Dual interleukin-2 / TNF receptor agonists for use in therapy - Patent Application 20070122997

Abstract

Provided herein are combinations of IL-2 molecules or mutant proteins with TNFR agonists, and conjugates comprising IL-2 / TNFR agonist molecules, such as Fc-linked IL-2 / TNFR agonist molecules, that preferentially expand and activate regulatory T cells and are suitable for large-scale production. Also provided herein are methods of making and using the compositions of this disclosure.

Description

[Background technology]

[0001] Regulatory T (Treg) cells, identified by expression of the transcription factor Foxp3, are a specialized subset of CD4 T cells tasked with limiting the activation and response of both innate and adaptive immune cells. Deletion or loss-of-function mutations in the Foxp3 gene cause an early-onset, multiorgan, and often fatal autoimmune disorder called IPEX (immunodysregulation, polyendocrinopathy, and enteropathy, X-linked) in both humans and mice. In Foxp3-deficient animals, various types of inflammatory responses are naturally deregulated, suggesting that Treg cells are required for maintaining normal immune homeostasis. Several human autoimmune disorders, such as type 1 diabetes (T1D), multiple sclerosis (MS), and systemic lupus erythematosus (SLE), exhibit defects in either the number or suppressive function of Treg cells isolated from peripheral blood. Because Treg cells can profoundly influence immune responses, positively targeting their number, function, or stability during autoimmunity represents an attractive therapeutic approach.

[0002] The maintenance of Treg cells is critically dependent on two major signaling pathways: T cell receptor (TCR) and IL-2 receptor signaling; the absence of either of these signaling pathways severely impairs Treg cell homeostasis and function. Compared to other cells, Treg cells express high levels of the high-affinity IL-2 receptor subunit (IL-2Rα, CD25). Based on this notion that IL-2 is a key cytokine for Treg cell differentiation, survival, and function, numerous studies have attempted to determine whether selective targeting of this pathway has therapeutic potential. In clinical settings, low-dose IL-2 has been evaluated for the treatment of several inflammatory diseases, such as chronic graft-versus-host disease (GVHD), T1D, and SLE, and has been shown to increase Treg cell numbers while also reducing disease activity. Therefore, improved methods for expanding Treg numbers are desirable. Summary of the Invention [Means for solving the problem]

[0003] Described herein are pharmacologically active human interleukin-2 (IL-2) chimeric molecules that can be produced in high yields, comprising a human IL-2 polypeptide comprising an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 1, and a tumor necrosis factor receptor (TNFR) agonist selected from the group consisting of anti-OX40, anti-DR3, and TNF conjugates. In efforts to generate such molecules for use as human therapeutics, several unexpected and unpredictable observations have occurred. The compositions and methods described herein are the result of this effort.

[0004] In some embodiments, the invention is an Fc fusion protein comprising an Fc, a human IL-2 polypeptide comprising an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO:1, and a tumor necrosis factor receptor (TNFR) agonist selected from the group consisting of anti-OX40, anti-DR3, and TNF.

[0005] In some embodiments, the invention provides a method of treating a subject suffering from an inflammatory disease or an autoimmune disease, comprising administering to the subject a therapeutically effective amount of a human interleukin-2 (IL-2) chimeric molecule comprising a human IL-2 polypeptide comprising an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO:1 and a tumor necrosis factor receptor (TNFR) agonist selected from the group consisting of anti-OX40, anti-DR3, and TNF conjugates, or an Fc fusion protein comprising an Fc, a human IL-2 polypeptide comprising an amino acid sequence at least 70% identical to the amino acid sequence set forth in SEQ ID NO:1, and a tumor necrosis factor receptor (TNFR) agonist selected from the group consisting of anti-OX40, anti-DR3, and TNF. [Brief explanation of the drawings]

[0006] [Figure 1]Figure 1 shows Treg proliferation upon stimulation with IL-2 in combination with TNFR agonists (anti-OX40, recombinant TNF, or anti-DR3). (A) Cell Trace Violet (CTV)-labeled human PBMCs were stimulated with the indicated reagents for 4 days and then subjected to flow cytometry analysis. Histograms are arranged in the following order (bottom to top): no stimulation, stimulation with 1 μg / ml anti-CD3, 20 U / ml IL-2, IgG, TNFR agonists (anti-OX40, recombinant TNF, anti-DR3), and TNFR agonists + IL-2. Histograms were gated on Treg cells (CD4+Foxp3+). Only the positive control anti-CD3 plot and the combination of TNFR agonists (anti-OX40, recombinant TNF, and anti-DR3) showed Treg proliferation. [Figure 2A] Histogram plots of anti-OX40 and IL-2 on PBMCs are shown. (A) CTV-labeled human PBMCs were stimulated with the indicated titrated doses of anti-OX40 (clone 15A9) for 4 days followed by flow cytometry analysis. Histograms were gated on Treg cells (CD4+Foxp3+). CD3-stimulated cells showed robust proliferation and served as a positive control for this assay. Stimulation with IL2 or control IgG did not result in any CTV dilution. Anti-OX40 alone did not result in proliferation at any of the indicated doses. However, combining stimulation with anti-OX40 and IL-2 led to Treg cell proliferation as seen by CTV dilution. (B) A summary of the data in (A). Graphs show three replicates from one donor per condition. T cell responses to these stimuli were also determined, and only very low levels of T cell proliferation were observed with high-dose anti-OX40 / IL-2 treatment. [Figure 2B]Histogram plots of anti-OX40 and IL-2 on PBMCs are shown. (A) CTV-labeled human PBMCs were stimulated with the indicated titrated doses of anti-OX40 (clone 15A9) for 4 days followed by flow cytometry analysis. Histograms were gated on Treg cells (CD4+Foxp3+). CD3-stimulated cells showed robust proliferation and served as a positive control for this assay. Stimulation with IL2 or control IgG did not result in any CTV dilution. Anti-OX40 alone did not result in proliferation at any of the indicated doses. However, combining stimulation with anti-OX40 and IL-2 led to Treg cell proliferation as seen by CTV dilution. (B) A summary of the data in (A). Graphs show three replicates from one donor per condition. T cell responses to these stimuli were also determined, and only very low levels of T cell proliferation were observed with high-dose anti-OX40 / IL-2 treatment. [Figure 3A] Histogram plots of TNF and IL-2 responses to PBMCs are shown. (A) Flow cytometry analysis was performed after stimulation of CTV-labeled human PBMCs with the indicated titrated doses of TNF for 4 days. Histograms were gated on Treg cells (CD4+Foxp3+). TNF alone did not induce proliferation at any of the indicated doses. However, combined stimulation with TNF and IL-2 led to Treg cell proliferation as seen by CTV dilution. (B) Summary of the data in (A). Graphs show three replicates from one donor per condition. T cell responses to these stimuli were also determined, and only very low levels of T cell proliferation were observed with TNF / IL-2 treatment at all doses. [Figure 3B]Histogram plots of TNF and IL-2 responses to PBMCs are shown. (A) Flow cytometry analysis was performed after stimulation of CTV-labeled human PBMCs with the indicated titrated doses of TNF for 4 days. Histograms were gated on Treg cells (CD4+Foxp3+). TNF alone did not induce proliferation at any of the indicated doses. However, combined stimulation with TNF and IL-2 led to Treg cell proliferation as seen by CTV dilution. (B) Summary of the data in (A). Graphs show three replicates from one donor per condition. T cell responses to these stimuli were also determined, and only very low levels of T cell proliferation were observed with TNF / IL-2 treatment at all doses. [Figure 4A] Histogram plots of anti-DR3 and IL-2 on PBMCs are shown. (A) CTV-labeled human PBMCs were stimulated with the indicated titrated doses of anti-DR3 for 4 days followed by flow cytometry analysis. Histograms were gated on Treg cells (CD4+Foxp3+). Anti-DR3 alone did not induce proliferation at any of the indicated doses. However, combined stimulation with anti-DR3 and IL-2 led to Treg cell proliferation. (B) Summary of the data in (A). Graphs show three replicates from one donor per condition. T cell responses to these stimuli were also determined, and very low levels of T cell proliferation were observed with anti-DR3 / IL-2 treatment at all doses. [Figure 4B]Histogram plots of anti-DR3 and IL-2 on PBMCs are shown. (A) CTV-labeled human PBMCs were stimulated with the indicated titrated doses of anti-DR3 for 4 days followed by flow cytometry analysis. Histograms were gated on Treg cells (CD4+Foxp3+). Anti-DR3 alone did not induce proliferation at any of the indicated doses. However, combined stimulation with anti-DR3 and IL-2 led to Treg cell proliferation. (B) Summary of the data in (A). Graphs show three replicates from one donor per condition. T cell responses to these stimuli were also determined, and very low levels of T cell proliferation were observed with anti-DR3 / IL-2 treatment at all doses. [Figure 5A] Histogram plots of anti-GITR and IL-2 on PBMCs are shown. (A) CTV-labeled human PBMCs were stimulated with the indicated titrated doses of anti-GITR for 4 days, followed by flow cytometry analysis. The histograms were gated on Treg cells (CD4+Foxp3+). Anti-GITR alone resulted in very low levels of proliferation. However, combined stimulation with anti-GITR and IL-2 led to more significant Treg cell proliferation. (B) Summary of the data in (A). Graphs show three replicates from one donor per condition. T cell responses to these stimuli were also determined, and only minimal levels of T cell proliferation were observed with anti-GITR / IL-2 treatment at all doses. [Figure 5B] Histogram plots of anti-GITR and IL-2 on PBMCs are shown. (A) CTV-labeled human PBMCs were stimulated with the indicated titrated doses of anti-GITR for 4 days, followed by flow cytometry analysis. The histograms were gated on Treg cells (CD4+Foxp3+). Anti-GITR alone resulted in very low levels of proliferation. However, combined stimulation with anti-GITR and IL-2 led to more significant Treg cell proliferation. (B) Summary of the data in (A). Graphs show three replicates from one donor per condition. T cell responses to these stimuli were also determined, and only minimal levels of T cell proliferation were observed with anti-GITR / IL-2 treatment at all doses. [Figure 6] 1 shows diagrams of chimeric OX-40 antibody and IL-2 molecules in several different formats. [Figure 7] 1 shows an in vivo mouse study using an OX-40 antibody and its binding IL-2 molecule to measure levels of Tregs, activated CD4+ and CD8+ T cells, and NK cells measured on days 4 and 15. The study shows mice treated with IL-2 alone, anti-OX-40 alone, or the chimeric molecule, as well as a control. DETAILED DESCRIPTION OF THE INVENTION

[0007] The section 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 by reference in their entirety.

[0008] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, tissue culture and transformation, protein purification, etc. 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 the specification. See, e.g., Sambrook et al., 2001, Molecular Cloning: A Laboratory Manuel, 3 rded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which is incorporated herein by reference for all purposes. 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 are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0009] 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 acts to stabilize the interaction between IL-2 and IL-2Rβγ. Signals transmitted by IL-2Rβγ include those of the PI3 kinase, Ras-MAP kinase, and STAT5 pathways.

[0010] Expression of CD25 is required for T cell responses to the low concentrations of IL-2 typically present in tissues. CD25-expressing T cells include both FOXP3+ regulatory 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. IL-2-stimulated STAT5 signaling is critical for normal T-reg cell growth and survival and for high FOXP3 expression.

