the art. Such methods include altering the sequence of the IL-2 mutein to include a peptide that binds to neonatal Fcγ receptors or to proteins with prolonged serum half-life, such as IgG or human serum albumin. In other embodiments, the IL-2 mutein is fused to a polypeptide that confers a prolonged half-life to the fusion molecule. Such polypeptides include IgG Fc or other polypeptides that bind to neonatal Fcγ receptors, human serum albumin, or polypeptides that bind to proteins with prolonged serum half-life. In a preferred embodiment, the IL-2 mutein is fused to an IgG Fc molecule.

[0054] The IL-2 mutein may be fused to the N-terminus or C-terminus of the IgG Fc region. As shown in the Examples, fusion to the C-terminus of the IgG Fc region maintains the activity of the IL-2 mutein to a greater extent than fusion to the N-terminus of the IgG Fc.

[0055] One embodiment of the present invention is directed to a dimer comprising two Fc fusion polypeptides made by fusing an IL-2 mutein to the Fc region of an antibody. The dimer can be made, 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 assembling the expressed fusion protein in a manner very similar to an antibody molecule, whereupon interchain bonds form between the Fc moieties to give rise to a dimer.

[0056] The term "Fc polypeptide" or "Fc region" as used herein includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Also included are truncated forms of such polypeptides containing the hinge region that promotes dimerization. In certain embodiments, the Fc region comprises the CH2 and CH3 domains of an antibody. Fusion proteins (and oligomers formed therefrom) that include an Fc portion offer the advantages of extended serum half-life as well as easy purification by affinity chromatography on Protein A or Protein G columns. Preferred Fc regions are derived from human IgG, including IgG1, IgG2, IgG3, and IgG4. As used herein, certain residues in Fc are identified by position. All Fc positions are based on the EU numbering scheme.

[0057] One of the functions of the Fc portion of an antibody is to signal to the immune system when the antibody binds to its target. This is considered an "effector function." Signaling can result in antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). ADCC and ADCP are mediated by binding of Fc to Fc receptors on the surface of cells of the immune system. CDC is mediated by binding of Fc to proteins of the complement system, such as C1q.

[0058] IgG subclasses differ in their ability to mediate effector functions. For example, IgG1 is much better than IgG2 and IgG4 in mediating ADCC and CDC. Therefore, in embodiments where effector functions are undesirable, IgG2 Fc is preferred. However, IgG2 Fc-containing molecules are known to be more difficult to manufacture and have less attractive biophysical properties, such as shorter half-life, compared to IgG1 Fc-containing molecules.

[0059] 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 Fcs engineered to increase effector function (U.S. Patent No. 7,317,091, and Strohl, Curr. Opin. Biotech., 20:685-691, 2009; both of which are incorporated herein by reference in their entireties). Exemplary IgG1 Fc molecules with high effector function include those with the following substitutions:

[0060] [Table 2]

[0061] Another way to increase the effector function of IgG Fc-containing proteins is by reducing the fucosylation of Fc. Removal of the core fucose from the biantennary complex oligosaccharides attached to Fc significantly increased the effector function of ADCC without altering the effector function of antigen binding or CDC. Several methods are known to reduce or abolish the fucosylation of Fc-containing molecules, such as antibodies. These include recombinant expression in certain mammalian cell lines, including FUT8 knockout cell lines, mutant CHO line Lec13, rat hybridoma cell line YB2 / 0, cell lines containing small interfering RNA specifically targeting the FUT8 gene, and cell lines co-expressing β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II. Alternatively, Fc-containing molecules may be expressed in non-mammalian cells, such as plant cells, yeast, or prokaryotic cells, e.g., E. coli.

[0062] In a preferred embodiment of the invention, the IL-2 mutein Fc fusion protein comprises an Fc engineered to reduce effector function. Exemplary Fc molecules with reduced effector function include molecules with the following substitutions:

[0063] [Table 3]

[0064] It is known that human IgG1 has a glycosylation site at N297 (EU numbering system) and that glycosylation contributes to the effector function of IgG1 antibodies. An exemplary IgG1 sequence is provided in SEQ ID NO: 3. The group mutated N297 in an effort to generate aglycosylated antibodies. The mutations focused on replacing N297 with amino acids similar to asparagine in physiochemical properties, such as glutamine (N297Q), or with alanine (N297A), which mimics asparagine without the polar group.

[0065] As used herein, "non-glycosylated antibody" or "non-glycosylated fc" refers to the glycosylation state of the residue at Fc position 297. An antibody or other molecule may contain glycosylation at one or more other locations and still be considered to be an non-glycosylated antibody or a non-glycosylated Fc fusion protein.

[0066] In an effort to generate an IgG1 Fc without effector function, it was discovered that mutation of amino acid N297 of human IgG1 to glycine, i.e., N297G, provides much better purification efficiency and physical properties than other amino acid substitutions at that residue. See Example 8. Thus, in a preferred embodiment, the IL-2 mutein Fc fusion protein comprises a human IgG1 Fc with an N297G substitution. An Fc with an N297G substitution is useful in any context in which the molecule comprises human IgG1 Fc, and is not limited to use in conjunction with IL-2 mutein Fc fusions. In a particular embodiment, the antibody comprises an Fc with an N297G substitution.

[0067] Fc also includes insertions, deletions, and substitutions, including human IgG1 Fc with N297G mutation. In a specific embodiment, human IgG1 Fc includes N297G substitution and is at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 3. In a particularly preferred embodiment, the C-terminal lysine residue is substituted or deleted. The amino acid sequence of human IgG1 including N297G substitution and deletion of C-terminal lysine is set forth in SEQ ID NO: 4.

[0068] It has been shown that glycosylated IgG1 Fc-containing molecules are less stable than glycosylated IgG1 Fc-containing molecules. The Fc region may be further engineered to increase the stability of the non-glycosylated molecule. In some embodiments, one or more amino acids are substituted with cysteine ​​to form disulfide bonds in the dimeric state. Residues V259, A287, R292, V302, L306, V323, or I332 of the amino acid sequence set forth in SEQ ID NO: 3 may be substituted with cysteine. In preferred embodiments, specific pairs of residues are substituted such that they selectively form disulfide bonds with each other, thus limiting or preventing recombination of disulfide bonds. Preferred pairs include, but are not limited to, A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C.

[0069] Provided herein are Fc-containing molecules in which one or more of residues V259, A287, R292, V302, L306, V323, or I332 are substituted with cysteine. Preferred Fc-containing molecules include molecules that contain A287C and L306C, V259C and L306C, R292C and V302C, or V323C and I332C substitutions.

[0070] Further mutations that may be made to IgG1 Fc include mutations that promote heterodimer formation among Fc-containing polypeptides. In some embodiments, the Fc region is engineered to form "knobs" and "holes" that promote heterodimer formation of two different Fc-containing polypeptide chains when co-expressed in a cell. U.S. Patent No. 7,695,963. In other embodiments, the Fc region is altered to use electrostatic steering to prevent homodimer formation of two different Fc-containing polypeptides while promoting heterodimer formation when co-expressed in a cell. WO09 / 089,004, which is incorporated herein by reference in its entirety. A preferred heterodimeric Fc is one in which one chain of the Fc contains D399K and E356K substitutions and the other chain of the Fc contains K409D and K392D substitutions. In another embodiment, one chain of the Fc comprises D399K, E356K, and E357K substitutions, and the other chain of the Fc comprises K409D, K392D, and K370D substitutions.

[0071] In certain embodiments, it may be advantageous for the IL-2 mutein Fc fusion protein to be monomeric, i.e., to contain only a single IL-2 mutein molecule. In such embodiments, the Fc region of the fusion protein may contain one or more mutations that promote the formation of a heterodimer. The fusion protein is co-expressed with an Fc region that has a reciprocal mutation in the Fc region in the IL-2 mutein Fc fusion polypeptide, but lacks the IL-2 mutein. The resulting protein, a heterodimer of two Fc-containing polypeptides, contains only a single IL-2 mutein.

[0072] Another way to make a monomeric IL-2 mutein Fc fusion protein is to fuse the IL-2 mutein to a monomeric Fc, i.e., an Fc region that does not dimerize. A stable monomeric Fc contains mutations that prevent dimerization and stabilize the monomeric form of the molecule. A preferred monomeric Fc is published in WO2011 / 063348, which is incorporated herein by reference in its entirety. In a specific embodiment, the IL-2 mutein Fc fusion protein comprises an Fc that contains negatively charged amino acids at positions 392 and 409, together with threonine substitutions at Y349, L351, L368, V397, L398, F405, or Y407.

[0073] In certain embodiments, the IL-2 mutein Fc fusion protein comprises a linker between the Fc and the IL-2 mutein. Many different linker polypeptides are known in the art and can be used in conjunction with the IL-2 mutein Fc fusion protein. In a preferred embodiment, the IL-2 mutein Fc fusion protein comprises one or more copies of a peptide consisting of GGGGS (SEQ ID NO:5), GGNGT (SEQ ID NO:6), or YGNGT (SEQ ID NO:7) between the Fc and the IL-2 mutein. In some embodiments, the polypeptide region between the Fc region and the IL-2 mutein region comprises a single copy of GGGGS (SEQ ID NO:5), GGNGT (SEQ ID NO:6), or YGNGT (SEQ ID NO:7). As shown herein, the linker GGNGT (SEQ ID NO:6) or YGNGT (SEQ ID NO:7) is glycosylated when expressed in an appropriate cell, and such glycosylation serves to stabilize the protein when administered in solution and / or in vivo. Thus, in certain embodiments, the IL-2 mutein fusion protein comprises a glycosylated linker between the Fc region and the IL-2 mutein region.

[0074] It is contemplated that glycosylation linkers may be useful when placed in relation to a polypeptide. Provided herein is a polypeptide comprising GGNGT (SEQ ID NO:6) or YGNGT (SEQ ID NO:7) inserted into or replacing one or more amino acids in the amino acid sequence of a polypeptide. In a preferred embodiment, GGNGT (SEQ ID NO:6) or YGNGT (SEQ ID NO:7) is inserted into a loop of the polypeptide tertiary structure. In another embodiment, one or more amino acids of the loop are replaced with GGNGT (SEQ ID NO:6) or YGNGT (SEQ ID NO:7).

[0075] The C-terminal portion of the Fc and / or the amino-terminal portion of the IL-2 mutein may contain one or more mutations that alter the glycosylation profile of the IL-2 mutein Fc fusion protein when expressed in a mammalian cell. In certain embodiments, the IL-2 mutein further comprises a T3 substitution, e.g., T3N or T3A. The IL-2 mutein may further comprise an S5 substitution, such as S5T.

[0076] Covalent modifications of IL-2 muteins and IL-2 mutein Fc fusion proteins are included within the scope of the present invention and are typically, but not necessarily, performed post-translationally. For example, some types of covalent modifications of IL-2 muteins or IL-2 mutein Fc fusion proteins are introduced into the molecule by reacting specific amino acid residues of the IL-2 mutein or IL-2 mutein Fc fusion protein with organic derivatizing agents that are capable of reacting with selected side chains or N- or C-terminal residues.

[0077] Cysteinyl residues most commonly are reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuric benzoate, 2-chloromercuric-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0078] Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

[0079] Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydrides; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pendanedione; and transaminase-catalyzed reactions with glyoxylic acid.

[0080] Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. a Because of the high pH, ​​derivatization of arginine residues requires that the reaction be carried out in alkaline conditions. Furthermore, these reagents can react with lysine groups as well as the arginine ε-amino group.

[0081] The specific modification of tyrosyl residues, with particular interest in introducing spectral labels into tyrosyl residues, can be made by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. To prepare labeled proteins suitable for use in the chloramine T method, a preferred radioimmunoassay method described above, 125 I or 131 Tyrosyl residues are iodized using I.

[0082] Carboxyl side groups (aspartyl or glutamine) are selectively modified by reaction with carbodiimides (R'-N=C=N--R'), where R and R' are optionally different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl and glutamine residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0083] Derivatization with bifunctional agents is useful for crosslinking antigen-binding proteins to water-insoluble support matrices or surfaces for use in a variety of methods. Commonly used crosslinking agents include bifunctional maleimides such as 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, such as esters with 4-azidosalicylic acid, homobifunctional imidoesters (including disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl propionate)), and bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate result in photoactivatable intermediates capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and reactive substrates described in U.S. Pat. Nos. 3,969,287, 3,691,016, 4,195,128, 4,247,642, 4,229,537, and 4,330,440 are used for protein immobilization.

[0084] Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues is within the scope of the invention.

[0085] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, pp. 79-86), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0086] Another type of covalent modification of an IL-2 mutein or IL-2 mutein Fc fusion protein that falls within the scope of the present invention involves altering the glycosylation pattern of the protein. As is known in the art, the glycosylation pattern can depend both on the sequence of the protein (e.g., the presence or absence of specific glycosylated amino acid residues, as described below), or on the host cell or organism in which the protein is produced. Specific expression systems are described below.

