PARTIAL AGONISTS OF INTERLEUKIN-2

Abstract

The present invention relates to a human interleukin 2 (hIL-2) mutein, wherein the hIL-2 mutein is a partial agonist of hIL-2 and has an amino acid sequence identical to SEQ ID NO: 1, except for: (i) the L18R and Q22E substitutions; (ii) an amino acid substitution at position Q126 selected from the group consisting of Q126H and Q126K; and (iii) an N-terminated deletion of 0, 1, 2, or 3 amino acids.

Description

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 62 / 589,497, filed November 21, 2017. The disclosures of the above-mentioned applications are expressly incorporated herein by reference in their entirety, including any drawings. STATEMENT REGARDING RESEARCH OR DEVELOPMENT SPONSORED BY THE FEDERAL GOVERNMENT

[0002] The research was conducted with government support under R37 AI051321 granted by the National Institutes of Health. The government has certain rights in the present invention. REFERENCE TO THE LIST OF SEQUENCES

[0003] This application is being submitted together with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled “Sequence_Listing_078430503001WO.txt”, created on November 14, 2018, which is approximately 32 KB in size. The information in the electronic Sequence Listing is incorporated herein by reference in its entirety. FIELD OF INVENTION

[0004] This document discloses, among other things, interleukin-2 mutains that exhibit the properties of partial agonism of the IL-2 receptor signal, as well as methods for using them for the treatment of autoimmune diseases. BACKGROUND OF THE INVENTION

[0005] Interleukin 2 (IL-2) is a pluripotent cytokine produced primarily by activated CD4+ T cells, which plays a key role in the production of a normal immune response. IL-2 promotes the proliferation and expansion of activated T lymphocytes, enhances the growth of B cells, and activates monocytes and natural killer cells.

[0006] In addition to its physiological role in the normal immune response, IL-2 can promote pathological responses, and a therapeutic goal is to maintain desired actions of this cytokine while blocking unwanted autoimmune or immunosuppressive responses. Two monoclonal antibodies (mAbs, monoclonal antibodies) to human IL-2, daclizumab and basiliximab, are approved by the Food and Drug Administration. Foods (FDA, Food and Drug Administration) and exhibit efficacy in kidney transplant rejection (Vincenti et al., N Engl J Med 338: 161 (1998)), heart transplantation (Hershberger et al., N Engl J Med 352: 2705 (2005)), multiple sclerosis (Gold et al., Lancet SQ : 2167 (2013)), and asthma (Bielekova et al., Proc. Nati. Acad. Sel. USA 101: 8705 (2004); and Busse et al., Am J Resplr Crit Care Med 178: 1002 (2008)) but do not block IL-2 signaling by means of intermediate-affinity IL-2Ry receptors expressed on NK and CD8+ memory cells (Tkaczuk et al., Am J Transplant 2: 31 (2002)). Although anti-human IL-2Rp mAb Mikpi can block the trans-presentation of IL-2 and IL-15 to cells expressing IL-2Ry receptors (Morris et al., Proc. Nati. Acad. Sel. USA 103: 401 (2006)), it is relatively ineffective at blocking cis signaling by IL-2 or IL-15 through their high-affinity heterotrimeric receptor complexes (Morris et al., Proc. Nati. Acad. Sel.USA 103: 401 (2006); and Waldmann et al., Blood 121: 476 (2013)). Consequently, new IL-2 mutains are needed that can promote the therapeutically beneficial effects of this cytokine but also block one or more IL-2 functions that are associated with unwanted autoimmune or immunosuppressive responses. BRIEF DESCRIPTION OF THE INVENTION

[0007] This disclosure generally pertains to the field of immunology and medicine, including compositions and methods for modulating the interleukin-2 (IL-2) mediated signal transduction pathway in a subject in need. More specifically, in some modalities, the disclosure provides novel IL-2 mutans with modulated affinity for at least one of the IL-2 receptors, e.g., interleukin-2 receptor alpha (IL-2Ra), interleukin-2 receptor beta (IL-2R3), interleukin-2 receptor gamma (IL-2Ry). Some modalities of the disclosure provide partial IL-2 agonists that do not promote the activation of immune cells responsible for adverse, unwanted autoimmune events, such as inflammation.Some aspects of the disclosure relate to compositions and methods useful for producing such IL-2 muteins, as well as methods for treating health conditions and disorders associated with signal transduction disturbances mediated by the IL-2 signaling pathway.

[0008] In one aspect, an interleukin 2 (IL-2) mutan is provided having: (a) reduced binding affinity for the interleukin 2 receptor (IL-2Rβ) compared to an IL-2 polypeptide encoded by ID of SEC NO: 2; and (b) 15-95% of Emax compared to the polypeptide encoded by ID of SEO NO: 2. In some embodiments, the IL-2 mutan includes: (i) one or more amino acid substitutions that increase the binding affinity of IL-2Rβ compared to the polypeptide encoded by ID of SEC NO: 1, selected from L80F, R81D, L85V, I86V, and I92F, numbered according to the amino acid sequence of ID of SEC NO: 1; and / or (i) one or more amino acid substitutions that reduce the binding affinity of the IL-2Ry receptor and result in 15-95% of Emax compared to the polypeptide encoded by ID of SEC NO: 2, selected from (A) L18R and Q22E; and (B) amino acid position 126, numbered according to the amino acid sequence of ID of SEC NO: 2.In some embodiments, the amino acid substitution at position 126 of the ID of SEC NO: 2 is selected from the group consisting of Q126A, Q126C, Q126D, Q126E, Q126G, Q126H, Q126I, Q126K, Q126M, Q126R, Q126S, or Q126T. In some embodiments, the IL-2 mutein includes the amino acid substitution Q126H, Q126K, or Q126M. In some embodiments, the IL-2 mutein includes an amino acid substitution Q126H.