[0011] At steady state, compared with other immune cells, Treg cells have also been found to express high surface levels of several TNF (tumor necrosis factor) receptor family members, most notably TNFR2, OX40, GITR, 4-1BB, CD30, and DR3. Expression of these TNF receptors, particularly OX40, GITR, and TNFR2, on Treg cells directly correlates with TCR signaling strength, and these receptors have been shown to enhance Treg cell development in the thymus by increasing sensitivity to IL-2 and providing costimulatory signals. IL-2 signaling also influences the expression of these TNFRs. Here, the inventors have discovered a synergistic effect between these two pathways.

[0012] The influence of TNFR signaling on Treg cells in the periphery remains unclear, as its modulation has diverse effects. For example, engagement of OX40 with Treg cells leads to loss of Foxp3 expression and reduced Treg cell function, whereas TNFR2 signaling has been implicated in maintaining Treg cell numbers and function. The present invention demonstrates that combining TNFR agonists, such as TNFR2, GITR, OX40, and DR3, with IL-2 stimulation enhances Treg cell proliferation. Because TNFR ligand expression is generally upregulated by antigen-presenting cells after sensing inflammatory signals, and IL-2 is secreted by T cells upon activation, these may cooperate to drive robust Treg cell proliferation during inflammation. Some embodiments of the present invention are chimeric fusion molecules between a TNFR agonist and IL-2, which may be selective for Treg cell proliferation. In some embodiments, the chimeric molecule is linked to an Fc molecule. In some embodiments, the invention is a method of expanding Tregs by co-administration of a TNFR agonist and an IL-2 molecule or mutein.

[0013] IL-2 The IL-2 molecules described herein include wild-type and variants of wild-type human IL-2. As used herein, "wild-type human IL-2," "wild-type IL-2," or "WT IL-2" shall mean a polypeptide having the following amino acid sequence: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFXQSIISTLT wherein X is C, S, V, or A (SEQ ID NO: 1).

[0014] Variants may contain one or more substitutions, deletions, or insertions in the wild-type IL-2 amino acid sequence, and include the IL-2 mutein variants described in WO2010085495, WO2014153111, WO2016164937, PCT / US2020 / 046202, WO1999060128, WO2002000243, WO2012107417, WO2005086798, WO2005086751, and WO2006089064, which are hereby incorporated by reference in their entireties. An example of an IL-2 mutein contains the mutation V91K, which has the following amino acid sequence: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINKIVLELKGSETTFMCEYADETATIVEFLNRWITFXQSIISTLT wherein X is C, S, V, or A (SEQ ID NO: 2).

[0015] Residues are designated herein by the single letter amino acid code followed by the IL-2 amino acid position, e.g., K35 is the lysine residue at position 35 of SEQ ID NO: 2. Substitutions are designated herein by the single letter amino acid code followed by the IL-2 amino acid position followed by the substituting single letter amino acid code, e.g., K35A is the substitution of the lysine residue at position 35 of SEQ ID NO: 2 with an alanine residue.

[0016] IL-2 mutant proteins Provided herein are human IL-2 molecules and muteins that, in combination with a TNFR agonist, preferentially stimulate regulatory T (Treg) cells. As used herein, "preferentially stimulates regulatory T cells" means that the mutein or antibody promotes the proliferation, survival, activation, and / or function of CD3+FoxP3+ T cells over CD3+FoxP3- T cells. The ability to preferentially stimulate Tregs can be measured by flow cytometry of peripheral blood leukocytes, observing an increased percentage of FOXP3+CD4+ T cells among total CD4+ T cells, an increased percentage of FOXP3+CD8+ T cells among total CD8+ T cells, an increased percentage of FOXP3+ T cells compared to NK cells, and / or a greater increase in the level of CD25 expression on the surface of FOXP3+ T cells compared to increased CD25 expression on other T cells. Additionally, preferential proliferation of Treg cells can be detected as increased expression of demethylated FOXP3 promoter DNA (i.e., Treg-specific demethylated region, or TSDR) compared to demethylated CD3 gene in DNA extracted from whole blood, as detected by sequencing polymerase chain reaction (PCR) products from genomic DNA treated with bisulfite (J. Sehouli, et al. 2011. Epigenetics 6:2, 236-246).

[0017] IL-2 molecules and mutant proteins that preferentially stimulate Treg cells in combination with a TNFR agonist 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%.

[0018] Examples of IL-2 muteins include, but are not limited to, IL-2 muteins including V91K, N30S, N30D, Y31H, Y31S, K35R, V69A, Q74P, V91K / D20L, D84R / E61Q, V91K / D20A / E61Q / M104T, N88K / M104L, V91H / M104L, V91K / H16E / M104V, V91K / H16R / M104V, V91K / H16R / M104T, V91K / D20A / M104T, V91K / H16E / M104T, V91K / H16E / E61Q / M104T, V91K / H16R / E61Q / M104T, V91K / H16E, V91H / D20A / M104T, H16E / V91H / M104V, V91H / D20A / E61Q / M104T, V91H / H16R / E16Q , V91K / D20A / M104V, H16E / V91H, V91H / D20A / M104V, H16E / V91H / M104T, H16 E / V91H / E61Q / M104T, V91K / E61Q / H16E, V91K / H16R / M104L, H16E / V91H / E16Q , V91K / E61Q / H16R, D20W / V91K / E61Q, V91H / H16R, V91K / H16R, D20W / V91K / E 61Q / M104T, V91K / D20A, V91H / D20A / E16Q, V91K / D20A / M104L, V91H / D20A, V9 1K / E61Q / D20A, V91H / M104T, V91H / M104V, V91K / E61Q, V91K / N88K / E61Q / M1 04T, V91K / N88K / E61Q, V91H / E61Q, V91K / N88K, D20A / H16E / M104T, D20A / M10 4T, H16E / N88K, D20A / M104V, D20A / M104L, H16E / M104T, H16E / M104V, N88K / M104V, N88K / E61Q, D20A / E61Q, H16R / D20A, D20W / E61Q, H16E / E61Q, H16E / M1 04L, N88K / M104T, D20A / H16E, D20A / H16E / E16Q, D20A / H16R / E16Q, V91K / D2 0W, V91A / H16A, V91A / H16D, V91A / H16E, V91A / H16S, V91E / H16A, V91E / H16D,V91E / H16E, V91E / H16S, V91K / H16A, V91K / H16D, V91K / H16S, V91S / H16E, L12G, L12K, L12Q, L12S, Q13G, E15A, E15G, E15S, H16A, H16D, H16G, H16K, H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D, L19E, L19G, L19N, L19R, L19S, L19T, L19V, D20A, D20E, D 20F, D20G, D20T, D20W, M23R, N30S, Y31H, K35R, V69A, Q74P, R81A, R81G, R81S, R81T, D84A, D84E, D84G, D84I, D84M, D84Q, D84R, D84S, D84T, S87R, N88A, N88D, N88E, N88F, N88G, N88M, N88R, N88S, N88V, N88W, V91D, V91E, V91G, V91S, I92K, I92R, and / or E95G substitutions. The IL-2 molecules and muteins of the invention optionally comprise a C125A substitution. Additionally, 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 containing truncations and / or additional insertions, deletions, and / or substitutions in addition to the following: V91K, N30S, N30D, Y31H, Y31S, K35R, V69A, Q74P, V91K / D20L, D84R / E61Q, V91K / D20A / E61Q / M104T, N88K / M104L, V91H / M104L, V91K / H16E / M104V, V91K / H16R / M104V, V91K / H16R / M104T , V91K / D20A / M104T, V91K / H16E / M104T, V91K / H16E / E61Q / M104T, V91K / H16R / E 61Q / M104T, V91K / H16E, V91H / D20A / M104T, H16E / V91H / M104V, V91H / D20A / E61Q / M104T, V91H / H16R / E16Q, V91K / D20A / M104V, H16E / V91H, V91H / D20A / M104V, H1 6E / V91H / M104T, H16E / V91H / E61Q / M104T, V91K / E61Q / H16E, V91K / H16R / M104L,H16E / V91H / E16Q, V91K / E61Q / H16R, D20W / V91K / E61Q, V91H / H16R, V91 K / H16R, D20W / V91K / E61Q / M104T, V91K / D20A, V91H / D20A / E16Q, V91K / D 20A / M104L, V91H / D20A, V91K / E61Q / D20A, V91H / M104T, V91H / M104V, V 91K / E61Q, V91K / N88K / E61Q / M104T, V91K / N88K / E61Q, V91H / E61Q, V91K / N88K, D20A / H16E / M104T, D20A / M104T, H16E / N88K, D20A / M104V, D20A / M104L, H16E / M104T, H16E / M104V, N88K / M104V, N88K / E61Q, D20A / E61Q , H16R / D20A, D20W / E61Q, H16E / E61Q, H16E / M104L, N88K / M104T, D20A / H16E, D20A / H16E / E16Q, D20A / H16R / E16Q, V91K / D20W, V91A / H16A, V91A / H16D, V91A / H16E, V91A / H16S, V91E / H16A, V91E / H16D, V91E / H16E, V9 1E / H16S, V91K / H16A, V91K / H16D, V91K / H16S, V91S / H16E, L12G, L12K, L 12Q, L12S, Q13G, E15A, E15G, E15S, H16A, H16D, H16G, H16K, H16M, H16N , H16R, H16S, H16T, H16V, H16Y, L19A, L19D, L19E, L19G, L19N, L19R, L19 S, L19T, L19V, D20A, D20E, D20F, D20G, D20T, D20W, M23R, N30S, Y31H, K35R, V69A, Q74P, R81A, R81G, R81S, R81T, D84A, D84E, D84G, D84I, D84M, D84Q, D84R, D84S, D84T, S87R, N88A, N88D, N88E, N88F, N88G, N88M, N88R, N88S, N88V, N88W, V91D, V91E, V91G, V91S, I92K, I92R, and / or E95G substitutions. However, this is limited to the case where the mutant protein maintains the activity of preferentially stimulating Tregs.Preferentially stimulates Treg cells, and V91K, N30S, N30D, Y31H, Y31S, K35R, V69A, Q74P, V91K / D20L, D84R / E61Q, V91K / D20A / E61Q / M104T, N88K / M104L, V91H / M104L, V91K / H16E / M104V, V91K / H16R / M104V, V91K / H16R / M104T, V91K / D20A / M104T, V91K / H16E / M104T, V91K / H16E / E61Q / M104T, V91K / H 16E, V91H / D20A / M104T, H16E / V91H / M104V, V91H / D20A / E61Q / M104T, V91H / H16R / E16Q, V91K / D20A / M104V, H16E / V91H, V91H / D20A / M104V, H16E / V91H / M104T, H16E / V91H / E61Q / M104T, V91K / E61Q / H16E, V91K / H16R / M104L, H16E / V91H / E16Q, V91K / E61Q / H16R, D20W / V91K / E61Q, V91H / H16R, V91K / H16R, D2 0W / V91K / E61Q / M104T, V91K / D20A, V91H / D20A / E16Q, V91K / D20A / M104L, V9 1H / D20A, V91K / E61Q / D20A, V91H / M104T, V91H / M104V, V91K / E61Q, V91K / N8 8K / E61Q / M104T, V91K / N88K / E61Q, V91H / E61Q, V91K / N88K, D20A / H16E / M10 4T, D20A / M104T, H16E / N88K, D20A / M104V, D20A / M104L, H16E / M104T, H16E / M104V, N88K / M104V, N88K / E61Q, D20A / E61Q, H16R / D20A, D20W / E61Q, H16E / E61Q, H16E / M104L, N88K / M104T, D20A / H16E, D20A / H16E / E16Q, D20A / H16R / E16Q, V91K / D20W, V91A / H16A, V91A / H16D, V91A / H16E, V91A / H16S, V91E / H1 6A, V91E / H16D, V91E / H16E, V91E / H16S, V91K / H16A, V91K / H16D, V91K / H16S,V91S / H16E, L12G, L12K, L12Q, L12S, Q13G, E15A, E15G, E15S, H16A, H16D, H16G , H16K, H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D, L19E, L19G, L19N , L19R, L19S, L19T, L19V, D20A, D20E, D20F, D20G, D20T, D20W, M23R, N30S, Y31 H, K35R, V69A, Q74P, R81A, R81G, R81S, R81T, D84A, D84E, D84G, D84I, D84M, D84 and / or E95G substitutions, and are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1. 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:1.