[0087] Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0088] Addition of glycosylation sites to an IL-2 mutein or IL-2 mutein Fc fusion protein can be conveniently accomplished by altering the amino acid sequence to contain one or more of the tripeptide sequences described above (for N-linked glycosylation sites). This alteration can also be accomplished by adding or substituting one or more serine or threonine residues to the start sequence (for O-linked glycosylation sites). For ease of use, the amino acid sequence of an IL-2 mutein or IL-2 mutein Fc fusion protein is altered preferably through alterations at the DNA level, specifically by mutating the DNA encoding the target polypeptide at preselected bases to generate codons that translate into the desired amino acids.

[0089] Another means of increasing the number of carbohydrate moieties on an IL-2 mutein or IL-2 mutein Fc fusion protein is by chemical or enzymatic coupling of glycosides to the protein. These procedures are advantageous in that they do not require production of the protein in a host cell with glycosylation capabilities for N- and O-linked glycosylation. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 87 / 05330 published Sep. 11, 1987, and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306.

[0090] Removal of carbohydrate moieties present on the starting IL-2 mutein or IL-2 mutein Fc fusion protein can be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine) while leaving the polypeptide intact. Chemical deglycosylation has been described by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52, and by Edge et al., 1981, Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties on the polypeptide can be achieved by the use of various endo- and exoglucosidases, as described by Thotakura et al., 1987, Meth. Enzymol. 138:350. Glycosylation at potential glycosylation sites can be prevented by the use of the compound tunicamycin, as described by Duskin et al., 1982, J. Biol. Chem. 257:3105. Tunicamycin prevents the formation of protein-N-glycosidic bonds.

[0091] Another type of covalent modification of an IL-2 mutein or IL-2 mutein Fc fusion protein involves coupling the IL-2 mutein or IL-2 mutein Fc fusion protein to various nonproteinaceous polymers, including, but not limited to, various polyols, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner described in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337. Additionally, amino acid substitutions may be made at various positions in the IL-2 mutein or IL-2 mutein Fc fusion protein to facilitate the addition of polymers such as PEG. Thus, embodiments of the present invention include PEGylated IL-2 muteins and IL-2 mutein Fc fusion proteins. Such PEGylated proteins may have a longer half-life and / or less immunogenicity than non-PEGylated proteins.

[0092] Polynucleotides encoding IL-2 muteins and IL-2 mutein Fc fusion proteins Nucleic acids encoding IL-2 muteins and IL-2 mutein-Fc fusion proteins are encompassed herein. Aspects of the invention include polynucleotide variants (e.g., due to degeneracy) that encode the amino acid sequences described herein. In a preferred embodiment, the polypeptide encoded by the isolated nucleic acid is a component of an IL-2 mutein-Fc fusion protein.

[0093] Nucleotide sequences corresponding to the amino acid sequences described herein, used as probes or primers for the isolation of nucleic acids or as query sequences for database searches, can be obtained by "reverse translation" from the amino acid sequences. Well-known polymerase chain reaction (PCR) procedures can be used to isolate and amplify DNA sequences encoding IL-2 muteins and IL-2 mutein-Fc fusion proteins. Oligonucleotides that define the desired termini of the combination of DNA fragments are used as 5' and 3' primers. The oligonucleotides can additionally contain recognition sites for restriction endonucleases to facilitate insertion of the amplified combination of DNA fragments into an expression vector. PCR techniques are described in Saiki et al., Science 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds., Academic Press, Inc., San Diego (1989), pp. 189-196; and PCR Protocols: A Guide to Methods and Applications, Innis et.al., eds., Academic Press, Inc. (1990).

[0094] The nucleic acid molecules of the present invention include DNA and RNA in both single-stranded and double-stranded form, as well as corresponding complementary sequences. "Isolated nucleic acid" refers to a nucleic acid that is isolated from adjacent gene sequences present in the genome of the organism from which the nucleic acid is isolated, in the case of a nucleic acid isolated from a naturally occurring source. In the case of a nucleic acid that is enzymatically or chemically synthesized from a template, such as a PCR product, a cDNA molecule, or an oligonucleotide, it is understood that the nucleic acid obtained from such a process is an isolated nucleic acid. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. In a preferred embodiment, the nucleic acid is substantially free of contaminating endogenous material. The nucleic acid molecule is preferably derived from isolated DNA or RNA at least once, in substantially pure form and in an amount or concentration that permits identification, manipulation, and recovery of its component nucleotide sequences by standard biochemical methods (such as those reviewed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). Such sequences are preferably provided and / or constructed in the form of an open reading frame, uninterrupted by internal untranslated sequences, or introns, that are typically present in eukaryotic genes. Sequences of untranslated DNA may be present 5' or 3' from the open reading frame, which do not interfere with manipulation or expression of the coding region.

[0095] The variants according to the invention are usually prepared by site-directed mutagenesis of nucleotides in the DNA encoding the IL-2 mutein or IL-2 mutein Fc fusion protein using cassette or PCR mutagenesis or other techniques well known in the art to produce DNA encoding the variant, followed by expression of the recombinant DNA in cell culture as outlined herein. However, IL-2 muteins and IL-2 mutein Fc fusions may also be prepared by in vitro synthesis using established techniques. The variants typically exhibit the same qualitative biological activity, e.g., proliferation of Tregs, as the naturally occurring analogues, although variants may be selected that have altered characteristics, as outlined in more detail below.

[0096] As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, a vast number of nucleic acids can be generated, all of which encode the IL-2 muteins and IL-2 mutein Fc fusion proteins of the present invention. Thus, upon identifying a particular amino acid sequence, one skilled in the art can generate any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the encoded protein.

[0097] The present invention also provides expression systems and constructs in the form of plasmids, expression vectors, transcription or expression cassettes comprising at least one of the polynucleotides described above. Additionally, the present invention provides host cells comprising such expression systems or constructs.

[0098] Typically, expression vectors used in any of the host cells contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to in certain embodiments as "flanking sequences", typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splicing sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element. Each of these sequences is described below.

[0099] Optionally, the vector may contain a "tag" coding sequence, i.e., an oligonucleotide molecule located at the 5' or 3' end of the IL-2 mutein or IL-2 mutein Fc fusion protein coding sequence, which encodes polyHis (such as hexaHis (SEQ ID NO: 21)) for which there are commercially available antibodies, or another "tag", e.g., FLAG, HA (influenza virus hemagglutinin), or myc. This tag is typically fused to the polypeptide upon expression and can serve as a means for affinity purification or detection of the IL-2 mutein from the host cell. Affinity purification may be achieved, for example, by column chromatography using antibodies against the tag as an affinity matrix. Optionally, the tag can be subsequently removed from the purified IL-2 mutein and IL-2 mutein Fc fusion protein by various means, such as using specific peptidases for cleavage.

[0100] Flanking sequences may be homologous (i.e., derived from the same species and / or strain as the host cell), heterologous (i.e., derived from a species other than the species or strain of the host cell), hybrid (i.e., a combination of flanking sequences from more than one source), synthetic, or natural. Thus, the source of the flanking sequences may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequences are functional in and capable of being activated by the host cell's machinery.

[0101] Flanking sequences useful in the vector of the present invention may be obtained by any of several methods well known in the art. Typically, flanking sequences useful herein have been previously identified by mapping and / or by restriction endonuclease digestion, and can therefore be isolated from suitable tissue sources using appropriate restriction endonucleases. In some cases, the full-length nucleotide sequence of the flanking sequence may be known. Flanking sequences herein may be synthesized using the methods described herein for nucleic acid synthesis or cloning.

[0102] Whether all or only a portion of the flanking sequence is known, the flanking sequence can be obtained using polymerase chain reaction (PCR) and / or by screening a genomic library with appropriate probes, such as oligonucleotides and / or flanking sequence fragments from the same or another species. If the flanking sequence is unknown, a fragment of DNA containing the flanking sequence may be isolated from a larger piece of DNA that may contain, for example, a coding sequence or even another gene(s). Isolation may be accomplished by digestion with a restriction endonuclease to generate the appropriate DNA fragment, followed by isolation using agarose gel purification, Qiagen® column chromatography (Chatsworth, Calif.), or other methods known to those of skill in the art. The selection of suitable enzymes to accomplish this purpose will be readily apparent to those of skill in the art.

[0103] Replication origin is typically part of commercially purchased prokaryotic expression vectors, and the origin is useful for amplifying the vector in host cells. If the vector selected does not contain a replication origin site, it may be chemically synthesized based on a known sequence and ligated into the vector. For example, the replication origin from plasmid pBR322 (New England Biolabs, Beverly, MA) is suitable for most Gram-negative bacteria, and various viral origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitis virus (VSV), or papillomavirus such as HPV or BPV) are useful for cloning vectors in mammalian cells. Generally, the replication origin component is not required for mammalian expression vectors (e.g., the SV40 origin is often used simply because it also contains the viral early promoter).

[0104] Transcription termination sequence is typically located at the 3' end of the polypeptide coding region and serves to terminate transcription.Usually, the transcription termination sequence of prokaryotic cells is a GC-rich fragment followed by a poly-T sequence.This sequence can be easily cloned from a library or even purchased commercially as part of a vector, but can also be easily synthesized using methods for nucleic acid synthesis such as those described herein.

[0105] A selectable marker gene encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium. Typical selectable marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, tetracycline, or kanamycin in the case of prokaryotic host cells, (b) complement auxotrophic deficiencies of the cells, or (c) supply critical nutrients unavailable from complex or defined media. Particular selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. Advantageously, a neomycin resistance gene can also be used for selection in both prokaryotic and eukaryotic host cells.

[0106] Other selectable genes may be used to amplify the gene to be expressed. Amplification is the process in which genes required for the production of a protein critical for proliferation or cell survival are repeated in tandem in the chromosome of successive generations of recombinant T cells. Examples of selectable markers suitable for mammalian cells include dihydrofolate reductase (DHFR) and promoterless thymidine kinase genes. Mammalian cell transformants are placed under selection pressure that is uniquely adapted so that only the transformants survive due to the selectable gene present in the vector. Selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of the selection agent in the medium is successively increased, thereby causing amplification of both the selectable gene and another gene, such as DNA encoding an IL-2 mutein or an IL-2 mutein Fc fusion protein. As a result, an increased amount of a polypeptide, such as an IL-2 mutein or an IL-2 mutein Fc fusion protein, is synthesized from the amplified DNA.

[0107] A ribosome binding site is usually necessary for translation initiation of mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is typically located 3' of the promoter and 5' of the coding sequence of the polypeptide to be expressed. In certain embodiments, one or more coding regions may be operably linked to an internal ribosome binding site (IRES), allowing translation of two open reading frames from a single RNA transcript.

[0108] In some cases, for example, when glycosylation in a eukaryotic host cell expression system is desired, various pre- or pro-sequences can be engineered to improve glycosylation or yield. For example, the peptidase cleavage site of a particular signal peptide may be altered or a pro-sequence may be added, which may affect glycosylation as well. The final protein product may have one or more additional amino acids upon expression at position -1 (relative to the first amino acid of the mature protein), which may not be completely removed. For example, the final protein product may have one or two amino acid residues found in the peptidase cleavage site attached to the amino terminus. Alternatively, the use of some enzyme cleavage sites may result in a slightly truncated form of the desired polypeptide when the enzyme cleaves such a region in the mature polypeptide.

[0109] The expression and cloning vectors of the invention typically contain a promoter that is recognized by the host organism and operably linked to the molecule encoding the IL-2 mutein or IL-2 mutein Fc fusion protein. A promoter is a non-transcribed sequence located upstream (i.e., 5') (usually within about 100-1000 bp) of the start codon of a structural gene that controls transcription of the structural gene. Traditionally, promoters are classified into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate high levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature. In contrast, constitutive promoters uniformly transcribe the gene to which they are operably linked, i.e., they exert little or no control over gene expression. A large number of promoters recognized by a variety of potential host cells are well known.

[0110] Suitable promoters for use in yeast hosts are also well known in the art. Yeast enhancers are advantageously used with yeast promoters. Suitable promoters for use in mammalian host cells are well known and include, but are not limited to, promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and most preferably simian virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and actin promoters.