[0019] For amino acid sequences, sequence identity and / or similarity are determined by standard techniques known in the art, such as, but not limited to, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. USA 85:2444, computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the BestFit alignment program described by Devereux et al., 1984, Nucl. Acid Res. 12:387-395, preferably using default settings, or by visual inspection. Preferably, percent identity is calculated by FastDB based on the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30, "Current Methods in Sequence Comparison and Analysis", Macromolecules Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149 (1988), Alan R. Liss, Inc.

[0020] One example of a useful algorithm is PILEUP. PILEUP uses progressive pairwise alignments to create a multiple sequence alignment from a group of related sequences. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplified version of the progressive alignment method of Feng & Doolittle, 1987, J. Mol. Evol. 35:351-360; the method is similar to that described by Higgins and Sharp, 1989, CABIOS 5:151-153. Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

[0021] Another example of a useful algorithm is the BLAST algorithm described in Altschul et al., 1990, J. Mol. Biol. 215:403410; Altschul et al., 1997, Nucleic Acids Res. 25:33893402; and Karin et al., 1993, Proc. Natl. Acad. Sci. USA 90:5873-5787. A particularly useful BLAST program is the WU-BLAST-2 program obtained from Altschul et al., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses several search parameters, most of which are set to default values. Adjustable parameters are set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 1. For alignment purposes, the claimed invention preferentially utilizes these parameters and BLAST as the alignment algorithm. 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 in which the sequence of interest is being searched; however, the values ​​may be adjusted to increase sensitivity.

[0022] An additional useful algorithm is Gapped BLAST, as 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 triggering ungapped extension, a charge of 10 + k for a gap length of k; and an X parameter set to 16. u , and X, which was set to 40 for the database search stage and 67 for the output stage of the algorithm. g Gapped alignments are triggered by scores corresponding to approximately 22 bits.

[0023] The site or region for introducing an amino acid sequence variation 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 may be conducted at the target codon or region, and the expressed IL-2 muteins screened for the optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis. Screening of mutants may be performed, for example, using the assays described herein.

[0024] Amino acid substitutions are typically of single residues; insertions will usually be on the order of about 1 to about 20 amino acid residues, although significantly larger insertions can be tolerated. Deletions range from about 1 to about 20 amino acid residues, although in some cases deletions may be much larger.

[0025] 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 alteration of the immunogenicity and specificity of the molecule, especially the antigen-binding protein. However, in certain circumstances, larger changes may be tolerated. Conservative substitutions are generally made according to the following chart shown as Table 1.

[0026] [Table 1]

[0027] Substantial changes in function or immunological identity can be made by selecting substitutions that are less conservative than those shown in Table 1. For example, substitutions can be made that have a greater effect on the structure of the polypeptide backbone, e.g., alpha-helical or beta-sheet structure, in the region of the change; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain. In general, the substitutions expected to produce the greatest changes in the properties of the polypeptide are those in which a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) cysteine ​​or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) a residue having no side chain, e.g., glycine.

[0028] Variants typically exhibit the same qualitative biological activity and elicit the same immune response as the naturally occurring analog, but variants are also selected to modify the characteristics of the IL-2 mutein, if desired. Alternatively, variants may be designed to alter the biological activity of the IL-2 mutein. For example, glycosylation sites may be altered or removed, as discussed herein.

[0029] TNFR agonists The tumor necrosis factor receptor (TNFR) superfamily is a family of cytokine receptors that bind tumor necrosis factor (TNF) through their extracellular cysteine-rich domains, forming trimeric complexes in the cell membrane. Some members of the TNFR family contain a death domain and are called death receptors.

[0030] The TNFR agonists of the present invention, in combination with the IL-2 molecules and muteins of the present invention, preferentially stimulate T regulatory (Treg) cells. TNFR agonists of the present invention include tumor necrosis factor receptor 1 (TNFR1); tumor necrosis factor receptor 2 (TNFR2); lymphotoxin beta receptor (LTBR); OX40; CD40; Fas receptor; decoy receptor 3; CD27; CD30; 4-1BB; death receptors 1, 2, 3, 4, 5, and 6; RANK, osteoprotegerin; TWEAK receptor; TACI; BAFF receptor; herpesvirus entry mediator; nerve growth factor receptor; B-cell maturation antigen; glucocorticoid-inducible TNFR-related protein; TROY; and ectodysplasin A2 receptor, as well as agonist antibodies against these receptors, such as OX40 agonist antibodies.

[0031] At baseline, Treg cells express several different TNFR family members, including GITR, 4-1BB (CD137), OX40, DR3, and TNFR2, at much higher levels than other immune cell populations. TNFR expression on different T cell subsets was determined from single-cell RNA data. For example, TNFR levels on various immune cell subsets can be extracted from human single-cell RNA sequencing data, such as those available at http: / / crc.cancer-pku.cn / index.php. In some embodiments, TNFR agonists of the present invention include anti-OX40, anti-DR3, and TNF. In some embodiments, the TNFR agonist may be a ligand of a TNFR member. These include TNF-α; lymphotoxin-β (TNF-C); OX40L; CD154; FasL; LIGHT; TL1A; CD70; Siva; CD153; 4-1BB ligand; TRAIL; RANKL; TWEAK; APRIL; BAFF; CAMLG; NGF; BDNF; NT-3; NT-4; GITR ligand; and EDA-A2.

[0032] Exemplary antibodies to OX40 include those that act as agonist antibodies found in WO2007062245A2, WO2010096418A2, WO2013008171A1, WO2013028231A1, WO2013038191A2, WO2013068563A2, WO2014148895A1, WO2015153513A1, WO2016057667A1, WO2016179517A1, WO2016196228A1, and WO2018112346A1. In some embodiments, the present invention is useful for antibodies against OX40, which can act as OX40 receptor agonists.Examples of antibodies against OX40 receptor include those found in International Publication No. 2003106498A2.Examples of OX40 ligands include those found in U.S. Patent No. 5,783,665A (all of which are incorporated by reference in their entirety).

[0033] In one embodiment, the anti-OX40 antibody has the following heavy chain sequence: [ka]

[0034] In one embodiment, the anti-OX40 antibody has the following light chain sequence: [ka]

[0035] In one embodiment, the anti-OX40 antibody has a heavy chain of SEQ ID NO:9 and a light chain of SEQ ID NO:10.

[0036] Examples of antibodies against death receptor 3 (DR3) include those found in WO2011106707A2 and WO2015152430A1. In some embodiments, antibodies against DR3 that can act as agonists of the DR3 receptor are useful in the present invention. Examples of DR3 ligands include TNF-like protein 1A (TL1A). All of the above references are incorporated by reference in their entirety.

[0037] Combinatorial molecules The combination of an IL-2 molecule or mutein and a TNFR agonist as provided herein can be administered in combination or as a single molecule. The IL-2 molecule or mutein in combination with a TNFR agonist provided herein may be constructed as a single molecular construct. In some examples, the IL-2 molecule or mutein and TNFR agonist of the present invention can be constructed as a single molecule, such as with an Fc molecule. In some embodiments, the Fc molecule has an IL-2 molecule or mutein in one arm and a TNFR agonist in the other arm, or as a fusion protein. In some examples, the Fc-bound IL-2 / TNFR agonist molecule extends the serum half-life of the Fc-bound IL-2 / TNFR agonist molecule. In some examples, this is done so as not to increase the risk that such an extension of half-life may increase the likelihood or intensity of side effects or adverse events in patients. Subcutaneous administration of such serum half-life-extended muteins can result in maximum systemic exposure (C max ) may allow for a prolonged target range with lower serum half-life. Extended serum half-life may allow for fewer or less frequent mutant protein dosing regimens.

[0038] The serum half-life of the IL-2 / TNFR agonist molecules provided herein may be extended by essentially any method known in the art. Such methods include altering the sequence of the IL-2 / TNFR agonist molecule to include a peptide that binds to neonatal Fcγ receptors or to bind to proteins with extended serum half-life, such as IgG or human serum albumin. In other embodiments, the IL-2 / TNFR agonist molecule is fused to a polypeptide that confers extended 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 extended serum half-life. In some preferred embodiments, the Fc-binding IL-2 / TNFR agonist molecule is fused to an IgG Fc molecule.

[0039] The IL-2 / TNFR agonist portion of the molecule may be fused to the N-terminus or C-terminus of the IgG Fc region.

[0040] One embodiment of the present invention relates to a dimer comprising two Fc fusion polypeptides created by fusing an IL-2 molecule or mutein to one Fc region of an antibody and a TNFR agonist to another region, which can be produced, for example, by inserting a gene fusion encoding the fusion protein into an appropriate expression vector, expressing the gene fusion in a host cell transformed with the recombinant expression vector, and allowing the expressed fusion protein to assemble in much the same manner as an antibody molecule, resulting in the formation of interchain bonds between the Fc portions to form a dimer.

[0041] As used herein, the term "Fc polypeptide" or "Fc region" includes native and mutein forms of polypeptides derived from the Fc region of an antibody, which may be part of either an IL-2 mutein fusion protein or an anti-IL-2 antibody of the present invention. 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. Along with extended serum half-life, fusion proteins (and oligomers formed therefrom) containing an Fc portion offer the advantage of ease of 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. Certain residues within the Fc region are identified herein by position. All Fc positions are based on the EU numbering scheme.

[0042] One of the functions of the Fc portion of an antibody is to communicate with the immune system once the antibody binds to its target. This is considered an "effector function." Communication can lead to antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). ADCC and ADCP are mediated through binding of Fc to Fc receptors on the surface of cells of the immune system. CDC is mediated by binding of Fc to proteins of the complement system, such as C1q.

[0043] IgG subclasses differ in their ability to mediate effector functions. For example, IgG1 is far superior to IgG2 and IgG4 in mediating ADCC and CDC. Thus, in embodiments where effector function is undesirable, IgG2 Fc may be preferred. However, IgG2 Fc-containing molecules are known to be more difficult to manufacture and to have less attractive biophysical properties, such as a shorter half-life, compared to IgG1 Fc-containing molecules.

[0044] 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 having an Fc engineered to enhance effector function (U.S. Pat. No. 7,317,091 and Strohl, Curr. Opin. Biotech., 20:685-691, 2009; both incorporated herein by reference in their entireties). Exemplary IgG1 Fc molecules with enhanced effector function include those with the following substitutions: S239D;S239E;S239K,F241A;V262A;V264D;V264L;V264A;V264S;D265A;D265S;D265V;F296A;Y296A;R301A;I332E;S239D / I332E;S 239D / A330S / I332E;S239D / A330L / I332E;S298A / D333A / K334A;P247I / A339D;P247I / A339Q;D280H / K290S;D280H / K290S / S298D;D2 80H / K290S / S298V;F243L / R292P / Y300L;F243L / R292P / Y300L / P396L;F243L / R292P / Y300L / V305I / P396L;G236A / S239D / I332E;K326A / E333A;K326W / E333S;K290E / S298G / T299A;K290N / S298G / T299A;K290E / S298G / T299A / K326E; and / or K290N / S298G / T299A / K326E.

[0045] Another approach to increasing the effector function of IgG Fc-containing proteins is by reducing Fc fucosylation. Removal of core fucose from biantennary complex oligosaccharides attached to Fc significantly increases ADCC effector function without altering antigen binding or CDC effector function. Several methods are known for reducing or abolishing fucosylation of Fc-containing molecules, such as antibodies. These methods 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 specific for the FUT8 gene, and cell lines coexpressing β-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II. Alternatively, Fc-containing molecules may be expressed in plant cells, yeast, or non-mammalian cells such as prokaryotic cells, e.g., E. coli.

[0046] In certain embodiments, the IL-2 mutein Fc fusion proteins or anti-IL-2 antibodies of the invention comprise an engineered Fc that has reduced effector function. Exemplary Fc molecules with reduced effector function include those with the following substitutions: N297A or N297Q (IgG1); L234A / L235A (IgG1); V234A / G237A (IgG2); L235A / G237A / E318A (IgG4); H268Q / V309L / A330S / A331S (IgG2); C220S / C226S / C229S / P238S (IgG1); C226S / C229S / E233P / L234V / L235A (IgG1); L234F / L235E / P331S (IgG1); or S267E / L328F (IgG1).

[0047] Human IgG1 has a glycosylation site at N297 (EU numbering system), and glycosylation is known to contribute to the effector function of IgG1 antibodies. An exemplary IgG1 sequence is provided in SEQ ID NO: 3: [ka]

[0048] The group has N297 mutated to form an aglycosylated antibody, focusing on replacing N297 with an amino acid similar in physiochemical properties to asparagine, such as glutamine (N297Q), or with an asparagine mimic without the polar group (N297A).

[0049] 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 positions and still be considered to be an non-glycosylated antibody or non-glycosylated Fc fusion protein.

[0050] In an attempt to create an effector-neutral IgG1 Fc, it was discovered that mutation of amino acid N297 of human IgG1 to glycine, i.e., N297G, provided significantly better purification efficiency and biophysical properties than other amino acid substitutions at that residue. See Example 8. Thus, in a preferred embodiment, an 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 the context of an IL-2 mutein Fc fusion. In a specific embodiment, the antibody comprises an Fc with an N297G substitution.

[0051] Fc, including human IgG1 Fc with an N297G mutation, may also contain further insertions, deletions, and substitutions. In certain embodiments, the human IgG1 Fc contains the N297G substitution and is at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 3. In particularly preferred embodiments, the C-terminal lysine residue is substituted or deleted. The amino acid sequence of human IgG1 containing the N297G substitution and the deletion of the C-terminal lysine is set forth in SEQ ID NO: 4, which has the following amino acid sequence: [ka]

[0052] Glycosylated IgG1 Fc-containing molecules have been shown to be less stable than glycosylated IgG1 Fc-containing molecules. The Fc region may be further engineered to increase the stability of unglycosylated molecules. In some embodiments, one or more amino acids are substituted with cysteine ​​to form disulfide bonds in the dimeric state. Residues V259, A287, R292, V302, L306, V323, or I332 of the amino acid sequence set forth in SEQ ID NO: 3 may be substituted with cysteine. In preferred embodiments, specific residue pairs are substituted such that they preferentially form disulfide bonds with each other, thereby limiting or preventing disulfide bond scrambling. Preferred pairs include, but are not limited to, A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C.

[0053] Provided herein are Fc-containing molecules in which one or more of residues V259, A287, R292, V302, L306, V323, or I332 have been substituted with cysteine, including those containing the following substitutions: A287C and L306C, V259C and L306C, R292C and V302C, or V323C and I332C.

[0054] Additional mutations that may be made to IgG1 Fc include those that promote heterodimer formation among Fc-containing polypeptides. In some embodiments, the Fc region is engineered to create "knobs" and "holes" that promote heterodimer formation between two different Fc-containing polypeptide chains when co-expressed in a cell. See U.S. Patent No. 7,695,963. In other embodiments, the Fc region is modified using electrostatic steering to promote heterodimer formation while preventing homodimer formation between two different Fc-containing polypeptides when co-expressed in a cell. See WO 09 / 089,004 (incorporated herein by reference in its entirety). A preferred heterodimeric Fc includes one Fc chain containing D399K and E356K substitutions and the other Fc chain containing K409D and K392D substitutions. In another embodiment, one chain of the Fc comprises the following substitutions: D399K, E356K, and E357K, and the other chain of the Fc comprises the following substitutions: K409D, K392D, and K370D.

[0055] In certain embodiments, the Fc-binding IL-2 / TNFR agonist molecule comprises a linker between the Fc and the IL-2 molecule or mutein and / or between the Fc and the TNFR agonist. Many different linker polypeptides are known in the art and can be used in the context of an Fc-binding IL-2 / TNFR agonist molecule. In preferred embodiments, the Fc-binding IL-2 / TNFR agonist molecule 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 and the IL-2 molecule or mutein and / or between the Fc and the TNFR agonist region comprises a single copy of GGGGS (SEQ ID NO: 5), GGNGT (SEQ ID NO: 6), or YGNGT (SEQ ID NO: 7). As provided 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 may help stabilize the protein in solution and / or upon administration in vivo. Thus, in certain embodiments, an IL-2 mutein fusion protein comprises a glycosylated linker between the Fc region and the IL-2 mutein region.

[0056] The C-terminal portion of the Fc and / or the amino-terminal portion of the IL-2 molecule or mutein may contain one or more mutations that alter the glycosylation profile of the Fc-binding IL-2 / TNFR agonist molecule when expressed in mammalian cells. In certain embodiments, the Fc-binding IL-2 / TNFR agonist molecule further comprises a T3 substitution, e.g., T3N or T3A. The Fc-binding IL-2 / TNFR agonist molecule may further comprise an S5 substitution, such as S5T.

[0057] Covalent modifications of Fc-binding IL-2 / TNFR agonist molecules are included within the scope of the present invention, which are generally, but not always, performed post-translationally. For example, several types of covalent modifications are introduced into a molecule by reacting certain of its amino acid residues with organic derivatizing agents capable of reacting with selected side chains or N- or C-terminal residues.

[0058] 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-imidazolyl)propionic acid, chloroacetylphosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0059] 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.

[0060] Lysinyl and amino-terminal residues react with succinic anhydride or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactions with glyoxylate.

[0061] Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues must be performed under alkaline conditions because of the pK of the guanidine functional group. a Furthermore, the reagent can react with the lysine groups and the arginine epsilon-amino group.

[0062] The specific modification of tyrosyl residues may be made, particularly for the introduction of spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues can be 125 I or 131 The previously described chloramine T method of iodination with 1 to prepare labeled proteins for use in radioimmunoassay is suitable.

[0063] Carboxyl side groups (aspartyl or glutamyl) 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 glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0064] Derivatization with bifunctional agents is useful for crosslinking antigen-binding proteins to water-insoluble support matrices or surfaces used in a variety of methods. Commonly used crosslinking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, such as esters with 4-azidosalicylic acid, homobifunctional imidoesters (including disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl propionate)), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatization agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are 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 have been used for protein immobilization.

[0065] Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Both forms of these residues are within the scope of this invention.

[0066] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-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.

[0067] Another type of covalent modification of an IL-2 mutein, IL-2 mutein Fc fusion, or anti-IL-2 antibody within the scope of the invention involves altering the glycosylation pattern of the protein. As is known in the art, glycosylation patterns can depend both on the sequence of the protein (e.g., the presence or absence of particular glycosylated amino acid residues, as discussed below), or on the host cell or organism in which the protein is produced. Specific expression systems are discussed below.

[0068] 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 the 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 can also be used.

[0069] Addition of glycosylation sites to an IL-2 mutein, IL-2 mutein Fc fusion, or anti-IL-2 antibody can be conveniently achieved by altering the amino acid sequence so that it contains one or more of the above tripeptide sequences (for N-linked glycosylation sites). Alterations may also be made by adding to or substituting one or more serine or threonine residues to the starting sequence (for O-linked glycosylation sites). To facilitate this, the amino acid sequence of the IL-2 mutein, IL-2 mutein Fc fusion, or anti-IL-2 antibody is preferably altered through changes at the DNA level, in particular by mutating the DNA encoding the target polypeptide at preselected bases to generate codons that will be translated into the desired amino acids.

[0070] Another means of increasing the number of carbohydrate moieties on an IL-2 mutein, IL-2 mutein Fc fusion, or anti-IL-2 antibody is by chemical or enzymatic coupling of glycosides to the protein. This procedure is advantageous in that it does not require production of the protein in a host cell capable of glycosylation for N-linked and O-linked glycosylation. Depending on the coupling mode used, sugars may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as those of cysteine, (d) free hydroxyl groups, such as those of serine, threonine, or hydroxyproline, (e) aromatic residues, such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 87 / 05330, published September 11, 1987, and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306.

[0071] Removal of carbohydrate moieties present on the starting IL-2 mutein, IL-2 mutein Fc fusion, or anti-IL-2 antibody can be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment cleaves most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine) while leaving the polypeptide intact. Chemical deglycosylation is described by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and Edge et al., 1981, Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of various endo- and exoglycosidases, 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 described by Duskin et al., 1982, J. Biol. Chem. 257:3105. Tunicamycin prevents the formation of protein-N-glycosidic bonds.

[0072] Another type of covalent modification of IL-2 / TNFR agonist molecules involves linking various nonproteinaceous polymers, including, but not limited to, various polyols such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, to the protein by methods described in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337. In addition, amino acid substitutions may be made at various positions within the IL-2 / TNFR agonist molecule to facilitate the addition of polymers such as PEG. Thus, embodiments of the present invention include PEGylated IL-2 / TNFR agonist molecules. Such PEGylated proteins may exhibit increased half-life and / or reduced immunogenicity compared to their non-PEGylated forms.

[0073] Polynucleotides encoding IL-2 / TNFR agonist molecules Included within the present invention are nucleic acids encoding IL-2 / TNFR agonist molecules. Aspects of the present invention include polynucleotide variants (e.g., due to degeneracy) that encode the amino acid sequences described herein.

[0074] Nucleotide sequences corresponding to the amino acid sequences described herein, to be used as probes or primers for nucleic acid isolation or as query sequences for database searches, can be obtained by "reverse translation" from the amino acid sequences. 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 the 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).

[0075] The nucleic acid molecules of the present invention include DNA and RNA in both single-stranded and double-stranded forms, as well as corresponding complementary sequences. An "isolated nucleic acid" is a nucleic acid isolated from a naturally occurring source, separated from adjacent gene sequences present in the genome of the organism from which the nucleic acid is isolated. In the case of nucleic acids synthesized enzymatically or chemically from a template, such as PCR products, cDNA molecules, or oligonucleotides, the nucleic acid resulting from such a process is understood to be an isolated nucleic acid. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. In a preferred embodiment, the nucleic acid is substantially free of contaminating endogenous materials. Nucleic acid molecules are 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 (e.g., 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, typically present within eukaryotic genes. Sequences of untranslated DNA can be present 5' or 3' from the open reading frame, where this does not interfere with manipulation or expression of the coding region.