[0111] Additional promoters of interest include, but are not limited to, the SV40 early promoter (Benoist and Chambon, 1981, Nature 290:304-310); the CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad. USA 81:659-663); the promoter contained in the 3' terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797); the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78:1444-1445); promoters and control sequences from metallothionein genes (Prinster et al., 1982, Nature 296:39-42); and prokaryotic promoters such as the β-lactamase promoter (Villa-Kamaroff et al., 1982, Nature 296:39-42). al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Also of interest are the following animal transcriptional control regions that exhibit tissue specificity and have been utilized in transgenic animals: the elastase I gene control region, active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulin gene control region, active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122); the immunoglobulin gene control region, active in lymphocytes (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.7:1436-1444); mouse mammary tumor virus control region active in testicular cells, breast cells, lymphocytes, and mast cells (Leder et al., 1986, Cell 45:485-495); albumin gene control region active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); alpha fetoprotein gene control region active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 253:53-58); alpha 1 antitrypsin gene control region active in liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); beta globin gene control region active in bone marrow cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94); the myelin basic protein gene control region active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); the myosin light chain-2 gene control region active in skeletal muscle (Sani, 1985, Nature 314:283-286); and the gonadotropin-releasing hormone gene control region active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

[0112] Enhancer sequences may be inserted into vectors to increase transcription by higher eukaryotes. Enhancers are cis-acting elements of DNA, usually about 10-300 bp in length, that act on promoters to increase transcription. Enhancers are relatively orientation- and position-independent and have been found at both 5' and 3' positions of transcription units. Several enhancer sequences available from mammalian genes are known (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). Typically, however, enhancers from viruses are used. SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, and adenovirus enhancer, known in the art, are exemplary enhancing elements for eukaryotic promoter activation. Enhancers may be located in vectors either 5' or 3' of the coding sequence, but are typically located at the 5' site of the promoter. To promote extracellular secretion of IL-2 muteins or IL-2 mutein Fc fusion proteins, sequences encoding suitable native or heterologous signal sequences (leader sequences or signal peptides) can be incorporated into expression vectors. The choice of signal peptide or leader depends on the type of host cell in which the protein is produced, and heterologous signal sequences can replace native signal sequences. Examples of signal peptides that are functional in mammalian host cells include the signal sequence of interleukin 7 (IL-7) described in U.S. Pat. No. 4,965,195; the signal sequence of interleukin 2 receptor described in Cosman et al., 1984, Nature 312:768; the interleukin 4 receptor signal peptide described in European Patent No. 0 367 566; the type I interleukin 1 receptor signal peptide described in U.S. Pat. No. 4,968,607; and the type II interleukin 1 receptor signal peptide described in European Patent No. 0 460 846.

[0113] A vector may contain one or more elements that promote expression when the vector is integrated into the host cell genome. Examples include EASE elements (Aldrich et al. 2003 Biotechnol Prog. 19:1433-38) and matrix attachment regions (MARs). MARs mediate the structural organization of chromatin and can protect the integrated vector from "position" effects. Thus, MARs are particularly useful when the vector is used to generate stable transformants. Many natural and synthetic MAR-containing nucleic acids are known in the art (e.g., U.S. Patent Nos. 6,239,328, 7,326,567, 6,177,612, 6,388,066, 6,245,974, 7,259,010, 6,037,525, 7,422,874, 7,129,062).

[0114] The expression vector of the present invention may be constructed from a starting vector, such as a commercially available vector. Such vector may or may not contain all of the desired flanking sequences. If one or more of the flanking sequences described herein are not already present in the vector, they may be obtained separately and linked to the vector. The method used to obtain each flanking sequence is well known to those skilled in the art.

[0115] After the vector has been constructed and the nucleic acid molecule encoding the IL-2 mutein or IL-2 mutein Fc fusion protein has been inserted into the appropriate site of the vector, the complete vector may be inserted into a suitable host cell for amplification and / or polypeptide expression. Transformation of the expression vector into the selected host cell may be accomplished by well-known methods including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran mediated transfection, or other known techniques. The method selected will be, in part, a function of the type of host cell used. These and other suitable methods are well known to those of skill in the art and are described, for example, in Sambrook et al., 2001 (see above).

[0116] When cultured under appropriate conditions, the host cells synthesize an IL-2 mutein or IL-2 mutein Fc fusion protein, which can subsequently be recovered from the culture medium (if the host cells secrete the protein into the medium) or directly from the host cells producing the protein (if the protein is not secreted). Selection of an appropriate host cell depends on a variety of factors, including the desired expression level, polypeptide modifications (such as glycosylation or phosphorylation) that are desired or necessary for activity, and the ease of folding into biologically active molecules. Host cells may be eukaryotic or prokaryotic.

[0117] Mammalian cell lines available as hosts for expression are well known in the art, including, but not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), and any cell line used in an expression system known in the art can be used to produce the recombinant polypeptides of the invention. In general, a host cell is transformed with a recombinant expression vector that contains DNA encoding the desired IL-2 mutein or IL-2 mutein-Fc fusion. Among the host cells that can be used are prokaryotes, yeast cells, or higher eukaryotic cells. Prokaryotes include gram-negative or gram-positive bacteria, such as E. coli or Bacillus. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cells include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or derivatives thereof, such as Veggie CHO and related cell lines grown in serum-free medium (Rasmussen et al., 1998, Cytotechnology 28:31), HeLa cells, the BHK (ATCC CRL 10) cell line, and the CVI / EBNA cell line (ATCC CCL 70) derived from the African green monkey kidney cell line CVI as described in McMahan et al., 1991, EMBO J. 10:2821, human embryonic kidney cells, such as 293, 293 EBNA or MSR. 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro culture of primary tissues, primary explants, HL-60, U937, HaK or Jurkat cells. Optionally, where it is desired to use the polypeptide in various signal transduction or reporter assays, mammalian cell lines such as, for example, HepG2 / 3B, KB, NIH 3T3, or S49 can be used for expression of the polypeptide.

[0118] Alternatively, it is possible to produce the polypeptides in lower eukaryotes such as yeast or prokaryotes such as bacteria. Suitable yeasts include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing a heterologous polypeptide. Suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing a heterologous polypeptide. If the polypeptide is made in yeast or bacteria, it may be desirable to modify the polypeptide produced therein, for example by phosphorylation or glycosylation of appropriate sites, to obtain a functional polypeptide. Such covalent attachment can be achieved using known chemical or enzymatic methods.

[0119] Polypeptides can also be produced by operably linking the isolated nucleic acids of the present invention to suitable control sequences in one or more insect expression vectors and using an insect expression system. Materials and methods for baculovirus / insect cell expression systems are commercially available in kit form, for example, from Invitrogen, San Diego, Calif., USA (MaxBac® kit), and such methods are well known in the art, as described by Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987) and Luckow and Summers, Bio / Technology 6:47 (1988). Cell-free translation systems may be used to produce polypeptides using RNA derived from the nucleic acid constructs disclosed herein. Appropriate cloning and expression vectors for use in bacterial, fungal, yeast, and mammalian cell hosts are described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985). A host cell containing an isolated nucleic acid of the invention, preferably operably linked to at least one expression control sequence, is a "recombinant host cell."

[0120] In certain embodiments, the invention includes an isolated nucleic acid encoding a human IL-2 mutein that selectively stimulates regulatory T cells and comprises a V91K substitution and an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1. The isolated nucleic acid may encode any of the exemplary IL-2 muteins provided herein.

[0121] Also included are isolated nucleic acids encoding any of the exemplary IL-2 mutein Fc fusion proteins described herein. In a preferred embodiment, the Fc portion of the antibody and the human IL-2 mutein are encoded within a single open reading frame, optionally with a linker encoded between the Fc region and the IL-2 mutein.

[0122] In another embodiment, provided herein is an expression vector comprising a nucleic acid encoding an IL-2 mutein or an IL-2 mutein-Fc fusion protein as described above, operably linked to a promoter.

[0123] In another aspect, a host cell is provided herein that comprises an isolated nucleic acid encoding the above-mentioned IL-2 mutein or IL-2 mutein Fc fusion protein. The host cell may be a prokaryotic cell, such as E. coli, or a eukaryotic cell, such as a mammalian cell. In a specific embodiment, the host cell is a Chinese Hamster Ovary (CHO) cell line.

[0124] In another aspect, provided herein is a method of making a human IL-2 mutein. The method comprises culturing a host cell under conditions in which a promoter operably linked to the human IL-2 mutein is expressed. The human IL-2 mutein is then recovered from the culture. The IL-2 mutein may be recovered from the culture medium and / or from a host cell lysate.

[0125] In another aspect, a method for producing a human IL-2 mutein Fc fusion protein is provided herein. The method comprises culturing a host cell under conditions in which a promoter operably linked to the human IL-2 mutein Fc fusion protein is expressed. The human IL-2 mutein Fc fusion protein is then recovered from the culture. The human IL-2 mutein Fc fusion protein may be recovered from the culture medium and / or from a host cell lysate.

[0126] Pharmaceutical Compositions In some embodiments, the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of an IL-2 mutein together with a pharma- ceutical effective diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant. In certain embodiments, the IL-2 mutein is contained within an IL-2 mutein Fc fusion protein. Pharmaceutical compositions of the present invention include, but are not limited to, liquid compositions, frozen compositions, and lyophilized compositions.

[0127] Preferably, formulation materials are nontoxic to recipients at the dosages and concentrations employed. In a specific embodiment, a pharmaceutical composition is provided that comprises a therapeutically effective amount of an IL-2 mutein, e.g., an IL-2 mutein Fc fusion, containing a therapeutic molecule.

[0128] In certain embodiments, a pharmaceutical composition may contain formulation materials to alter, maintain, or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, proline, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite); buffers (such as boric acid, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta cyclodextrin, or hydroxypropyl-beta cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polysaccharides (such as polyvinylpyrrolidone); peptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEGs, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapol, etc.); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol, etc.); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants.See REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company.

[0129] In certain embodiments, optimal pharmaceutical compositions are determined by those of skill in the art depending, for example, on the intended route of administration, delivery form, and desired dosage. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES (supra). In certain embodiments, such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the antigen binding proteins of the invention. In certain embodiments, the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, saline, or artificial cerebrospinal fluid, possibly supplemented with other materials commonly found in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In specific embodiments, the pharmaceutical composition comprises a Tris buffer of about pH 7.0-8.5 or an acetate buffer of about pH 4.0-5.5, and may further include sorbitol or a suitable substitute therefor. In certain embodiments of the invention, the IL-2 mutein compositions can be prepared for storage in the form of lyophilized cakes or aqueous solutions by mixing selected compositions having the desired degree of purity with optional formulation substances (REMINGTON'S PHARMACEUTICAL SCIENCES, supra). Furthermore, in certain embodiments, the IL-2 mutein products may be formulated as lyophilizates using appropriate excipients such as sucrose.

[0130] The pharmaceutical compositions of the present invention can be selected for parenteral delivery. Alternatively, the compositions may be selected for inhalation or for delivery through the digestive tract, e.g., oral delivery. Preparation of such pharma-ceutically acceptable compositions is within the skill of the art. The formulation components are preferably present in concentrations that are acceptable to the site of administration. In certain embodiments, a buffer is used to maintain the composition at or slightly below physiological pH, typically within a pH range of about 5 to about 8.

[0131] When parenteral administration is contemplated, the therapeutic composition used in the present invention may be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired IL-2 mutein composition in a pharma- ceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the IL-2 mutein composition is formulated as a sterile isotonic solution and appropriately stored. In certain embodiments, the preparation may include formulating the desired molecule with substances such as injectable microparticles, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes that may provide controlled or sustained release of the product, which may be delivered, for example, by depot injection. In certain embodiments, hyaluronic acid may also be used, which has the effect of promoting sustained duration in the circulation. In certain embodiments, the desired IL-2 mutein composition may be introduced using an implantable drug delivery device.

[0132] Additional pharmaceutical compositions will be apparent to those skilled in the art, including formulations that include IL-2 mutein compositions in sustained or controlled delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot-injections, are also known to those skilled in the art. See, for example, International Patent Application No. PCT / US93 / 00829, which is incorporated by reference and describes controlled release of porous polymeric microparticles for delivery of pharmaceutical compositions. Sustained release preparations may include semipermeable polymer matrices, for example, in the form of shaped articles such as films or microcapsules. The sustained release matrix may comprise polyester, hydrogel, polylactide (as disclosed in U.S. Pat. No. 3,773,919 and European Patent Application Publication No. EP 058481, each of which is incorporated by reference), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15:167-277, and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinyl acetate (Langer et al., 1981, supra), or poly-D(-)-3-hydroxybutyric acid (European Patent Application Publication No. EP 133,988). The sustained release composition may also comprise liposomes, which may be prepared by any of several methods known in the art. See, e.g., Eppstein et al., 1985, Proc. Natl. Acad. Sci. USA 82:3688-3692; European Patent Application Publication Nos. EP 036,676; EP 088,046 and EP 143,949, which are incorporated by reference.