[0076] IL-2 muteins according to the invention are typically prepared by site-directed mutagenesis of nucleotides in DNA encoding the IL-2 / TNFR agonist molecule using cassette or PCR mutagenesis, or other techniques well known in the art, to generate DNA encoding the variant, followed by expression of the recombinant DNA in cell culture as outlined herein. However, IL-2 / TNFR agonist molecules may also be prepared by in vitro synthesis using established techniques. Variants typically exhibit the same qualitative biological activity, e.g., Treg proliferation, as the naturally occurring analog, although variants may be selected with altered characteristics, as outlined in more detail below.

[0077] As will be appreciated by those skilled in the art, due to the degeneracy of the genetic code, each IL-2 / TNFR agonist molecule of the present invention is encoded by a large number of nucleic acids, each of which is within the scope of the present invention and can be generated using standard techniques. Thus, having identified 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.

[0078] 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 foregoing polynucleotides. In addition, the present invention provides host cells comprising such expression systems or constructs.

[0079] Typically, expression vectors used in any host cell will contain sequences for plasmid maintenance and sequences for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as "flanking sequences," will, in certain embodiments, 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 splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element. Each of these sequences is discussed below.

[0080] In some cases, the vector may contain a "tag" coding sequence, i.e., an oligonucleotide molecule located at the 5' or 3' end of the IL-2 / TNFR agonist molecule coding sequence; the oligonucleotide sequence encodes poly-His (such as hexa-His: HHHHHH (SEQ ID NO: 8)) for which commercially available antibodies exist, or another "tag," e.g., FLAG, HA (influenza virus hemagglutinin), or myc. This tag is typically fused to the polypeptide immediately after expression and can serve as a means for affinity purification or detection of the polypeptide from host cells. Affinity purification can be achieved, for example, by column chromatography using antibodies against the tag as an affinity matrix. In some cases, the tag can then be removed by various means, for example, using a specific peptidase for cleavage.

[0081] 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 host cell species or strain), hybrid (i.e., a combination of flanking sequences from two or more sources), synthetic, or natural. Thus, the source of the flanking 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 machinery.

[0082] Flanking sequences useful in the vectors of the present invention can be obtained by any of several methods well known in the art. Typically, flanking sequences useful herein will have been previously identified by mapping and / or restriction endonuclease digestion and can be isolated from a suitable tissue source using appropriate restriction endonucleases. In some cases, the entire nucleotide sequence of the flanking sequence may be known. In this case, the flanking sequence can be synthesized using the methods described herein for nucleic acid synthesis or cloning.

[0083] Whether all or only a portion of a flanking sequence is known, the flanking sequence can be obtained using polymerase chain reaction (PCR) and / or by screening a genomic library with suitable probes, such as oligonucleotides and / or flanking sequence fragments from the same or another species. When the flanking sequence is unknown, a fragment of DNA containing the flanking sequence can be isolated from a larger piece of DNA that may contain, for example, a coding sequence or even one or more additional genes. Isolation may be achieved by digestion with restriction endonucleases to generate the appropriate DNA fragment, followed by isolation using agarose gel purification, Qiagen® column chromatography (Chatsworth, CA), or other methods known to those skilled in the art. The selection of appropriate enzymes to accomplish this purpose will be readily apparent to those skilled in the art.

[0084] Origins of replication are typically a part of commercially available prokaryotic expression vectors; the origin aids in the amplification of the vector within a host cell. If the vector of choice does not contain an origin of replication site, one may be chemically synthesized based on a known sequence and ligated into the vector. For example, the origin of replication from the 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 origin of replication component is not needed for mammalian expression vectors (e.g., the SV40 origin is often used only because it also contains the viral early promoter).

[0085] A transcription termination sequence is typically located 3' to the end of a polypeptide coding region and functions to terminate transcription. In prokaryotic cells, a transcription termination sequence is usually a GC-rich fragment followed by a poly-T sequence. While such sequences can be easily cloned from a library or even purchased commercially as part of a vector, they can also be easily synthesized using nucleic acid synthesis methods, such as those described herein.

[0086] A selectable marker gene encodes a protein essential for the survival and growth of host cells grown in selective media. Typical selectable marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, tetracycline, or kanamycin, on prokaryotic host cells; (b) complement auxotrophic deficiencies of the cells; or (c) supply critical nutrients unavailable from complex or defined media. Specific 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.

[0087] Other selection genes may be used to amplify the gene to be expressed. Amplification is a process in which genes required for the production of proteins important for growth or cell survival are tandemly repeated in the chromosomes of recombinant cells in successive generations. Examples of suitable selectable markers for mammalian cells include the dihydrofolate reductase (DHFR) gene and the promoterless thymidine kinase gene. Mammalian cell transformants are placed under selection pressure to which only the transformants are uniquely adapted to survive due to the selection gene present in the vector. Selection pressure is imparted by culturing the transformed cells under conditions in which the concentration of the selection agent in the medium is successively increased, thereby leading to amplification of both the selectable gene and, ultimately, the gene encoding the desired polypeptide, such as an IL-2 / TNFR agonist molecule. As a result, increased quantities of the polypeptide are synthesized from the amplified DNA.

[0088] 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' to the promoter and 5' to 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.

[0089] In cases where glycosylation is desired in eukaryotic host cell expression systems, various pre- or pro-sequences may be manipulated to improve glycosylation or yield. For example, the peptidase cleavage site of a particular signal peptide may be altered or a pro-sequence may be added, which may also affect glycosylation. The final protein product may have one or more additional amino acids at position -1 (relative to the first amino acid of the mature protein) that are involved in expression and may not be completely removed. For example, the final protein product may have one or two amino acid residues found within the peptidase cleavage site attached to the amino terminus. Additionally, the use of some enzyme cleavage sites may result in slightly truncated forms of the desired polypeptide if the enzyme is cleaved at such a region within the mature polypeptide.

[0090] The expression and cloning vectors of the present invention will typically contain a promoter recognized by the host organism and operably linked to the molecule encoding the IL-2 / TNFR agonist molecule. A promoter is a non-transcribed sequence located upstream (i.e., 5') of the start codon of a structural gene (generally within about 100-1000 bp) that controls transcription of the structural gene. Traditionally, promoters are grouped into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased 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. Constitutive promoters, on the other hand, transcribe the gene to which they are operably linked uniformly, i.e., with little or no control over gene expression. Numerous promoters recognized by a variety of potential host cells are known.

[0091] Suitable promoters for use in yeast hosts are also well known in the art. Advantageously, yeast enhancers are used in conjunction with yeast promoters. Suitable promoters for use in mammalian host cells are well known and include, but are not limited to, those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus type 2), bovine papilloma virus, 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.

[0092] 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' long 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); the promoter and regulatory sequences from the metallothionein gene (Prinster et al., 1982, Nature 296:39-42); and prokaryotic promoters such as the beta-lactamase promoter (Villa-Kamaroff et al., 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, which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulin gene control region, which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122); and the immunoglobulin gene control region, which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Hepatology 7:425-515). al., 1987, Mol. Cell. Biol.7:1436-1444); mouse mammary tumor virus control region, which is active in testicular, breast, lymphoid, and mast cells (Leder et al., 1986, Cell 45:485-495); albumin gene control region, which is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); alpha-fetoprotein gene control region, which is 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, which is active in liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); beta-globin gene control region, which is 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, which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); the myosin light chain 2 gene control region, which is active in skeletal muscle (Sani, 1985, Nature 314:283-286); and the gonadotropin-releasing hormone gene control region, which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

[0093] Enhancer sequences may be inserted into vectors to increase transcription in higher eukaryotes. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp in length, that act on promoters to increase transcription. Enhancers are relatively orientation- and position-independent and have been found both 5' and 3' to transcription units. Several enhancer sequences are known from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein, and insulin). However, enhancers of viral origin are typically used. The SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, and adenovirus enhancers known in the art are exemplary enhancing elements for activating eukaryotic promoters. Enhancers can be located either 5' or 3' to the coding sequence in a vector, but are typically located 5' from the promoter. A sequence encoding an appropriate native or heterologous signal sequence (leader sequence or signal peptide) can be incorporated into the expression vector to promote extracellular secretion of the IL-2 / TNFR agonist molecule. The choice of signal peptide or leader depends on the type of host cell in which the protein will be produced, and a heterologous signal sequence may replace the native signal sequence. Examples of signal peptides functional in mammalian host cells include the interleukin-7 (IL-7) signal sequence described in U.S. Pat. No. 4,965,195; the interleukin-2 receptor signal sequence described in Cosman et al., 1984, Nature 312:768; the interleukin-4 receptor signal peptide described in European Patent No. 0367566; the type I interleukin-1 receptor signal peptide described in U.S. Pat. No. 4,968,607; and the type II interleukin-1 receptor signal peptide described in European Patent No. 0460846.

[0094] 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. Therefore, MARs are particularly useful when the vector is used to generate stable transfectants. Several natural and synthetic MAR-containing nucleic acids are known in the art, e.g., U.S. Pat. Nos. 6,239,328; 7,326,567; 6,177,612; 6,388,066; 6,245,974; 7,259,010; 6,037,525; 7,422,874; and 7,129,062.

[0095] The expression vector of the present invention may be constructed from a starting vector, such as a commercially available vector. Such a vector may or may not contain all of the desired flanking sequences. If one or more of the flanking sequences described herein are not originally present in the vector, they may be individually obtained and ligated into the vector. The methods used to obtain each of the flanking sequences are well known to those skilled in the art.

[0096] After the vector has been constructed and the nucleic acid molecule encoding the IL-2 / TNFR agonist molecule 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, such as transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran-mediated transfection, or other known techniques. The method selected will depend, in part, on the type of host cell to be used. These and other suitable methods are well known to those of skill in the art and are set forth, for example, in Sambrook et al., 2001, supra.

[0097] When cultured under appropriate conditions, the host cells will synthesize IL-2 / TNFR agonist molecules, which can then be collected from the culture medium (if the host cells secrete them into the medium) or directly from the producing host cells (if they are not secreted). The selection of an appropriate host cell will depend on various factors, such as the desired expression level, polypeptide modifications (such as glycosylation or phosphorylation) that are desirable or necessary for activity, and the ease of folding into a biologically active molecule. The host cell can be eukaryotic or prokaryotic.

[0098] Mammalian cell lines available as expression hosts are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC). Any cell line used in an expression system known in the art can be used to produce the recombinant polypeptides of the present invention. Generally, host cells are transformed with a recombinant expression vector containing DNA encoding the desired IL-2 / TNFR agonist molecule. Host cells that may be used include prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes include gram-negative or gram-positive organisms, such as E. coli or Bacilli. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL1651) (Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC CCL163), Chinese hamster ovary (CHO) cells, or derivatives thereof, such as Veggie CHO and related cell lines grown in serum-free medium (Rasmussen et al., 1998, Cytotechnology 28:31), HeLa cells, the BHK (ATCC CRL10) cell line, and the African green monkey kidney cell line CVI (ATCC CRL1651) as described in McMahan et al., 1991, EMBO J. 10:2821. Examples of suitable cell lines include CVI / EBNA cell lines derived from human ovarian tumor cells (e.g., CCL70), human embryonic kidney cells such as 293, 293EBNA, or MSR293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro culture of primary tissue, primary explants, HL-60 cells, U937 cells, HaK cells, or Jurkat cells. In some cases, mammalian cell lines such as HepG2 / 3B, KB, NIH3T3, or S49 may be used to express the polypeptide when it is desired to use the polypeptide in various signal transduction or reporter assays.