[0133] Pharmaceutical compositions used for in vivo administration are typically provided as sterile preparations. Sterilization can be achieved by filtration through a sterile filtration membrane. If the composition is lyophilized, sterilization using this method can be performed either before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in a solution. Parenteral compositions are generally placed in a container with a sterile access port, for example, an intravenous infusion bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0134] An embodiment of the present invention includes self-buffering IL-2 mutein formulations that can be used as pharmaceutical compositions, as described in International Patent Application No. WO06138181A2 (PCT / US2006 / 022599), which is incorporated herein by reference in its entirety.

[0135] As mentioned above, certain embodiments provide pharmaceutical IL-2 mutein compositions, particularly IL-2 mutein Fc fusion proteins, that in addition to the IL-2 mutein compositions include one or more excipients, such as those illustratively described in this section and elsewhere herein. Excipients in this regard can be used in the present invention for a variety of purposes, such as to adjust the physical, chemical, or biological properties of the formulations (e.g., adjusting viscosity) and / or processes of the invention, improve efficacy, and / or stabilize such formulations and processes against degradation and damage due to stresses that occur, for example, during manufacture, shipping, storage, preparation prior to use, administration, and thereafter.

[0136] Various descriptions of protein stabilization, and formulation materials and methods useful therefor, are available, e.g., Arakawa et al., "Solvent interactions in pharmaceutical formulations," Pharm Res. 8(3):285-91 (1991); Kendrick et al., "Physical stabilization of proteins in aqueous solution," in: RATIONAL DESIGN OF STABLE PROTEIN FORMULATIONS: THEORY AND PRACTICE, Carpenter and Manning, eds. Pharmaceutical Biotechnology. 13:61-84 (2002); and Randolph et al., "Surfactant-protein interactions," Pharm Biotechnol. 13:159-75 (2002), each of which is incorporated herein by reference in its entirety, particularly in the portions relating to excipients and processes for self-buffering protein formulations according to the present invention, and particularly with respect to protein pharmaceutical products and processes for veterinary and / or human medical use.

[0137] Salts may be used in accordance with certain embodiments of the invention, for example, to adjust the ionic strength and / or tonicity of the formulation and / or to enhance the solubility and / or physical stability of proteins or other components of compositions according to the invention.

[0138] As is well known, ions can stabilize the native state of proteins by binding to charged residues on the surface of proteins, as well as by shielding charged and polar groups in proteins to reduce the strength of their electrostatic, attractive, and repulsive interactions. Ions can also stabilize the denatured state of proteins, particularly by binding to denatured peptide bonds (--CONH) of proteins. In addition, ionic interactions with charged and polar groups in proteins can also reduce intermolecular electrostatic interactions, thereby avoiding or reducing protein aggregation and insolubility.

[0139] Ionic species vary significantly in their effects on proteins. A number of categorical permutations of ions and their effects on proteins have been developed that can be used in formulating pharmaceutical compositions according to the invention. One example is the Hofmeister series, which ranks ionic and polar non-ionic solutes by their effect on the conformational stability of proteins in solution. Stabilizing solutes are termed "kosmotropic". Destabilizing solutes are termed "chaotropic". Cosmotropes are commonly used at high concentrations (e.g., >1 molar ammonium sulfate) to precipitate ("salt out") proteins from solution. Chaotropes are commonly used to denature and / or solubilize ("salt out") proteins. The relative effectiveness of ions to "salt in" and "salt out" clarifies their place in the Hofmeister series.

[0140] Free amino acids can be used in IL-2 mutein formulations according to various embodiments of the present invention as bulking agents, stabilizers, and antioxidants, as well as other standard uses. Lysine, proline, serine, and alanine can be used to stabilize proteins in the formulation. Glycine is useful in lyophilization to ensure proper cake structure and properties. Arginine can be useful to inhibit protein aggregation in both liquid and lyophilized formulations. Methionine is useful as an antioxidant.

[0141] Polyols include sugars such as mannitol, sucrose, and sorbitol, and polyhydric alcohols such as glycerol and propylene glycol, and for the purposes of the discussion herein, polyethylene glycol (PEG) and related substances. Polyols are cosmotropic. They are useful stabilizers in both liquid and lyophilized formulations to protect proteins from physical and chemical degradation processes. Polyols are also useful for adjusting the tonicity of the formulation.

[0142] A polyol useful in selected embodiments of the present invention is mannitol, which is commonly used to ensure the structural stability of the cake in lyophilized formulations. Mannitol ensures the structural stability of the cake. Mannitol is commonly used with a lyoprotectant, such as sucrose. Sorbitol and sucrose are preferred agents for adjusting tonicity and stabilizers for protection against freeze-thaw stress during transportation or bulk preparation during manufacturing processes. Reducing sugars (containing free aldehyde or ketone groups), such as glucose and lactose, can glycate surface lysine and arginine residues. Therefore, they are generally not among the preferred polyols for use according to the present invention. Furthermore, sugars that form such reactive species, such as sucrose, which is hydrolyzed to fructose and glucose under acidic conditions, resulting in glycation, are also not among the preferred polyols of the present invention in this respect. PEG is useful for stabilizing proteins and as a cryoprotectant and can be used in the present invention in this respect.

[0143] An embodiment of the IL-2 mutein formulation further comprises a surfactant. Protein molecules may be susceptible to adsorption on surfaces, as well as denaturation at air-liquid, solid-liquid, and liquid-liquid interfaces, and resulting aggregation. These effects generally scale inversely with protein concentration. These deleterious interactions generally scale inversely with protein concentration, and are typically exacerbated by physical agitation, such as that occurring during product shipping and handling.

[0144] Surfactants are routinely used to prevent, minimize, or reduce surface adsorption. Surfactants useful in the present invention in this regard include polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan polyethoxylate, and poloxamer 188.

[0145] Detergents are also commonly used to control the conformational stability of proteins: any given detergent will typically stabilize some proteins and destabilize others, and thus their use in this regard is protein specific.

[0146] Polysorbates are susceptible to oxidative degradation and, when supplied, often contain sufficient amounts of peroxides to cause oxidation of protein residue side chains, especially methionine. As a result, polysorbates should be used with caution and, when used, at their lowest effective concentrations. In this respect, polysorbates illustrate the principle that excipients should be used at their lowest effective concentrations.

[0147] The embodiment of the IL-2 mutein formulation further comprises one or more antioxidants. The harmful oxidation of proteins in pharmaceutical formulations can be prevented to some extent by maintaining appropriate levels of ambient oxygen and temperature, and by avoiding exposure to light. Antioxidant excipients can also be used to prevent oxidative degradation of proteins. Among the antioxidants useful in this regard are reducing agents, oxygen / free radical scavengers, and chelating agents. The antioxidants used in therapeutic protein formulations according to the present invention are preferably water-soluble and maintain their activity throughout the shelf life of the product. In this regard, EDTA is the preferred antioxidant according to the present invention.

[0148] Antioxidants may damage proteins. For example, reducing agents, such as glutathione, may disrupt intramolecular disulfide bonds. Therefore, the antioxidants used in the present invention are selected, inter alia, to eliminate or substantially reduce the possibility that they themselves may damage proteins in the formulation.

[0149] The formulations according to the invention may contain metal ions, which are protein cofactors and necessary to form protein coordination complexes, such as zinc, which is necessary to form certain insulin suspensions. Metal ions can also inhibit some processes that break down proteins. However, metal ions also catalyze physical and chemical processes that break down proteins.

[0150] Magnesium ions (10-120 mM) can be used to inhibit the isomerization of aspartic acid to isoaspartic acid. +2 ions (up to 100 mM) can increase the stability of human deoxyribonuclease. However, Mg +2 , Mn +2 , and Zn +2 can destabilize rhDNase. +2 and Sr +2 can stabilize factor VIII, which is +2 , Mn+2 and Zn +2 , Cu +2 and Fe +2 The aggregation can be destabilized by Al +3 It can be increased by ions.

[0151] The IL-2 mutein formulation embodiment further comprises one or more preservatives. Preservatives are necessary when developing parenteral formulations for multiple doses, which involves more than one withdrawal from the same container. Their main function is to inhibit microbial growth and ensure sterility of the product throughout the shelf life or use of the drug product. Commonly used preservatives include benzyl alcohol, phenol, and m-cresol. Although preservatives have a long history of use with small molecule parenteral agents, developing protein formulations containing preservatives can be difficult. Preservatives almost always have a destabilizing effect on proteins (aggregation), which is the main factor limiting their use in protein formulations for multiple doses. To date, most protein drugs have been formulated for single use only. However, when multiple dose formulations are possible, they have the added benefit of allowing for increased patient convenience and marketability. A case in point is that of human growth hormone (hGH), where the development of preserved formulations led to the commercialization of more convenient multi-use injection pens. At least four such pen devices containing preserved formulations of hGH are currently available on the market: Norditropin (liquid, Novo Nordisk), Nutropin AQ (liquid, Genentech), and Genotropin (lyophilized, dual-chamber cartridge, Pharmacia & Upjohn) contain phenol, while Somatrope (Eli Lilly) is formulated with m-cresol.

[0152] In one embodiment, the IL-2 mutein or Fc fusion of an IL-2 mutein, e.g., Fc.IL-2(V91K) or Fc.IL-2(N88D), is formulated at 10 mg / mL in 10 mM L-glutamic acid, 3.0% (w / v) L-proline, pH 5.2. In another embodiment, the IL-2 mutein or Fc fusion of an IL-2 mutein, e.g., Fc.IL-2(V91K) or Fc.IL-2(N88D), is formulated in 10 mM KPi, 161 mM L-arginine, pH 7.6.

[0153] During the formulation and development of a preserved dosage form, several aspects must be considered: The effective preservative concentration in the drug product must be optimized, which requires testing a given preservative in a range of concentrations in the dosage form that imparts antimicrobial effectiveness without sacrificing protein stability.

[0154] In another embodiment, the present invention provides an IL-2 mutein or an Fc fusion of an IL-2 mutein as a lyophilized formulation. The lyophilized product can be lyophilized without preservatives and reconstituted with a diluent containing a preservative at the time of use. This reduces the time that the preservative is in contact with the protein and greatly minimizes the associated stability risks. In liquid formulations, the preservative's effectiveness and stability should be maintained throughout the shelf life of the product (approximately 18-24 months). An important point to note is that the preservative's effectiveness should be demonstrated in the final formulation containing the active drug and all excipient components.

[0155] An IL-2 mutein formulation will generally be designed for a particular route and method of administration, for a particular dosage and frequency of administration, for a particular treatment of a particular disease, with, among other things, a range of bioavailability and duration. Thus, formulations may be designed in accordance with the present invention for delivery by any suitable route, including, but not limited to, oral, aural, ocular, rectal, and vaginal, as well as by parenteral routes, including intravenous and intraarterial injection, intramuscular injection, and subcutaneous injection.

[0156] Once the pharmaceutical composition is formulated, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. Such formulations can be stored either in a ready-to-use form or in a form that is reconstituted (e.g., lyophilized) before administration. The present invention also provides kits for producing single-dose units. The kits of the present invention may each include both a first container with a dried protein and a second container with an aqueous formulation. In certain embodiments of the present invention, kits are provided that include single-chamber and multi-chamber pre-filled syringes (e.g., liquid syringes and lyophilized syringes).

[0157] The therapeutically effective amount of the pharmaceutical composition containing the IL-2 mutein used will depend, for example, on the nature and purpose of the treatment. Those skilled in the art will appreciate that the appropriate dosage level for treatment will depend in part on the molecule being delivered, the indication for which the IL-2 mutein is being used, the route of administration, and the size (weight, body surface or organ size) and / or condition (age and general health) of the patient. In certain embodiments, the clinician may titrate the dosage and modify the route of administration to obtain the optimal therapeutic effect. Typical dosages may range from about 0.1 μg / kg up to about 1 mg / kg, or more, depending on the factors mentioned above. In specific embodiments, dosages may range from 0.5 μg / kg up to about 100 μg / kg, optionally from 2.5 μg / kg up to about 50 μg / kg.

[0158] A therapeutically effective amount of an IL-2 mutein preferably results in a reduction in the severity of disease symptoms, an increase in the frequency or duration of symptom-free periods of the disease, or prevention of damage or disability due to the affliction of the disease.

[0159] Pharmaceutical compositions can be administered using medical device.The example of medical device for administering pharmaceutical compositions is described in U.S. Patent No. 4,475,196, U.S. Patent No. 4,439,196, U.S. Patent No. 4,447,224, U.S. Patent No. 4,447,233, U.S. Patent No. 4,486,194, U.S. Patent No. 4,487,603, U.S. Patent No. 4,596,556, U.S. Patent No. 4,790,824, U.S. Patent No. 4,941,880, U.S. Patent No. 5,064,413, U.S. Patent No. 5,312,335, U.S. Patent No. 5,312,335, U.S. Patent No. 5,383,851 and U.S. Patent No. 5,399,163, all of which are incorporated herein by reference.

[0160] Methods for treating autoimmune or inflammatory diseases In a specific embodiment, the IL-2 muteins of the invention are used to treat autoimmune or inflammatory diseases, hi a preferred embodiment, an IL-2 mutein Fc fusion protein is used.

[0161] Disorders that are particularly suitable for treatment with the IL-2 muteins disclosed herein include, but are not limited to, inflammation, autoimmune diseases, atopic diseases, paraneoplastic autoimmune diseases, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), dermatomyositis, psoriatic arthritis inflammation, scleroderma, vasculitis, myositis, polymyositis, dermatomyositis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus, Still's disease, systemic lupus erythematosus (SLE), severe These include: myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, sinusitis, sinusitis with polyposus, eosinophilic esophagitis, eosinophilic bronchitis, Guillain-Barre disease, type I diabetes, thyroiditis (e.g. Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD, transplant rejection, nephropathy, Hepatitis C induced vasculitis, spontaneous abortion, etc.

[0162] In preferred embodiments, the autoimmune or inflammatory disease is lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type I diabetes, multiple sclerosis, spontaneous abortion, atopic disease, and inflammatory bowel disease.

[0163] In another embodiment, a patient having or at risk of developing an autoimmune or inflammatory disease is treated with an IL-2 mutein (e.g., an IL-2 mutein disclosed herein, e.g., an IL-2 mutein Fc fusion disclosed herein, or another IL-2 mutein or wild-type IL-2 known in the art (optionally as part of an Fc fusion molecule of the type disclosed herein)) and the patient's response to the treatment is monitored. The patient response monitored may be any detectable or measurable patient response to the treatment, or any combination of such responses. For example, the response may be a change in the patient's physiological state, such as body temperature or fever, appetite, sweating, headache, nausea, fatigue, hunger, dry mouth, mental alertness, etc. Alternatively, the response may be a change in the amount of a cell type or gene product (e.g., a protein, peptide, or nucleic acid), for example, in a peripheral blood sample taken from the patient. In one embodiment, the patient's treatment plan is altered if the patient has a detectable or measurable response, or if such response exceeds a certain threshold. The change may be a reduction or increase in the frequency of administration, a reduction or increase in the amount of IL-2 mutein administered per dose, or a "break" from administration (i.e., a temporary interruption of treatment, either for a specified period of time or until the attending physician determines that treatment should be continued or until monitored patient response indicates that treatment should or can be resumed), or the end of treatment. In one embodiment, the response is a change in the patient's body temperature or CRP level. For example, the response may be an increase in the patient's body temperature, or an increase in the CRP level in a peripheral blood sample, or both. In a particular embodiment, if the patient's body temperature increases by at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1, 1.5, 2, or 2.5° C. during the course of treatment, the patient's treatment is reduced, suspended, or discontinued. In another specific embodiment, a patient's treatment is reduced, suspended, or discontinued if the concentration of CRP in the patient's peripheral blood sample increases by at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1, 1.5, or 2 mg / mL during the course of treatment.Other patient responses that can be monitored and used in determining whether to change, reduce, suspend, or discontinue treatment include the onset or worsening of capillary leak syndrome (hypotension and cardiovascular instability), neutrophil dysfunction (e.g., resulting in or detecting the onset or worsening of infection), thrombocytopenia, thrombotic vascular disease, injection site reactions, vasculitis (vasculitis due to Hepatitis C), or inflammatory conditions or diseases. Additional patient responses that can be monitored and used in determining whether to change, reduce, increase, suspend, or discontinue treatment include NK cells, Treg cells, FOXP3. - CD4T cells, FOXP3 + The increase in the number of CD4 T cells, FOXP3-CD8 T cells, or eosinophils can be detected, for example, as an increase in the number of such cells per unit of peripheral blood (e.g., expressed as an increase in cells per milliliter of blood) or as an increase in the proportion of such a cell type compared to another type of cell or cells in the blood sample. Another patient response that can be monitored is the increase in the number of CD25 T cells, FOXP3-CD8 T cells, or eosinophils in the patient's peripheral blood sample. + This is an increase in the amount of IL-2 mutein bound to the cell surface of the cells.

[0164] Methods for expanding Treg cells The IL-2 mutein or IL-2 mutein Fc fusion protein can be used to expand Treg cells in a subject or sample. Provided herein is a method for increasing the ratio of Treg to non-regulatory T cells. The method comprises contacting a population of T cells with an effective amount of a human IL-2 mutein or IL-2 mutein Fc fusion. The ratio can be measured by determining the ratio of CD3+FOXP3+ cells to CD3+FOXP3- cells in the population of T cells. The typical Treg frequency in human blood is 5-10% of the total CD4+CD3+ T cells, although in the diseases listed above, this percentage may be lower or less. In a preferred embodiment, the percentage of Treg is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000%. The maximum fold increase of Treg may vary for each specific disease: however, the maximum Treg frequency that may be obtained by IL-2 mutein treatment is 50% or 60% of total CD4+CD3+T cells. In a specific embodiment, an IL-2 mutein or an IL-2 mutein Fc fusion protein is administered to a subject to increase the ratio of regulatory T cells (Treg) to non-regulatory T cells in the peripheral blood of the subject.

[0165] Because IL-2 muteins and IL-2 mutein Fc fusion proteins selectively expand Tregs over other cell types, they are also useful for increasing the ratio of regulatory T cells (Tregs) to natural killer (NK) cells in the peripheral blood of a subject, which can be measured by determining the ratio of CD3+FOXP3+ cells to CD16+ and / or CD56+ lymphocytes that are CD19- and CD3-.

[0166] It is contemplated that the IL-2 mutein or IL-2 mutein Fc fusion protein may have a therapeutic effect on a patient's disease or disorder without significantly increasing the ratio of Treg to non-regulatory T cells or NK cells in the patient's peripheral blood. The therapeutic effect may be due to the local activity of the IL-2 mutein or IL-2 Fc fusion protein at the site of inflammation or autoimmunity. EXAMPLES

[0167] The following examples, both actual and prophetic, are provided for the purpose of illustrating certain embodiments or features of the present invention and are not intended to limit its scope.

[0168] Example 1 - A small number of mutations that confer high affinity for CD25 IL-2 muteins with high affinity for CD25 and low signaling strength through IL-2Rβγ selectively promote Treg proliferation and function. To reduce potential immunogenicity, we sought to determine the minimum number of mutations required to achieve high affinity for CD25. The crystal structure of IL-2 in complex with its three receptors (PDB code-2B5I) shows V69A and Q74P located within the helical structure that interacts with CD25. This may explain why V69A and Q74P were frequently isolated in two independent IL-2 mutagenesis screens for high binding affinity to CD25 (Rao et al. 2005; Thanos et al. 2006). In this example, we explore which of the other mutations in IL-2 mutein "2-4" identified in the Rao et al. screen are most important for increasing affinity above that observed with V69A and Q74P alone. The following proteins were screened by flow cytometry for binding to CD25 on the surface of activated T cells. All constructs also contained C-terminal FLAG and poly-His tags for purification and detection. Specific mutations are provided in parentheses.

[0169] [Table 4]

[0170] HaMut7D bound CD25 with nearly the same affinity as the original isolate "2-4" (~200 pM), suggesting that the mutation N71R was able to increase affinity significantly above that observed with V69A,Q74P alone (HaMut1D, ~2 nM). The other constructs had similar or slightly higher affinities than HaMut1D, with the exception of HaMut8D, whose affinity was only slightly higher than that of WT IL-2.

[0171] Example 2 - IL-2 mutein fused to IgG1-Fc domain for improved half-life To reduce the dosing frequency required to achieve enrichment of Tregs with IL-2 muteins, various fusions between IL-2 and the IgG1-Fc domain were evaluated. The Fc domain contained point mutations that abolished effector functions such as IgG1-mediated target cell lysis. The Fc effector function mutations used were either A327Q, Ala Ala (L234A+L235A), or N297G. Because Treg-selective IL-2 muteins partially reduce the potency of IL-2, it was important to fuse IL-2 to Fc in a manner that did not significantly affect IL-2R signaling. Therefore, IL-2 muteins with and without Fc fusion were examined for activation of IL-2R.

[0172] To determine whether dimerization of IL-2 by fusion of Fc increases IL-2R signaling strength due to increased binding activity to IL-2R, a weaker IL-2 mutein (haD5) (US20110274650) was fused to the amino terminus of Fc separated by a GGGGS (SEQ ID NO: 5) linker sequence. This mutein had three mutations (E15Q, H16N, N88D) that affect IL-2R signaling, eight mutations (N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P) that confer high affinity for CD25 (Rao et al. 2005), and C125S to prevent cysteine ​​mispairing and aggregation. Binding to Fc in such a manner completely abolishes the biological activity of haD5, while enhancing its high affinity binding to cell surface CD25, presumably due to increased avidity upon dimerization.

[0173] The IL-2 mutein was also fused to either the N- or C-terminus of the Fc heterodimer, such that only one chain of the Fc dimer carried the IL-2 domain. Electrostatic interactions between a lysine introduced on one Fc chain and an aspartic acid introduced on the other promoted heterodimer pairing between the two asymmetric Fc chains. When one configuration was preferred, the IL-2 mutein haD6 was fused to the N-terminus of one or the other Fc chain, resulting in two protein constructs designated haD6.FcDD and haD6.FcKK. The mutein haMut7D was also fused to the C-terminus of the Fc heterodimer using one or two GGGGS (SEQ ID NO: 5) linkers (FcKK(G4S)haMut7D, FcKK(G4S)2haMut7D). Fusion of the IL-2 mutein haD6 to the N-terminus of the Fc heterodimer resulted in a partial loss of activity compared to free haD6 in both pSTAT5 and T cell proliferation experiments. In contrast, fusion of haMut7D to the C-terminus of the Fc heterodimer using one or two GGGGS (SEQ ID NO:5) linkers did not alter the potency of haMut7D.

[0174] Fusion of IL-2 muteins to the C-terminus of the Fc homodimer was also investigated. Total PBMCs were activated at 300 million cells per 100 ml with 100 ng / ml anti-CD3 (OKT3) in T75 tissue culture flasks. On day 3 of culture, cells were washed three times and rested in fresh medium for 3 days. Cells were then stimulated with 10x dose-escalated IL-2 variants ranging from 1 pM to 10 nM in a final volume of 50 μl. STAT5 phosphorylation levels were measured using the BD Phosflow buffer kit. Briefly, stimulation was stopped by adding 1 ml of BD Lysis / Fixation Phosflow buffer. Cells were fixed for 20 min at 37°C and stained for CD4, CD25, FOXP3, and pSTAT5 after permeabilization with 1x BD Phosflow permeabilization buffer on ice.

[0175] As can be seen in Figure 1, the biological activity of the muteins haMut1D and haMut7D was not altered by fusion to the C-terminus of the Fc homodimer. Thus, the fusion between the N-terminus of IL-2 and the C-terminus of Fc did not impair the agonistic activity of the IL-2 muteins, even in the context of the Fc.IL-2 homodimer. The C125A mutation was used in these constructs instead of C125S to improve manufacturability.

[0176] Example 3 - Tailoring the potency of IL-2 muteins to achieve favorable Treg expansion The first panel of IL-2 muteins contained N88D alone or one or two additional mutations affecting IL-2R signaling. The second panel of muteins was designed to all have single point mutations with the goal of identifying muteins with similar or slightly higher potency than those in the N88D series. A panel of 24 signaling mutations was identified based on predicted IL-2Rβ interacting amino acids (crystal structure, PDB code-2B5I). Specific substitutions were selected based on predicted reduction in binding free energy between the muteins and IL-2Rβ. Binding free energies were calculated using the EGAD computational algorithm (Handel's Laboratory, University of California, San Diego, USA). The binding free energies of the mutants were expressed as △△G mut =μ(△G mut -△G wt ) where μ (typically = 0.1) is a scale factor used to normalize the predicted change in binding affinity to have a slope of 1 when compared to the experimental energy (Pokala and Handel 2005). The free energy of dissociation (ΔG) is the energy of dissociation of the complex state (ΔG bound ) and the free state (△G free The dissociation energy ΔGmut was calculated for each substitution.

[0177] A panel of IL-2 muteins with the following substitutions (H16E, H16Q, L19K, D20R, D20K, D20H, D20Y, M23H, D84K, D84H, S87Y, N88D, N88K, N88I, N88H, N88Y, V91N, V91K, V91H, V91R, I92H, E95K, E95R, or E95I) were expressed as C-terminal fusions to the Fc heterodimer. These constructs also contained haMut7 mutations (V69A, N71R, Q74P) for high CD25 binding affinity and C125A for efficient folding.