[0099] Alternatively, polypeptides can be produced in lower eukaryotes, such as yeast, or in prokaryotes, such as bacteria. Suitable yeasts include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous polypeptides. Suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous polypeptides. If the polypeptide is formed in yeast or bacteria, it may be desirable to modify the polypeptide produced in yeast or bacteria, for example, by phosphorylation or glycosylation of appropriate sites, to obtain a functional polypeptide. Such covalent attachments can be achieved using known chemical or enzymatic methods.

[0100] Polypeptides can also be produced using insect expression systems by operably linking the isolated nucleic acids of the present invention to suitable control sequences in one or more insect expression vectors. 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 in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), and Luckow and Summers, Bio / Technology 6:47 (1988). Cell-free translation systems can also be used to produce polypeptides using RNA derived from the nucleic acid constructs disclosed herein. Cloning and expression vectors suitable for use in bacterial, fungal, yeast, and mammalian cell hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985). A host cell containing an isolated nucleic acid of the invention, preferably operably linked to at least one expression control sequence, is a "recombinant host cell."

[0101] Also included are isolated nucleic acids encoding any of the exemplary IL-2 / TNFR agonist molecules described herein. In preferred embodiments, the Fc portion and the IL-2 / TNFR agonist molecule are encoded within a single open reading frame, optionally with a linker encoded between the Fc region and the IL-2 / TNFR agonist molecule.

[0102] In another embodiment, provided herein is an expression vector comprising an IL-2 / TNFR agonist molecule-encoding nucleic acid operably linked to a promoter.

[0103] In another aspect, provided herein is a host cell comprising an isolated nucleic acid encoding the above-described IL-2 / TNFR agonist molecule. 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.

[0104] In another aspect, provided herein is a method of forming an IL-2 / TNFR agonist molecule. The method comprises culturing host cells under conditions in which a promoter operably linked to an IL-2 / TNFR agonist molecule Fc fusion protein is expressed. The IL-2 / TNFR agonist molecule Fc fusion protein is then harvested from the culture. The IL-2 / TNFR agonist molecule Fc fusion protein may be harvested from the culture medium and / or host cell lysate.

[0105] Pharmaceutical Composition In some embodiments, the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of an IL-2 / TNFR agonist molecule together with a pharmaceutically effective diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant. In certain embodiments, the IL-2 mutein is in the context of an IL-2 / TNFR agonist molecule Fc fusion protein. Pharmaceutical compositions of the present invention include, but are not limited to, liquid compositions, frozen compositions, and lyophilized compositions.

[0106] Preferably, the formulation materials are non-toxic to recipients at the dosages and concentrations used. In certain embodiments, pharmaceutical compositions are provided that include a therapeutically effective amount of an IL-2 / TNFR agonist molecule-containing therapeutic molecule, e.g., an IL-2 / TNFR agonist molecule Fc fusion.

[0107] In certain embodiments, pharmaceutical compositions may contain formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or permeability of the composition. In such embodiments, suitable formulation materials include amino acids (such as glycine, glutamine, asparagine, arginine, proline, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavoring agents, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming agents. These include, but are not limited to, ions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, 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 chloride or potassium chloride, mannitol, sorbitol, etc.); delivery vehicles; diluents; excipients, and / or pharmaceutical adjuvants. See REMINGTON'S PHARMACEUTICAL SCIENCES, 18” Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company.

[0108] In certain embodiments, the optimal pharmaceutical composition will be determined by one of skill in the art depending, for example, on the intended route of administration, delivery format, and desired dosage. See, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certain embodiments, such compositions may influence the physical state, stability, in vivo release rate, and in vivo clearance rate of the antigen-binding proteins of the present invention. In certain embodiments, the 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 common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, the pharmaceutical composition comprises Tris buffer of about pH 7.0-8.5 or acetate buffer of about pH 4.0-5.5, and may further include sorbitol or a suitable substitute thereof. In certain embodiments of the invention, IL-2 mutein or anti-IL-2 antibody compositions may be prepared for storage in the form of a lyophilized cake or aqueous solution by mixing a selected composition having the desired purity with optional formulating agents (REMINGTON'S PHARMACEUTICAL SCIENCES, supra). Furthermore, in certain embodiments, the IL-2 mutein or anti-IL-2 antibody product may be formulated as a lyophilizate using appropriate excipients such as sucrose.

[0109] The pharmaceutical compositions of the present invention can be selected for parenteral delivery. Alternatively, the compositions may be selected for inhalation or for delivery via the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art. The formulation components are preferably present in concentrations acceptable to the site of administration. In certain embodiments, a buffer is used to maintain the composition at physiological pH or a slightly lower pH, typically within a pH range of about 5 to about 8.

[0110] When intended for parenteral administration, the therapeutic compositions used in the present invention may be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired IL-2 / TNFR agonist molecule composition in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, in which the IL-2 / TNFR agonist molecule composition is formulated as a sterile, isotonic solution and properly maintained. In certain embodiments, the preparation may involve formulating the desired molecule with an agent capable of achieving controlled or sustained release of the product, such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, which can be delivered via depot injection. In certain embodiments, hyaluronic acid, which has the effect of promoting sustained release in the circulatory system, may be used. In certain embodiments, an implantable drug delivery device may be used to introduce the IL-2 / TNFR agonist molecule.

[0111] Additional pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising IL-2 / TNFR agonist molecule compositions in sustained- or controlled-delivery formulations. Techniques for formulating various 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 Application PCT / US93 / 00829 (incorporated by reference), which describes the controlled release of porous polymeric microparticles for delivering pharmaceutical compositions. Sustained-release preparations may include semipermeable polymer matrices in the form of shaped articles, e.g., films or microcapsules. Sustained-release matrices may include polyesters, hydrogels, polylactides (disclosed in U.S. Pat. No. 3,773,919 and 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-hydroxybutyrate (EP 133,988). Sustained-release compositions may also include liposomes, which can 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; EP 036,676; EP 088,046; and EP 143,949, all of which are incorporated by reference).

[0112] Pharmaceutical compositions for in vivo administration are typically provided as sterile preparations.Sterilization can be achieved by filtration through sterile filtration membranes.When lyophilizing compositions, sterilization using this method can be carried out either before or after lyophilization and reconstitution.Compositions for parenteral administration can be stored in lyophilized form or in solution.Parenteral compositions are generally placed in a container with a sterile access port, for example, an intravenous solution bag or vial, with a stopper that can be pierced by a hypodermic injection needle.

[0113] Embodiments of the present invention include self-buffering IL-2 / TNFR agonist molecule formulations, which can be used as pharmaceutical compositions as described in WO 06138181(A2) (PCT / US2006 / 022599), which is incorporated herein by reference in its entirety.

[0114] As discussed above, certain embodiments provide pharmaceutical IL-2 / TNFR agonist molecule compositions, particularly IL-2 / TNFR agonist molecule Fc-fusion proteins, that include, in addition to the IL-2 / TNFR agonist molecule composition, one or more excipients, such as those illustratively described in this section and elsewhere herein. Excipients can be used in the present invention for a wide range of purposes, including adjusting the physical, chemical, or biological properties of the formulation, such as adjusting viscosity, and / or processes of the invention to improve efficacy and / or stabilize such formulations, as well as processes against deterioration and spoilage due to stresses that occur during and after manufacture, transportation, storage, preparation prior to use, and administration.

[0115] Various explanations are available for protein stabilization and formulation materials and methods useful in this regard, 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 sections relating to excipients and processes for self-buffering protein formulations according to the present invention, particularly with respect to protein pharmaceutical products and processes for veterinary and / or human medical use.

[0116] 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 improve the solubility and / or physical stability of proteins or other components of compositions in accordance with the invention.

[0117] As is well known, ions can stabilize proteins in their native state by binding to charged residues on the surface of the protein and by shielding charged and polar groups in the protein, reducing the strength of their electrostatic, attractive, and repulsive interactions. Ions can also stabilize proteins in their denatured state, particularly by binding to the denatured peptide bond (--CONH) of the protein. Furthermore, ionic interactions with charged and polar groups in proteins can also reduce intermolecular electrostatic interactions, thereby preventing or reducing protein aggregation and insolubilization.

[0118] Ionic species vary significantly in their effects on proteins. Several methods have been developed to rank ions and their effects on proteins into categories and can be used in formulating pharmaceutical compositions of the present invention. One example is the Hofmeister series, which ranks ionic solutes and polar nonionic solutes by their effect on the conformational stability of proteins in solution. Stabilizing solutes are called "kosmotropics." Destabilizing solutes are called "chaotropics." Kosmotropes are generally used at high concentrations (e.g., greater than 1 molar ammonium sulfate) to precipitate proteins from solution ("salting out"). Chaotropes are generally used to denature and / or solubilize proteins ("salting in"). The relative effectiveness of an ion for "salting in" and "salting out" defines its position on the Hofmeister series.

[0119] Free amino acids can be used in IL-2 / TNFR agonist molecule 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 in inhibiting protein aggregation in both liquid and lyophilized formulations. Methionine is useful as an antioxidant.

[0120] Polyols include sugars such as mannitol, sucrose, and sorbitol, and polyhydric alcohols such as glycerol and propylene glycol, and for purposes of discussion herein, polyethylene glycol (PEG) and related substances. Polyols are kosmotropic. Polyols are useful stabilizers in both liquid and lyophilized formulations to protect proteins from physical and chemical degradation processes. Polyols are also useful for adjusting the tonicity of the formulation.

[0121] Among polyols, mannitol is useful in select embodiments of the present invention, and is commonly used to ensure cake structural stability in lyophilized formulations. Mannitol ensures cake structural stability. Mannitol is commonly used in conjunction with a lyoprotectant, such as sucrose. Among the preferred agents, sorbitol and sucrose are preferred stabilizers for adjusting tonicity and protecting against freeze-thaw stress during transportation or bulk preparation during the manufacturing process. Reducing sugars (containing free aldehyde or ketone groups), such as glucose and lactose, can glycate surface lysine and arginine residues. Therefore, reducing sugars are generally not among the preferred polyols for use in accordance with the present invention. Additionally, sugars that form such reactive species, such as sucrose, are also not among the preferred polyols of the present invention, since they are hydrolyzed to fructose and glucose under acidic conditions, resulting in glycation. PEG is useful for stabilizing proteins and as a lyoprotectant, and in this regard can be used in the present invention.

[0122]

[0003] Embodiments of the IL-2 / TNFR agonist molecule formulation further include a surfactant. Protein molecules can be susceptible to adsorption to surfaces and to denaturation and resulting aggregation at air-liquid, solid-liquid, and liquid-liquid interfaces. These effects are generally inversely proportional to protein concentration. These adverse interactions are generally inversely proportional to protein concentration and are typically exacerbated by physical agitation that occurs, for example, during product transportation and handling.

[0123] Surfactants are routinely used to prevent, minimize, or reduce surface adsorption. In this regard, surfactants useful in the present invention include polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan polyethoxylate, and poloxamer 188.