[0178] The panel was screened for potency in the T cell STAT5 phosphorylation assay of Example 2 and found that H16E, D84K, V91N, V91K, and V91R had lower activity than wild-type IL-2 and higher activity than N88D (Figure 2).

[0179] H16E, D84K, V91N, V91K, and V91R had lower activity than wild-type IL-2 and higher activity than N88D.

[0180] Selected muteins were also examined in T cell proliferation assays and NK proliferation assays.

[0181] For T cell assays, total PBMCs were activated at 3 million / ml with 100 ng OKT3. On day 2, cells were washed 3 times and rested in fresh medium for 5 days. Cells were then labeled with CFSE and further cultured in 24-well plates at 0.5 million / well in IL-2-containing medium for 7 days before FACS analysis. Proliferation of T cell subsets is shown in Figure 3 as CFSE dilution (median CFSE fluorescence).

[0182] For NK cell assays, MACS-sorted CD16+ NK cells were cultured in IL-2-containing medium at 100,000 / well in 96-well plates for 3 days. During the final 18 h of incubation, 0.5 μCi 3 H-thymidine was added, and the results are shown in FIG.

[0183] The H16E, D84K, V91N, V91K, and V91R mutants were able to stimulate Treg proliferation similar to WT IL-2, but were approximately 10x less potent against other T cells (Figure 3) and approximately 100x less potent against NK cells (Figure 4).

[0184] Another panel of Fc.IL-2 fusion proteins was designed in which the distance between the Fc heterodimer and the mutein haMut7 (V69A, N71R, Q74P, C125A) was reduced by a series of individual amino acid truncations.

[0185] [Table 5]

[0186] As described for Figures 2, 3, and 4, Trunc1-Trunc4 were equipotent to the full-length parental construct Fc.haMut7, as measured by STAT5 phosphorylation and T and NK cell proliferation. Trunc5 and Trunc6 stimulated weaker responses, but stronger than those stimulated by the N88D mutations (haD and haMut7D) and very similar to those stimulated by V91K. Trunc7 was weaker than the N88D mutein, and Trunc8 showed negligible activity. However, Trunc5 and Trunc6 were more potent agonists than V91K when tested on NK cells, suggesting that Treg selectivity was more easily achieved by signaling mutations rather than steric hindrance by the proximal Fc domain.

[0187] Example 4 - CD25 high affinity mutations associated with Fc homodimers Mutations that conferred high CD25 binding affinity were considered beneficial because they increased tropism for CD25-high T cells, promoted prolonged CD25::IL-2 mutein association, and prolonged signaling. However, reducing the number of muteins may reduce the potential for immunogenicity. N88D or V91K muteins with and without the haMut1 high affinity mutations V69A and Q74P were expressed as fusions to the C-terminus of Fc homodimers and compared for biological activity. In pSTAT5 stimulation assays, homodimerization did not affect signal strength compared to the monomeric muteins. Reversion of the high affinity mutations V69A and Q74P also did not affect pSTAT5 signaling. In T cell proliferation assays, the high affinity mutations reduced activity for conventional CD4 and CD8 T cells, but not for regulatory T cells (Figure 5). The high-affinity mutations also did not alter the proliferative response in NK cells (Fig. 6 ).

[0188] To determine whether the high affinity mutations affected T cell responses in vivo, humanized mice (NOD.SCID.Il2rg null mice reconstituted with human CD34+ hematopoietic stem cells) were administered Fc.IL-2 mutein fusion proteins and Treg proliferation was monitored. Seven-week-old NOD.SCID.Il2rg-null (NSG) mice (Jackson Labs, Bar Harbor, ME) were irradiated (180 rads) and transfected with 94,000 human fetal liver CD34 + Mice were reconstituted with hematopoietic stem cells. At week 21, mice were divided into six groups based on the evenly distributed percentage of chimerism (determined by flow cytometry of PBLs) and injected subcutaneously with 1 μg of the indicated Fc.mutein fusion protein or PBS on days 0 and 7. The frequency of T cell subsets in the blood was determined by flow cytometry on day 11. At doses as low as 1 μg per animal, the high affinity mutations did not improve Treg proliferation above that observed with the N88D or V91K mutations alone (Figure 7).

[0189] FOXP3- CD4 + The expansion of Tregs was selective in that T cells were not increased in abundance relative to total peripheral blood leukocytes (PBLs), which contain a mixture of human B and T cells and mouse myeloid cells. Furthermore, at higher doses, the high affinity mutant CD25 + FOXP3 - It promotes T cell expansion and therefore reduces Treg selectivity, and therefore high affinity mutations were not found to be necessary to promote selective Treg expansion in the context of Fc homodimers.

[0190] [Table 6]

[0191] Example 5 - Prolonged association of Fc.IL-2 muteins with cell surface CD25 An unexpected result from the humanized mouse studies was that despite their reduced signaling capacity, the muteins induced more robust Treg enrichment compared to Fc.WT IL-2. At a dose of 1 μg / mouse (FIG. 7) and even lower at 0.5 μg / mouse (FIG. 8), higher Treg enrichment and FOXP3 upregulation was observed compared to that seen with Fc.WT. This increased potency in vivo may be due to reduced consumption by T cells, making more Fc.IL-2 muteins available for prolonged signaling.

[0192] However, in vitro and in vivo PK studies failed to demonstrate a significantly increased persistence of Fc.V91K or Fc.N88D compared to Fc.WT in supernatants from activated T cell cultures or in serum from treated mice. Because the Fc fusions carry two IL-2 mutein domains, increased endosomal recycling may result in prolonged cell surface association due to increased binding activity to CD25. Indeed, we found that after short-term exposure of the fusion proteins, Fc.V91K and Fc.N88D remained more efficiently on the surface of previously activated T cells than Fc.WT (Figures 9A and B).

[0193] Primary PBMCs were pre-stimulated with 100ng / ml OKT3 for 2 days. Cells were harvested, washed 4 times and rested overnight in medium. Cells were then pulsed with 400pM Fc.IL-2 for 30 minutes at 37°C. After pulsing, cells were either washed once and then harvested at T0 or washed an additional 3 times in 12ml warm medium and cultured for 4 hours. To detect cell-associated Fc.IL-2, cells were stained with anti-human IgG-FITC (Jackson Immunoresearch, West Grove, PA) and anti-CD25-APC (Figure 9A).

[0194] Persistent IL-2R signaling was observed with Fc.V91K and Fc.N88D compared to Fc.WT by intracellular immunodetection of phospho-STAT5 at the same time points. Phospho-STAT5 MFI in FOXP3+CD4+ T cells is shown (Figure 9B).

[0195] Example 6 - Optimization of fusion sequences In preclinical studies in mice, the Fc.IL-2 muteins showed differential exposure when serum concentrations of the intact molecule were compared to those of the human Fc portion alone, suggesting circulating human Fc degradation products. To optimize the in vivo stability and pharmacokinetics of the Fc.IL-2 muteins, modifications of the fusion sequence were characterized for their effect on the proteolysis of the Fc.IL-2 muteins during systemic circulation and recycling through the reticuloendothelial system. The following constructs were evaluated for proteolysis in vitro and in vivo:

[0196] [Table 7]

[0197] Stability was measured by quantitative immunoassay comparing the concentration of total human Fc over time to that of intact Fc.IL-2 mutein. Proteolysis of the Fc.IL-2 mutein was confirmed by Western blot analysis using anti-IL-2 and anti-human Fc antibodies followed by immunocapture and mass spectrometric characterization of the degradation products. Mass spectrometric characterization of the degradation products of (Ala_Ala)_G4S from in vitro and in vivo samples identified the C-terminal Lys of the Fc domain as the proteolytic cleavage site. C-terminal lysine deletion or mutation of the Fc domain ((N297G_delK)_G4S and (N297G_KtoA)_AAPT) resulted in prolonged in vitro stability in mouse serum at 37°C compared to Fc constructs containing the C-terminal lysine ((Ala_Ala)_G4S). This prolonged in vitro serum stability resulted in higher exposure in mice as measured by the area under the Fc.IL-2 mutein serum concentration versus time curve (AUC). This long-term stability of the Fc.IL-2 mutein lacking the C-terminal Fc lysine was also observed in vitro in serum from cynomolgus monkeys and humans. The Thr-3 to Ala ((N297G_KtoA)_AAPA) mutation of IL-2 resulted in decreased in vitro stability at 37°C (compared to (N297G_KtoA)_AAPT) in mouse serum as well as in separate incubations with recombinant human cathepsins D and L. This decreased in vitro serum stability resulted in lower in vivo mouse exposure (AUC) to (N297G_KtoA)_AAPA compared to (N297G_KtoA)_AAPT. Characterization of degradation products of (N297G_KtoA)_AAPA from in vitro and in vivo samples by mass spectrometry identified Lys 8 and Lys 9 in the IL-2 mutein domain as residues susceptible to proteolysis that were not observed in comparable samples of (N297G_KtoA)_AAPT. The reduced stability of (N297G_KtoA)_AAPA relative to that of (N297G_KtoA)_AAPT at 37°C was also observed in vitro in serum from cynomolgus monkeys and humans.

[0198] Because of the importance of glycosylation in this region, and to potentially improve the manufacturability of the fusion protein, the fusion sequence was altered as follows to promote N-linked rather than O-linked glycosylation.

[0199] [Table 8]

[0200] Example 7 - Cynomolgus Monkey PK / PD Determination Standard IL-2 immune stimulation therapy requires a drug holiday (no exposure) between dosing cycles to avoid unwanted side effects. In contrast, Treg expansion or stimulation therapy requires sustained drug trough levels (serum C min ) but below the drug levels that cause immune activation. max This example demonstrates that prolonged exposure to serum C is below the drug levels contemplated to be required for proinflammatory immune activation. max ) while maintaining long-term target coverage (serum C min ) shows the administration strategy of the half-life extended mutein in cynomolgus monkeys.

[0201] Cynomolgus monkeys were dosed with Fc.V91K (IgG1Fc(N297G_delK)::G4S::huIL-2(V91K,C125A) in four groups (A-D): three groups (A-C) were administered subcutaneously and one group (D) was administered intravenously. Four biologically naive male cynomolgus monkeys per group were dosed according to the dosing strategy outlined below. Subcutaneous administration of the extended half-life mutein may allow for greater lymphatic absorption, resulting in a lower maximum exposure (serum C max) and / or more robust pharmacological responses (Treg expansion). The dosing strategy for Group A consisted of three consecutive doses of 10 micrograms per kilogram on days 0, 2, and 4 in cycle 1, and 10 micrograms per kilogram on day 14, resulting in a lower maximum exposure (serum C max The dosing strategy for arm B is 50 micrograms per kilogram administered on days 0 and 14 to compare with arm A. The dosing strategy for arm C is 50 micrograms per kilogram administered on days 0 and 28 to allow for the determination of whether trough coverage is necessary to maintain Treg enrichment or whether a rest period between dosing cycles is beneficial. The dosing strategy for intravenous arm arm D is 50 micrograms per kilogram administered on day 0 to achieve similar long-term target coverage while maintaining maximum exposure (C max ) and Treg enrichment can be compared with subcutaneous administration.

[0202] For each designated dose group, pharmacokinetics (quantitative immunoassays of intact molecule and total human Fc), anti-drug antibodies, shed soluble CD25, and serum cytokines (IL-1β, TNF-α, IFN-γ, IL-10, IL-5, IL-4, and IL-13) were measured at the following time points: Group A: Predose (1st cycle; dose 1), 48 (1st cycle predose; dose 2), 96 (1st cycle predose; dose 3), 100, 104, 120, 168, 216, 264, 336 (2nd cycle predose), 340, 344, 360, 408, 456, 504, 576, 672, 744, 840, and 1008 hours. Group B: pre-dose (first cycle), 4, 8, 24, 72, 120, 168, 240, 336 (second pre-dose cycle), 340, 344, 360, 408, 456, 504, 576, 672, 744, 840, and 1008 hours. Group C: pre-administration (first cycle), 4, 8, 24, 72, 120, 168, 240, 336, 408, 504, 672 (second pre-administration cycle), 676, 680, 696, 744, 792, 840, 912, 1008, 1080, and 1176 hours. Group D: pre-dose (first cycle), 0.25, 1, 4, 8, 24, 72, 120, 168, 240, 336, 408, 504, and 672 hours.

[0203] Pharmacokinetics (immunophenotyping and enumeration of peripheral blood Treg, non-regulatory CD4 and CD8 T cells, and NK cells) were measured for each designated dose group at the following time points: Group A: predose (1st cycle; dose 1), 96 (1st cycle predose; dose 3), 168, 336 (2nd cycle predose), 456, and 576 hours. Group B: pre-dose (first cycle), 120, 240, 336 (second pre-dose cycle), 456, and 576 hours. Group C: pre-dose (first cycle), 120, 240, 672 (second pre-dose cycle), 792, and 912 hours. Group D: pre-administration (first cycle), 120 and 240 hours.