[0124] Detergents are also commonly used to control the conformational stability of proteins. In this regard, detergent use is protein-specific, as any given detergent will typically stabilize some proteins and destabilize others.

[0125] Polysorbates are susceptible to oxidative degradation and often contain sufficient peroxide content to cause oxidation of protein residue side chains, particularly methionine, if provided. As a result, polysorbates should be used with caution and, if used, at the lowest effective concentration. In this regard, polysorbates exemplify the principle that excipients should be used at the lowest effective concentration.

[0126] Embodiments of the IL-2 / TNFR agonist molecule formulation further include one or more antioxidants. 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. Useful antioxidants in this regard include reducing agents, oxygen / free radical scavengers, and chelating agents. Antioxidants used in therapeutic protein formulations according to the present invention are preferably water-soluble and maintain activity throughout the shelf life of the product. In this regard, EDTA is a preferred antioxidant according to the present invention.

[0127] Antioxidants can damage proteins. For example, reducing agents, such as glutathione, can disrupt intramolecular disulfide bonds. Therefore, the antioxidants used in the present invention are selected specifically to eliminate or sufficiently reduce the possibility of damaging proteins in the formulation.

[0128] The formulations of the present invention may contain metal ions that are protein cofactors and essential for forming protein coordination complexes, such as zinc, which is essential for forming certain insulin suspensions. Metal ions can also inhibit some processes that degrade proteins. However, metal ions also catalyze physical and chemical processes that degrade proteins.

[0129] Magnesium ions (10-120 mM) can be used to inhibit the isomerization of aspartate to isoaspartate. +2 ions (up to 100 mM) can increase the stability of human deoxyribonuclease. +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.

[0130] Embodiments of IL-2 / TNFR agonist molecule formulations further include one or more preservatives. Preservatives are essential when developing multi-dose parenteral formulations involving two or more extractions from the same container. Their primary function is to inhibit microbial growth and ensure product sterility throughout the drug product's shelf life or usage period. Commonly used preservatives include benzyl alcohol, phenol, and m-cresol. While preservatives have a long history of use in small molecule parenteral drugs, developing protein formulations containing preservatives can be challenging. Preservatives almost always have a destabilizing effect on proteins (aggregation), which is a major factor limiting their use in multi-dose protein formulations. To date, most protein drugs have only been formulated for single-use. However, the possibility of multi-dose formulations offers the added benefit of enabling patient convenience and increased marketability. A good example is 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.

[0131] Several aspects must be considered during the formulation and development of a preservative dosage form. The effective preservative concentration in the drug product must be optimized. This requires testing a given preservative in a dosage form over a range of concentrations that impart antimicrobial efficacy without compromising protein stability.

[0132] In another aspect, the present invention provides IL-2 / TNFR agonist molecules or Fc fusions of IL-2 / TNFR agonist molecules in lyophilized formulations. The lyophilized product can be lyophilized without preservatives and reconstituted with a preservative-containing diluent at the time of use. This reduces the time the preservative is in contact with the protein, significantly minimizing associated stability risks. For liquid formulations, the preservative's effectiveness and stability should be maintained throughout the product's shelf life (approximately 18-24 months). It is important to note that preservative effectiveness must be demonstrated in the final formulation containing the active drug and all excipient components.

[0133] IL-2 / TNFR agonist molecule formulations 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, and for a range of bioavailability and duration, among other things. 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, and by parenteral routes, including intravenous and intraarterial, intramuscular, and subcutaneous injections.

[0134] 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 in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted before administration. The present invention also provides kits for producing single-dose administration units. Each kit of the present invention may contain both a first container with a dried protein and a second container with an aqueous formulation. In certain embodiments of the present invention, kits containing single-chamber and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes) are provided.

[0135] The therapeutically effective amount of a pharmaceutical composition containing an IL-2 / TNFR agonist molecule to be used will depend, for example, on the context and purpose of the treatment. Those skilled in the art will understand that dosage levels appropriate for treatment will vary, depending in part, on the molecule being delivered, the indication for which the IL-2 / TNFR agonist molecule is being used, the route of administration, and the patient's size (weight, body surface, or organ size) and / or condition (age and health). In certain embodiments, a clinician may titrate the dosage and modify the route of administration to obtain optimal therapeutic effect. Typical dosages can range from about 0.1 μg / kg up to about 1 mg / kg or more, depending on the factors described above. In certain embodiments, dosages can range from 0.5 μg / kg up to about 100 μg / kg, and in some cases from 2.5 μg / kg up to about 50 μg / kg.

[0136] A therapeutically effective amount of an IL-2 / TNFR agonist molecule preferably results in a decrease in the severity of disease symptoms, an increase in the frequency or duration of disease symptom-free periods, or prevention of disability or inconvenience resulting from the affliction of the disease.

[0137] Pharmaceutical compositions can be administered using medical device.The example of medical device for administering pharmaceutical compositions is described in United States Patent (USP) 4,475,196; United States Patent (USP) 4,439,196; United States Patent (USP) 4,447,224; United States Patent (USP) 4,447,233; United States Patent (USP) 4,486,194; United States Patent (USP) 4,487,603; United States Patent (USP) 4,596,556; United States Patent (USP) 4,790,824; United States Patent (USP) 4,941,880; United States Patent (USP) 5,064,413; United States Patent (USP) 5,312,335; United States Patent (USP) 5,312,335; United States Patent (USP) 5,383,851; and United States Patent (USP) 5,399,163 (all are incorporated herein by reference).

[0138] In one embodiment, a pharmaceutical composition comprising:

[0139] Methods of treating autoimmune or inflammatory disorders In a specific embodiment, the IL-2 / TNFR agonist molecules of the invention are used to treat autoimmune or inflammatory disorders. In a preferred embodiment, an IL-2 / TNFR agonist molecule Fc fusion protein is used.

[0140] Disorders that are particularly suitable for treatment with the IL-2 muteins or anti-IL-2 antibodies disclosed herein include inflammation, autoimmune diseases, atopic diseases, paraneoplastic autoimmune diseases, cartilage inflammation, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, small-articular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic-onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile rheumatoid arthritis, and juvenile rheumatoid arthritis. 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, small-articular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic-onset rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myelitis, multiple myelitis Myositis, 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), myasthenia gravis, inflammatory bowel disease (IBD) BD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, rhinosinusitis, rhinosinusitis with polyposis, eosinophilic esophagitis, eosinophilic bronchitis, Guillain-Barré disease, type 1 diabetes, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD, transplant rejection, nephropathy, hepatitis C-induced vasculitis, and spontaneous abortion.

[0141] In preferred embodiments, the autoimmune or inflammatory disorder is systemic lupus erythematosus (SLE), graft-versus-host disease, hepatitis C-induced vasculitis, type I diabetes, rheumatoid arthritis, multiple sclerosis, spontaneous abortion, atopic disease, and inflammatory bowel disease, including ulcerative colitis and celiac disease.

[0142] In another embodiment, a patient suffering from or at risk of developing an autoimmune or inflammatory disorder is treated with an IL-2 / TNFR agonist molecule (e.g., an IL-2 / TNFR agonist molecule disclosed herein, e.g., an IL-2 / TNFR agonist molecule Fc fusion as disclosed herein), and the patient's response to the treatment is monitored. The patient response monitored can be any detectable or measurable response of the patient to the treatment, or any combination of such responses. For example, a response can be a change in the patient's physiological state, e.g., body temperature or fever, appetite, sweating, headache, nausea, fatigue, hunger, dry mouth, mental acuity, etc. Alternatively, a response can be, for example, a change in the amount of a cell type or gene product (e.g., a protein, peptide, or nucleic acid) in a sample of peripheral blood taken from the patient. In one embodiment, the patient's treatment regimen is altered if the patient has a detectable or measurable response to the treatment, or if such response exceeds a certain threshold. This change can be a decrease or increase in dosing frequency, or a decrease or increase in the amount of IL-2 / TNFR agonist molecule administered per dose, or a "break" in dosing (i.e., a temporary interruption of treatment, either for a specified period of time or until the treating physician determines that treatment should be continued or until monitored patient response indicates that treatment should or can be resumed), or termination of treatment. In one embodiment, the response is a change in the patient's body temperature or CRP level. For example, the response can be an increase in the patient's body temperature, or an increase in the CRP level in a sample of peripheral blood, or both. In a particular embodiment, the patient's treatment is reduced, suspended, or terminated 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. In another specific embodiment, a patient's treatment is reduced, suspended, or terminated if the concentration of CRP in a sample of the patient's peripheral blood increases by at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1, 1.5, or 2 mg / mL during the course of treatment.Other patient responses that may be monitored and used in deciding whether to modify, reduce, pause, or terminate treatment include the development or worsening of capillary leak syndrome (hypotension and cardiovascular instability), neutrophil dysfunction (e.g., the development or worsening of an infection occurs or is detected), thrombocytopenia, thrombotic vasculopathy, injection site reactions, vasculitis (such as hepatitis C virus vasculitis), or inflammatory symptoms or disease. Further patient responses that may be monitored and used in deciding whether to modify, reduce, increase, pause, or terminate treatment include NK cell, Treg cell, and 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 percentage of such cell types compared to other cell types in the blood sample. Another patient response that can be monitored is the increase in the number of CD25 T cells in a sample of the patient's peripheral blood. + This is an increase in the amount of cell surface-bound IL-2 / TNFR agonist molecules on the cells.

[0143] Methods for expanding Treg cells IL-2 / TNFR agonist molecules or IL-2 / TNFR agonist molecule Fc fusion proteins may be used to expand Treg cells in a subject or sample. Provided herein are methods for increasing the ratio of Tregs to non-regulatory T cells. The method involves contacting a population of T cells with an effective amount of a human IL-2 / TNFR agonist molecule or IL-2 / TNFR agonist molecule Fc fusion. The ratio can be measured by determining the ratio of CD3+FOXP3+ cells to CD3+FOXP3- cells in the T cell population. The typical Treg frequency in human blood is 5-10% of the total CD4+CD3+ T cells. However, in the diseases listed above, this percentage may be lower or higher. In preferred embodiments, the percentage of Tregs 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 in Tregs may vary for specific diseases; however, the maximum Treg frequency potentially achievable by IL-2 mutein treatment is 50% or 60% of total CD4+CD3+ T cells. In specific embodiments, an IL-2 / TNFR agonist molecule or an IL-2 / TNFR agonist molecule Fc fusion protein is administered to a subject to increase the ratio of regulatory T cells (Tregs) to non-regulatory T cells in the subject's peripheral blood.

[0144] Because IL-2 / TNFR agonist molecules, IL-2 / TNFR agonist molecule Fc fusion proteins, and other combinations of IL-2 and TNFR preferentially expand Tregs over other cell types, they are also useful for increasing the ratio of regulatory T cells (Tregs) to natural killer (NK) cells and / or the ratio of Tregs to cytotoxic T cells (Tcon) in a subject's peripheral blood. This ratio can be measured by determining the ratio of CD3+ FOXP3+ cells to CD16+ and / or CD56+ lymphocytes that are CD19- and CD3-. In addition, it has been surprisingly discovered that IL-2 / TNFR agonist molecules, IL-2 / TNFR agonist molecule Fc fusion proteins, and combinations of IL-2 and TNFR not only preferentially expand Tregs over other cell types, but also reduce the levels of other cell types, including Tcon cells, e.g., CD4+ and / or CD8+ Tcon cells and / or NK cells. In some embodiments, the levels of Tcon, e.g., CD4+ and / or CD8+ Tcon and / or NK cells, are lower than the levels of those cells after administration of IL-2 alone. In some embodiments, this reduction is 10%, 20%, 30%, 40%, 50%, 60%, 70% or more. In some embodiments, the levels of Tcon, e.g., CD4+ and / or CD8+ Tcon and / or NK cells, are lower than baseline levels (e.g., the control in Example 2 and FIG. 6). In some embodiments, this reduction is 10%, 20%, 30%, 40%, 50%, 60%, 70% or more.