[0204] Hematology and clinical chemistry were evaluated for all animals and dose groups pre-dose and 24 hours after the first dose for each dose group. Hematology: ·White blood cell count (total and absolute differential) ·Red blood cell count ·hemoglobin Hematocrit Mean corpuscular hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration (calculated values) Absolute reticulocyte count Platelet count ·Blood cell morphology ·Red blood cell distribution width ·Average platelet volume Clinical chemistry: Alkaline phosphatase Total bilirubin (or direct bilirubin if total bilirubin is greater than 1 mg / dL) Aspartate aminotransferase Alanine aminotransferase γ-glutamyltransferase Urea nitrogen Creatinine Total Protein ·albumin Globulin and A / G (albumin / globulin) ratio (calculated value) ·glucose Total cholesterol Triglycerides Electrolytes (sodium, potassium, chloride) ·calcium Phosphorus

[0205] Example 8 - Non-glycosylated IgG1 Fc Naturally occurring IgG antibodies have a glycosylation site in the constant domain 2 (CH2) of the heavy chain. For example, human IgG1 antibodies have a glycosylation site located at Asn297 (EU numbering). Strategies to generate non-glycosylated antibodies to date include substituting the Asn residue with an amino acid similar to Asn in physicochemical properties (e.g., Gln) or with an Ala residue that mimics the Asn side chain without a polar group. This example demonstrates the benefits of substituting Asn with glycine (N297G). N297G Fc is a non-glycosylated molecule with better biophysical properties and manufacturability (e.g., recovery rate during purification).

[0206] Examination of several known crystal structures of Fc fragments and IgG antibodies reveals considerable structural flexibility around the glycosylated loop segments, especially at the glycosylated Asn297 position. In many of the known crystal structures, Asn297 conforms to a positive main chain dihedral angle. Gly has a strong tendency to conform to a positive main chain dihedral angle due to the lack of a side chain atom. Therefore, based on this conformational and structural reasons, Gly may be a better replacement for Asn than N297Q or N297A.

[0207] Mutation of Asn297 to Gly resulted in a non-glycosylated molecule with much improved recovery (or efficiency) and biophysical properties in the purification process. For example, the recovery (final yield) from the Protein A pool was 82.6% for the N297G mutation compared to 45.6% for N297Q and 39.6% for N297A. SPHP column analysis revealed that the lower recovery of the N297Q and N297A mutants was due to tailing peaks representing molecular weight aggregates and / or misfolded species. This result was reconfirmed in a larger 2L scale experiment.

[0208] In the biopharmaceutical industry, molecules that potentially require large-scale production, e.g., have the potential to be sold as drugs, are evaluated for many attributes to mitigate the risk that the molecule is not suitable for large-scale production and purification. In the manufacturability evaluation, N297G demonstrated robustness against pH changes. N297G had no aggregation issues, whereas N297Q and N297A showed 20% and 10% increased aggregation, respectively. N297G had better manufacturability, but was similar to N297Q and N297A in all functional assays examined. For example, in the ADCC assay, N297G did not show cytotoxicity, similar to N297Q and N297A.

[0209] Example 9 - Stabilized non-glycosylated IgG1 Fc This example describes a method to improve the stability of an IgG antibody backbone by introducing engineered disulfide bond(s). Naturally occurring IgG antibodies are stable molecules. However, for certain therapeutic applications, it may be necessary to perform mutations or create non-glycosylated molecules. For example, non-glycosylated IgG molecules may be used in therapeutic indications where ADCC and binding to Fcγ receptors need to be avoided. However, non-glycosylated IgG1 has a much lower melting temperature than glycosylated IgG1 (melting temperature of the CH2 domain is reduced by about 10°C; 70°C to 60°C). The observed lower melting temperature adversely affects various biophysical properties of non-glycosylated IgG1. For example, non-glycosylated IgG1 has increased levels of aggregation at low pH compared to glycosylated IgG1.

[0210] To engineer disulfide bonds, a structure-based approach involving calculation of distances between Cα atoms was first used to identify 54 residue pairs in the Fc region for mutation to Cys. These 54 sites were further narrowed down to 4 residue pairs (V259C-L306C, R292C-V302C, A287C-L306C, and V323C-I332C). Criteria used included (i) location within the CH2 domain, (ii) distance from loops, turns, and carbohydrates, (iii) distance from Fcγ receptor and FcRn interaction sites, and (iv) solvent accessibility (preferably buried positions).

[0211] Paired cysteine ​​substitutions were made in the context of the non-glycosylated N297G Fc. Non-reduced peptide mapping analysis revealed that three of the four engineered sites formed disulfide bonds, as expected and designed in such a situation. The V259C-L306C mutation did not correctly form a disulfide bond, but rather formed an unpaired pair with a native disulfide already present in the CH2 domain. The other three designs, R292C-V302C, A287C-L306C, and V323C-I332C, correctly formed disulfide bonds as predicted and designed. The addition of a disulfide bond to the N297G mutation resulted in an improvement in thermostability of approximately 15° C. over the N297G mutation alone. Among the R292C-V302C, A287C-L306C, and V323C-I332C disulfide mutants, R292C-V302C and A287C-L306C showed favorable pharmacokinetics when administered to rats (t 1 / 2 ) This is a previously published 5-day 1 / 2 This is in contrast to the pharmacokinetic profile observed in rats with a CH2 domain disulfide bond (Gong et al., J. Biol. Chem. 2009 284:14203-14210), which showed

[0212] Engineering disulfide bonds within the CH2 domain improves the stability of nonglycosylated molecules comparable to glycosylated IgG1 molecules (10°C-15°C improvement in melting temperature as determined by differential scanning calorimetry). The engineered sites described herein do not cause disulfide scrambling, with disulfides forming predictably in approximately 100% of the population. More importantly, unlike published disulfide bond sites in the CH2 domain, the disulfide bonds described herein do not affect PK in rats.

[0213] Example 10 The effects of the V91K and N88D mutations on T cell and NK cell responses from cynomolgus monkeys and humans were compared in vitro.+ CD25 + T cells gated on CD25 in whole blood pSTAT5 responses), the effect of the V91K mutation on cyno IL-2R signaling was minimal compared to the reduced activity it exerts on the human IL-2R. - In both T cell and NK cell proliferation gated by β-gated IL-2R, the V91K mutation more significantly reduced cynomolgus IL-2R signaling. In contrast, Fc.N88D reduced CD25 expression in cynomolgus whole blood. + Although it shows reduced signaling in T cells, it is more similar to the signaling effects of Fc.V91K in T cells in human whole blood. The in vitro data summarized in Table 2 suggest that the therapeutic window observed with the weaker agonist Fc.N88D in cynomolgus monkeys is a precursor to the effects of Fc.V91K in human subjects.

[0214] [Table 9]

[0215] Example 11 Two in vivo studies were performed in cynomolgus monkeys. The first cynomolgus study was designed to compare 2-week and 4-week dosing intervals of Fc.V91K to determine whether a complete or partial pharmacokinetic (PK) and pharmacodynamic (PD) trough altered the magnitude of response to the second dose (Figure 10A and B). The first dose was predicted to produce a strong Treg response (50 μg / kg) and the second dose was used to explore the lower end of the therapeutic window (10 μg / kg). Because it was unclear whether 10 μg / kg was too low, doses were administered on days 1, 3, and 5 to increase the likelihood of a response. This dosing schedule provided the same exposure after 5 days as achieved with a single 50 μg / kg subcutaneous (SC) dose, but with a lower C-max. A 50 μg / kg intravenous (IV) group was also included to explore potential differences in PD dependent on higher drug exposure in lymphatic versus blood components. The results of this study demonstrated that each dose level induced strong Treg proliferative responses without adverse events (AEs) or Teff or NK proliferation, and responses to a second dose on either day 14 or day 28 were equivalent.

[0216] [Table 10]

[0217] The second cynomolgus monkey study was designed to explore the margin of the therapeutic window at Fc.V91K doses (SC) of 1, 3, 100, and 200 μg / kg and compare this with the weaker agonist Fc.N88D at doses (SC) of 3, 10, 100, and 200 μg / kg and PROLEUKIN® (SC QDx5) at 3, 10, 30, and 100 μg / kg. The PROLEUKIN® dose was selected based on published human and non-human primate studies (Hartemann et al., 2013, Lancet Diabetes Endocrin 1:295-305; Saadoun et al., 2011, NEJM 365:2067-77; Aoyama et al., 2012, Am J Transplantation 12:2532-37) and was administered QDx5 to mimic low-dose IL-2 clinical trials in HCV vasculitis and type 1 diabetes (T1D).

[0218] [Table 11]

[0219] 11A-F show the kinetics of cellular responses, body temperature, and serum CRP. The time series on the X-axis starts from day 0 instead of day 1 as the day of the first administration.

[0220] The two cynomolgus monkey studies together demonstrated that the IL-2 muteins induced higher Treg enrichment with a broader therapeutic window than was achieved with PROLEUKIN® (Figures 12A and B). In the case of PROLEUKIN®, Treg enrichment was paralleled by NK and eosinophil proliferation. Without being bound to any particular theory, eosinophil proliferation is a well-known response to IL-2 therapy and is associated with increased expression of CD25 +This is likely a result of IL-2-induced induction of IL-5 from innate lymphoid cells. Expansion of CD4 and CD8 Teff occurred at doses that increased Tregs to 25-35% of CD4 T cells. In contrast, Fc.V91K and Fc.N88D induced Treg expansion with selectivity over NK cells and eosinophils, and doses that promoted Teff expansion were higher than doses that enriched Tregs to >40% of CD4 T cells.

[0221] It has been reported in the literature that the first AEs observed in low-dose IL-2 clinical trials were flu-like symptoms and fever. Thus, in addition to comparing therapeutic windows, the aim of this study was to discover biomarkers that precede fever. As shown in Figure 12C, for the two higher doses of PROLEUKIN®, it was found that CRP levels paralleled body temperature. For Fc.V91K, a moderate increase in body temperature was detected at the highest dose, and a slight increase in CRP was observed at the next lower dose. Thus, CRP can be used to monitor the subject's response to treatment with the molecules of the present invention and / or to define the upper limit of dose escalation in patients.

[0222] Specific toxicities were also observed in animals treated with PROLEUKIN® that were less pronounced or absent in animals treated with Fc.V91K or Fc.N88D (Figure 12D). Platelet, neutrophil, and albumin levels were all found to be reduced by treatment with PROLEUKIN®, while doses of either Fc.V91K or Fc.N88D that produced similar or higher enrichment of Tregs caused only slight or no reduction in these parameters. Taken together, these data suggest that the therapeutic window for treatment of patients with either Fc.V91K or Fc.N88D is expected to be significantly larger than that with PROLEUKIN®.

[0223] Example 12 At selected time points, serum from the first cynomolgus study of Example 11 was tested for anti-drug antibodies (ADA) (Figure 13). ADA signal / noise data for samples in which Fc.V91K specificity was confirmed by competition is shown. Time points tested for ADA are indicated by vertical lines above the x-axis. In group 1, one animal produced ADA at least 15 days after the last dose, in group 2, no animals were positive for ADA, and in group 3, ADA consistently appeared in three animals 15 days or more after the first dose. On day 162, groups 1 and 2 were given a repeat dose of 50 μg / kg, and no further animals were positive for ADA 4 weeks later (day 190). The two animals in group 3 that produced the strongest ADA signals (210, 212) showed a reduced PD response, consistent with the decrease in C-max observed in these animals after the second dose. No animals in Group 4 (50 μg / kg IV) were positive for ADA. ADA was specific for both the IL-2 and Fc domains and is expected to be due to the eight amino acid differences between cynomolgus IL-2 and human IL-2 (V91K, C125A). ADA neutralizing activity was not examined.

Claims

1. A human interleukin-2 (IL-2) mutein comprising a V91K substitution and an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:1, wherein the IL-2 mutein selectively stimulates regulatory T cells.

2. 2. The human IL-2 mutein of claim 1, wherein the mutein comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:

1.

3. The human IL-2 mutein according to claim 1 or 2, wherein the mutein comprises the amino acid sequence set forth in SEQ ID NO:

1.

4. 4. The human IL-2 mutein of claim 1, 2, or 3, wherein position 125 is alanine.

5. 4. The human IL-2 mutein of claim 1, 2, or 3, wherein position 125 is a cysteine.

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

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

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

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

10. 10. The Fc fusion protein of claim 9, wherein the substitution at N297 is N297G.

11. The Fc fusion protein according to any one of claims 7 to 10, comprising a substitution or deletion of the C-terminal lysine of the human IgG Fc.