[0145] It is contemplated that an IL-2 / TNFR agonist molecule or IL-2 / TNFR agonist molecule Fc fusion protein may have a therapeutic effect on a disease or disorder in a patient without significantly increasing the ratio of Tregs to non-regulatory T cells or NK cells in the patient's peripheral blood. This therapeutic effect may be due to local activity of the IL-2 / TNFR agonist molecule or IL-2 / TNFR agonist molecule Fc fusion protein at the site of inflammation or autoimmunity. [Example]

[0146] The following examples, both real and hypothetical, are provided for the purpose of illustrating specific embodiments or features of the present invention and are not intended to limit its scope.

[0147] Example 1 - Combining TNFR and IL-2R agonism promotes Treg cell expansion To determine the effects of TNFR and IL-2R stimulation, human peripheral blood mononuclear cells were labeled with cell trace violet, treated with various TNFR agonists (anti-OX40, anti-DR3, TNF) in conjunction with IL-2, and analyzed 4 days later. Cells stimulated with anti-CD3 showed robust proliferation and served as a positive control for this assay. Stimulation with IL2 or control IgG did not result in any CTV dilution. No proliferation was observed with any of the indicated TNFR agonists alone. Combining stimulation with anti-OX40 and IL2 led to Treg cell expansion as seen by CTV dilution.

[0148] PBMCs from healthy human donors were labeled with cell trace violet (Invitrogen) according to the manufacturer's instructions and cultured in x-vivo 15 medium (Lonza). Reagents were obtained from the following suppliers: TNF (R&D systems), anti-DR3 (Biolegend), and anti-OX40 (having a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO: 10). Samples were analyzed on a Symphony flow cytometer (BD biosciences), and data were analyzed using Flojo software.

[0149] Figure 1 shows Treg proliferation upon stimulation with IL-2 in combination with TNFR agonists (anti-OX40, recombinant TNF, or anti-DR3). (A) Cell Trace Violet (CTV)-labeled human PBMCs were stimulated with the indicated reagents for 4 days and then subjected to flow cytometry analysis. Histograms are arranged in the following order (bottom to top): no stimulation, 1 μg / ml anti-CD3, 20 U / ml IL-2, IgG, TNFR agonists (anti-OX40, recombinant TNF, anti-DR3), and TNFR agonist + IL-2 stimulation. Histograms were gated on Treg cells (CD4+Foxp3+). Only the positive control anti-CD3 plot and the combination of TNFR agonists (anti-OX40, recombinant TNF, and anti-DR3) demonstrated Treg proliferation. Figure 2 shows histogram plots of anti-OX40 and IL-2 on PBMCs. Figure 3 shows histogram plots of TNF and IL-2 against PBMCs, Figure 4 shows histogram plots of anti-DR3 and IL-2 against PBMCs, and Figure 5 shows histogram plots of anti-GITR and IL-2 against PBMCs.

[0150] Example 2 - In vivo study of combined TNFR and IL-2R agonism promoting Treg cell expansion C57 / Bl6 mice (n = 6) received 1 mg / kg of either murine IL-2 muteins, anti-OX40, or anti-OX40-IL2, and the spleens, lymph nodes, and lungs were removed on day 4 (n = 3) or day 15 (n = 3). Examples of molecules created for this study are shown in Figure 6. Molecule #3 was used for this specific study. PBS-treated mice were used as controls. The effects of these treatments on the frequencies of the indicated cell populations were examined. Tregs were identified as CD4+Foxp3+, activated T cells were identified as CD4+Foxp3-CD25+, activated CD8 cells were identified as CD8+CD44+, and NK / ILCs were identified as CD4-CD8-NK1.1+. Results are representative of two independent experiments. Data are presented as fold expansion relative to the control (value set at 1).

[0151] The results are shown in Figure 7. Treatment with IL2 alone resulted in the expansion of Treg cells in some tissues; however, increases in the frequencies of activated CD4 and CD8 cells and NK cells were also observed. Anti-OX40 treatment also resulted in Treg expansion of a similar magnitude compared to IL2, without significant effects on other immune cell types. Anti-OX40-IL2 fusion administration led to a much higher fold expansion of Treg cells compared to IL2 or anti-OX40 alone, while having minimal effects on other cells. Furthermore, anti-OX40-IL2 fusion-mediated Treg cell expansion persisted for a longer period of time compared to other treatments, demonstrating a supra-additive effect to IL2 or OX40 alone in both spleen and lung tissues.

Claims

1. A human interleukin-2 (IL-2) chimeric molecule comprising a human IL-2 polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:1, and an OX40 agonist antibody.

2. The human IL-2 polypeptide is a human IL-2 polypeptide mutein, and the IL-2 mutein is selected from the group consisting of V91K, N30S, N30D, Y31H, Y31S, K35R, V69A, Q74P, V91K / D20L, D84R / E61Q, V91K / D20A / E61Q / M104T, N88K / M104L, V91H / M104L, V91K / H16E / M104V, V91K / H16R / M104V, V91K / H16R / M104T, V91K / D20A / M104T, V91K / H16E / M104T, and V91K / H16E / E61Q / M104T, V91K / H16R / E61Q / M104T, V91K / H16E, V91H / D20A / M104T, H16E / V91 H / M104V, V91H / D20A / E61Q / M104T, V91H / H16R / E16Q, V91K / D20A / M104V, H16 E / V91H, V91H / D20A / M104V, H16E / V91H / M104T, H16E / V91H / E61Q / M104T, V9 1K / E61Q / H16E, V91K / H16R / M104L, H16E / V91H / E16Q, V91K / E61Q / H16R, D20W / V91K / E61Q, V91H / H16R, V91K / H16R, D20W / V91K / E61Q / M104T, V91K / D20A, V91H / D20A / E16Q, V91K / D20A / M104L, V91H / D20A, V91K / E61Q / D20A, V91H / M1 04T, V91H / M104V, V91K / E61Q, V91K / N88K / E61Q / M104T, V91K / N88K / E61Q, V 91H / E61Q, V91K / N88K, D20A / H16E / M104T, D20A / M104T, H16E / N88K, D20A / M1 04V, D20A / M104L, H16E / M104T, H16E / M104V, N88K / M104V, N88K / E61Q, D20A / E61Q, H16R / D20A, D20W / E61Q, H16E / E61Q, H16E / M104L, N88K / M104T, D20A / H16E, D20A / H16E / E16Q, D20A / H16R / E16Q, V91K / D20W, V91A / H16A, V91A / H1 6D, V91A / H16E, V91A / H16S, V91E / H16A, V91E / H16D, V91E / H16E, V91E / H16S,V91K / H16A, V91K / H16D, V91K / H16S, V91S / H16E, L12G, L12K, L12Q, L12S, Q13G, E15A, E15G, E15S, H16A, H16D, H16G, H16K, H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D, L19E, L19G, L19N, L19R, L19S, L19T, L19V, D20A, D20E, D20F, D20G, D20T, D20W, M23R, N30S, The human IL-2 chimeric molecule of claim 1, which has at least one mutation selected from Y31H, K35R, V69A, Q74P, R81A, R81G, R81S, R81T, D84A, D84E, D84G, D84I, D84M, D84Q, D84R, D84S, D84T, S87R, N88A, N88D, N88E, N88F, N88G, N88M, N88R, N88S, N88V, N88W, V91D, V91E, V91G, V91S, I92K, I92R, and / or E95G.

3. A human IL-2 chimeric molecule as described in claim 2, wherein the human IL-2 polypeptide further comprises a substitution at C125A.

4. 2. The human IL-2 chimeric molecule of claim 1, wherein the anti-OX40 antibody has a heavy chain antibody sequence of SEQ ID NO:9 and a light chain amino acid sequence of SEQ ID NO:

10.

5. The human IL-2 chimeric molecule according to any one of claims 1 to 4, wherein a linker connects the anti-OX40 agonist antibody and the human IL-2 polypeptide portion of the human IL-2 chimeric molecule.

6. The human IL-2 chimeric molecule of claim 5, wherein the linker is GGGGS (SEQ ID NO: 5), GGNGT (SEQ ID NO: 6), or YGNGT (SEQ ID NO: 7).

7. An isolated nucleic acid encoding the human IL-2 chimeric molecule of any one of claims 1 to 6.

8. 8. An expression vector comprising the isolated nucleic acid of claim 7 operably linked to a promoter.

9. 9. A host cell comprising the isolated nucleic acid of claim 7 or the expression vector of claim 8.

10. 10. A method for producing a human IL-2 chimeric molecule, comprising culturing the host cell of claim 9 under conditions in which the promoter is expressed, and recovering the human IL-2 chimeric molecule from the culture.

11. 10. An in vitro method for increasing the ratio of regulatory T cells (Treg) to non-regulatory T cells within a T cell population or in peripheral blood of a subject, the method comprising contacting the T cell population with a human IL-2 chimeric molecule according to any one of claims 1 to 6.

12. 12. The method of claim 11, wherein the ratio of CD3+FoxP3+ cells to CD3+FoxP3- cells is increased.

13. An in vitro method for increasing the ratio of regulatory T cells (Treg) to natural killer (NK) cells in the peripheral blood of a subject, the method comprising contacting the T cell population with a human IL-2 chimeric molecule described in any one of claims 1 to 6.

14. The method of claim 13, wherein the ratio of CD3+FoxP3+ cells to CD3-CD19- lymphocytes expressing CD56 and / or CD16 is increased.

15. A pharmaceutical composition for treating a subject suffering from an inflammatory disease or an autoimmune disease, the pharmaceutical composition comprising a therapeutically effective amount of a human IL-2 chimeric molecule according to any one of claims 1 to 6.

16. The inflammatory disease or autoimmune disease is selected from the group consisting of inflammation, autoimmune disease, atopic disease, paraneoplastic autoimmune disease, 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 (serum Negative reaction, 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, dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myelitis, polymyositis, dermatomyositis, polyarteritis nodosa, ulcerative colitis ... Egener'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), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, 16. The pharmaceutical composition of claim 15, wherein the disease is ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, rhinosinusitis, rhinosinusitis with polyposis, eosinophilic esophagitis, eosinophilic bronchitis, Guillain-Barré disease, type 1 diabetes, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, graft-versus-host disease, transplant rejection, kidney damage, or hepatitis C-induced vasculitis.

17. 16. The pharmaceutical composition of claim 15, wherein the inflammatory disease or autoimmune disease is systemic lupus erythematosus (SLE), graft-versus-host disease, hepatitis C-induced vasculitis, type I diabetes, rheumatoid arthritis, multiple sclerosis, spontaneous abortion, atopic disease, inflammatory bowel disease, ulcerative colitis, celiac disease, lupus, type II diabetes, alopecia areata, atherosclerosis, psoriasis, transplant organ rejection, Sjogren's syndrome, Behcet's disease, or asthma.

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