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

13. The Fc fusion protein of any one of claims 6 to 12, wherein a linker connects the Fc and human IL-2 mutein portions of the protein.

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

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

16. 16. The Fc-fusion protein of any of claims 6 to 15, wherein the IL-2 mutein further comprises an amino acid addition, substitution, or deletion that alters the glycosylation of the Fc-fusion protein when expressed in a mammalian cell.

17. The Fc fusion protein of claim 16, wherein the IL-2 mutein comprises a T3 substitution.

18. The Fc fusion protein of claim 17, wherein the IL-2 mutein comprises a T3N or T3A substitution.

19. The Fc fusion protein of claim 18, wherein the IL-2 mutein comprises a T3N substitution.

20. 20. The Fc fusion protein of claim 19, wherein the IL-2 mutein further comprises an S5 mutation.

21. The Fc fusion protein of claim 20, wherein the IL-2 mutein further comprises an S5T mutation.

22. 22. The Fc fusion protein of claim 6, wherein the Fc fusion protein comprises an Fc dimer.

23. The Fc-fusion protein of claim 22, wherein the Fc-fusion protein comprises two IL-2 muteins.

24. The Fc-fusion protein of claim 22, wherein the Fc-fusion protein comprises a single IL-2 mutein.

25. An isolated nucleic acid encoding a human IL-2 mutein according to any one of claims 1 to 5.

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

27. 27. The isolated nucleic acid of claim 26, wherein the Fc portion of an antibody and the human IL-2 mutein are encoded within a single open reading frame.

28. 28. The isolated nucleic acid of claim 26 or 27, wherein the Fc is a human IgG1 Fc.

29. 29. The isolated nucleic acid of claim 28, wherein the human IgGl Fc comprises one or more mutations that alter an effector function of the Fc.

30. 30. The isolated nucleic acid of claim 29, wherein the human IgG1 comprises a substitution at N297.

31. 29. The isolated nucleic acid of claim 28, wherein the substitution at N297 is N297G.

32. 32. The isolated nucleic acid of any one of claims 28 to 31, encoding a C-terminal lysine substitution or deletion of said human IgG Fc.

33. 33. The isolated nucleic acid of claim 32, wherein the C-terminal lysine of the human IgG Fc is deleted.

34. The isolated nucleic acid of any one of claims 26 to 33, further encoding a linker connecting said Fc portion of an antibody and said human IL-2 mutein.

35. 35. The isolated nucleic acid of claim 34, wherein the linker is GGGGS (SEQ ID NO: 5), GGNGT, or (SEQ ID NO: 6), and YGNGT (SEQ ID NO: 7).

36. 36. The isolated nucleic acid of claim 35, wherein the linker is GGGGS (SEQ ID NO:5).

37. 37. The isolated nucleic acid of any of claims 26-36, wherein the IL-2 mutein further comprises an amino acid addition, substitution, or deletion that alters glycosylation of a protein containing the IL-2 mutein when expressed in a mammalian cell.

38. 38. The isolated nucleic acid of claim 37, wherein the IL-2 mutein comprises a T3 substitution.

39. 39. The isolated nucleic acid of claim 38, wherein the IL-2 mutein comprises a T3N or T3A substitution.

40. 40. The isolated nucleic acid of claim 39, wherein the IL-2 mutein comprises a T3N substitution.

41. 41. The isolated nucleic acid of claim 40, wherein the IL-2 mutein further comprises an S5 mutation.

42. 42. The isolated nucleic acid of claim 41, wherein the IL-2 mutein further comprises an S5T mutation.

43. 43. An expression vector comprising the isolated nucleic acid of any one of claims 25 to 42 operably linked to a promoter.

44. A host cell comprising the isolated nucleic acid of any one of claims 25 to 42.

45. 45. The host cell of claim 44, wherein the isolated nucleic acid is operably linked to a promoter.

46. 46. ​​The host cell of claim 44 or 45, wherein the host cell is a prokaryotic cell.

47. The host cell of claim 46 , wherein the host cell is E. coli.

48. 46. ​​The host cell of claim 44 or 45, wherein the host cell is a eukaryotic cell.

49. 49. The host cell of claim 48, wherein the host cell is a mammalian cell.

50. 50. The host cell of claim 49, wherein the host cell is a Chinese Hamster Ovary (CHO) cell line.

51. 51. A method for producing a human IL-2 mutein, comprising culturing a host cell according to any one of claims 45 to 50 under conditions in which the promoter is expressed, and recovering the human IL-2 mutein from the culture.

52. 51. A method for producing an Fc-fusion protein, comprising culturing a host cell according to any one of claims 45 to 50 under conditions in which the promoter is expressed, and recovering the Fc-fusion protein from the culture.

53. A method for increasing the ratio of regulatory T cells (Treg) to non-regulatory T cells in a population of T cells, comprising contacting the population of T cells with an effective amount of a human IL-2 mutein according to any one of claims 1 to 5.

54. 54. The method of claim 53, wherein the ratio of CD3+FoxP3+ cells to CD3+FoxP3- cells is increased.

55. 55. The method of claim 54, wherein the ratio of CD3+FoxP3+ cells to CD3+FoxP3- cells is increased by at least 50%.

56. A method for increasing the ratio of regulatory T cells (Treg) to non-regulatory T cells in a population of T cells, comprising contacting the population of T cells with an effective amount of an Fc fusion protein of any one of claims 6 to 24.

57. 57. The method of claim 56, wherein the ratio of CD3+FoxP3+ cells to CD3+FoxP3- cells is increased.

58. 58. The method of claim 57, wherein the ratio of CD3+FoxP3+ cells to CD3+FoxP3- cells is increased by at least 50%.

59. A method for increasing the ratio of regulatory T cells (Treg) to non-regulatory T cells in the peripheral blood of a subject, comprising administering an effective amount of a human IL-2 mutein according to any one of claims 1 to 5.

60. 60. The method of claim 59, wherein the ratio of CD3+FoxP3+ cells to CD3+FoxP3- cells is increased.

61. 61. The method of claim 60, wherein the ratio of CD3+FoxP3+ cells to CD3+FoxP3- cells is increased by at least 50%.

62. A method for increasing the ratio of regulatory T cells (Treg) to non-regulatory T cells in the peripheral blood of a subject, comprising administering an effective amount of an Fc fusion protein according to any one of claims 6 to 24.

63. 63. The method of claim 62, wherein the ratio of CD3+FoxP3+ cells to CD3+FoxP3- cells is increased.

64. 64. The method of claim 63, wherein the ratio of CD3+FoxP3+ cells to CD3+FoxP3- cells is increased by at least 50%.

65. A method for increasing the ratio of regulatory T cells (Treg) to natural killer (NK) cells in the peripheral blood of a subject, comprising administering an effective amount of a human IL-2 mutein according to any one of claims 1 to 5.

66. The method of claim 65, wherein the ratio of CD3+FoxP3+ cells to CD3-CD19- lymphocytes expressing CD56 and / or CD16 is increased.

67. 67. The method of claim 66, wherein the ratio of CD3+FoxP3+ cells to CD3-CD19- lymphocytes expressing CD56 and / or CD16 is increased by at least 50%.

68. A method for increasing the ratio of regulatory T cells (Treg) to natural killer (NK) cells in the peripheral blood of a subject, comprising administering an effective amount of an Fc fusion protein according to any one of claims 6 to 24.

69. The method of claim 68, wherein the ratio of CD3+FoxP3+ cells to CD3-CD19- lymphocytes expressing CD56 and / or CD16 is increased.

70. The method of claim 63, wherein the ratio of CD3+FoxP3+ cells to CD3-CD19- lymphocytes expressing CD56 and / or CD16 is increased by at least 50%.

71. A method of treating a patient suffering from an inflammatory or autoimmune disease, comprising administering to said subject a therapeutically effective amount of an IL-2 mutein according to any one of claims 1 to 5.

72. A method for treating a patient suffering from an inflammatory or autoimmune disease, comprising administering to said subject a therapeutically effective amount of an Fc fusion protein according to any one of claims 6 to 21.

73. 73. A method of treating a patient suffering from an inflammatory or autoimmune disease according to claim 71 or 72, wherein administration results in the alleviation of at least one symptom of the disease.

74. 74. The method of claim 73, wherein the ratio of regulatory T cells (Treg) to non-regulatory T cells in the peripheral blood of the subject is increased following administration.

75. 74. The method of claim 73, wherein the ratio of regulatory T cells (Treg) to non-regulatory T cells in the subject's peripheral blood remains essentially the same following administration.

76. 76. The method of any of claims 71 to 75, wherein the inflammatory or autoimmune disease is lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type I diabetes, multiple sclerosis, spontaneous abortion, atopic disease, or inflammatory bowel disease.

77. A polypeptide comprising an Fc region of a human IgG1 antibody, said Fc region comprising an N297G mutation, said Fc region of human IgG1 having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:

3.

78. The polypeptide of claim 77, wherein the Fc region of human IgG1 has at least 95% identity to the amino acid sequence set forth in SEQ ID NO:

3.

79. The polypeptide of claim 77, wherein the Fc region of human IgG1 comprises the amino acid sequence set forth in SEQ ID NO:

3.

80. 78. The polypeptide of claim 77, wherein the Fc region of human IgG1 further comprises one or more mutations to stabilize the polypeptide.

81. 81. The polypeptide of claim 80, wherein one or more amino acids set forth in SEQ ID NO:3 are substituted with cysteine.

82. 82. The polypeptide of claim 81, wherein V259, A287, R292, V302, L306, V323, or I332 of the amino acid sequence set forth in SEQ ID NO:3 is substituted with cysteine.

83. 83. The polypeptide of claim 82, wherein the Fc region comprises A287C and L306C substitutions in the amino acid sequence set forth in SEQ ID NO:

3.

84. 83. The polypeptide of claim 82, wherein the Fc region comprises a V259C and L306C substitution in the amino acid sequence set forth in SEQ ID NO:

3.

85. 83. The polypeptide of claim 82, wherein the Fc region comprises an R292C and a V302C substitution in the amino acid sequence set forth in SEQ ID NO:

3.

86. 83. The polypeptide of claim 82, wherein the Fc region comprises a V323C and I332C substitution in the amino acid sequence set forth in SEQ ID NO:

3.

87. An antibody comprising an Fc region described in any one of claims 77 to 86.

88. An Fc fusion protein comprising the Fc region of any one of claims 77 to 86.

89. A polypeptide comprising a linker, said linker being GGNGT (SEQ ID NO: 6) or YGNGT (SEQ ID NO: 7).

90. The polypeptide of claim 89, wherein the linker comprises an N-glycosylation.

91. 90. The polypeptide of claim 89, wherein the linker is inserted into a loop within the polypeptide structure or replaces the structure.

92. 1. A method of making an aglycosylated IgG1 Fc-containing molecule, comprising: a) expressing in a mammalian cell culture a nucleic acid encoding a polypeptide according to any one of claims 77 to 86; b) recovering said non-glycosylated IgG1 Fc-containing molecules from said culture.

93. 1. A method of making an IgG1 Fc-containing molecule that is aglycosylated upon expression in a mammalian cell, comprising the step of mutating the codon for N297 in the Fc region to a glycine codon.

94. An Fc fusion protein consisting of the sequence of SEQ ID NO: 18 or SEQ ID NO:

20.

95. A nucleic acid encoding the Fc fusion of claim 94.

96. A cell comprising the nucleic acid of claim 95.

97. 97. A method of making an Fc-fusion protein comprising incubating a cell of claim 96 under conditions that allow the cell to express the Fc-fusion protein.

98. 95. A method of treating an inflammatory or autoimmune condition in a subject comprising administering to the subject an effective amount of the Fc-fusion protein of claim 94.

99. 99. The method of claim 98, wherein the inflammatory or autoimmune condition is graft-versus-host disease.

100. 10. A method for monitoring a subject's response to treatment with a human interleukin-2 (IL-2) mutein of claim 1, an Fc-fusion protein of claim 6, or an Fc-fusion protein of claim 94, comprising detecting a change in the subject, said change comprising: a) Increase in body temperature, b) an elevation of CRP in the peripheral blood of said subject; c.) a decrease in platelets in the peripheral blood of said subject; d) a decrease in peripheral blood neutrophils in the subject; or e) a decrease in albumin in the peripheral blood of the subject; After said alteration is detected, said treatment is stopped, suspended, reduced in frequency, or reduced in dosage.

101. The change is a) an increase in body temperature of at least 0.5°C; b) an elevation of CRP in the peripheral blood of said subject of at least 0.2 mg / mL; c) a decrease in peripheral blood platelets in said subject of at least 0.8 fold; d) a decrease in peripheral blood neutrophils in said subject of at least 0.8-fold; or The method of claim 100, comprising: e) a decrease in albumin in the peripheral blood of the subject of at least 0.4-fold.

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