[0009] In some formulations, the IL-2 mutein is structurally modified to increase its half-life. In some formulations, the modification includes one or more modifications selected from the group consisting of fusion to a human antibody Fe fragment, fusion to albumin, and PEGylation. In some formulations, the IL-2 mutein promotes Trecy cell expansion and does not promote the expansion of potentially inflammatory T cells and natural killer (NK) cells or granulocytes. In some formulations, the IL-2 mutein induces less proliferation of potentially inflammatory T cells compared to the polypeptide encoded by ID SEC NO: 1 or ID SEO NO: 2. In some formulations, the IL-2 mutein increases Leg cell proliferation by at least 3-fold and / or induces less IFNγ secretion compared to the polypeptide encoded by ID SEC NO: 1 or ID SEC NO: 2.In some modalities, the potentially inflammatory T cells are CD4+IFNγ+ T cells or CD8+IFNγ+ T cells. In some modalities of any of the modalities disclosed herein, the mutein does not induce IFNγ secretion from CD8+ T cells and / or other subsets of inflammatory immune cells. In some modalities, the mutein has 70–95% of the Emax compared to the polypeptide encoded by the SEC NO:1 ID.

[0010] In one aspect, this document provides an interleukin 2 (IL-2) mutein having (a) reduced binding affinity for the interleukin receptor (IL-2Ry); and (b) 15-95% Emaxen compared to a polypeptide encoded by ID of SEC NO: 1. In some embodiments, the mutein includes one or more amino acid substitutions that reduce the IL-2Ry receptor binding affinity and result in 15-95% Emaxen compared to a polypeptide encoded by ID of SEC NO: 1, selected from (A) L18R and Q22E; and (B) amino acid position 126, numbered according to the amino acid sequence of ID SEC NO: 1. In some embodiments, the IL-2 mutein includes an amino acid substitution at position 126 of ID SEC NO: 1 selected from the group consisting of Q126A, Q126C, Q126D, Q126E, Q126G, Q126H, Q126I, Q126K, Q126M, Q126R, Q126S, or Q126T. In some embodiments, the IL-2 mutein includes an amino acid substitution of Q126H, Q126K, or Q126M.In some formulations, the IL-2 mutein includes a Q126H amino acid substitution. In some formulations, the IL-2 mutein is structurally modified to increase its half-life. In some formulations, the modification includes one or more modifications selected from the group consisting of fusion to a human antibody Fe fragment, fusion to albumin, and PEGylation. In some formulations, the IL-2 mutein of the disclosure enhances regulatory T cell (Treg) proliferation and / or induces minimal proliferation of potentially inflammatory T cells. In some formulations, the IL-2 mutein promotes Trecy cell expansion and does not promote the expansion of potentially inflammatory T cells and natural killer (NK) cells or granulocytes. In some formulations, the IL-2 mutein induces less proliferation of potentially inflammatory T cells compared to the polypeptide encoded by ID SEC NO: 1 or ID SEC NO: 2.In some modalities, potentially inflammatory T cells are CD4+IFNγ+ T cells or CD8+IFNγ+ T cells. In some modalities, the IL-2 mutan increases Treg cell proliferation by at least 3-fold and / or induces less IFNγ secretion compared to the polypeptide encoded by ID SEC NO: 1 or ID SEO NO: 2. In some modalities, the mutan does not induce IFNγ secretion from CD8+ T cells and / or other subsets of inflammatory immune cells. In some modalities, the IL-2 mutan of the disclosure has 70-95% of Emax compared to the polypeptide encoded by ID SEC NO: 1.

[0011] In additional respects, this document provides (i) nucleic acids encoding any of the IL-2 muteins disclosed herein, (ii) vectors including the nucleic acids, (iii) host cells including the vectors or nucleic acids, and (iv) sterile pharmaceutical compositions including any of the IL-2 muteins disclosed herein, and / or any of the nucleic acids or vectors disclosed herein and a pharmaceutically acceptable excipient.

[0012] In additional aspects, this document also provides syringes and catheters including a syringe containing any of the IL-2 muteins disclosed herein, any of the nucleic acids or vectors disclosed herein, and / or any of the pharmaceutical compositions disclosed herein.

[0013] In still other respects, this document provides kits that include: (i) any of the IL-2 muteins disclosed herein, (ii) any of the nucleic acids or vectors disclosed herein, (ii) any of the syringes or catheters disclosed herein, and / or any of the pharmaceutical compositions disclosed herein as well as written instructions for using them.

[0014] In another respect, this document provides methods for treating an autoimmune disease in an individual in need. The method includes administering a therapeutically effective amount of (i) any of the IL-2 mutains disclosed herein, (ii) any of the nucleic acids or vectors disclosed herein, and / or (ii) any of the pharmaceutical compositions disclosed herein to the individual. In some modalities, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, insulin-dependent diabetes mellitus, hemolytic anemias, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, multiple sclerosis, alopecia areata, psoriasis, vitiligo, dystrophic epidermolysis bullosa, systemic lupus erythematosus, and graft-versus-host disease. In some modalities, the autoimmune disease is graft-versus-host disease.In some embodiments of the methods disclosed herein, the method further includes administering any of the IL-2 muteins or pharmaceutical compositions disclosed herein in combination with an antibody that directs the mutein to a specific cell type. In some embodiments, the cell type is a regulatory T cell (Treg). In some embodiments, the antibody is either covalently or non-covalently bound to the IL-2 mutein.

[0015] In other respects, this document provides methods for producing any of the muteins disclosed herein. The method includes culturing any of the host cells disclosed herein under conditions suitable for mutein production. In other embodiments, the method further includes isolating and / or purifying the mutein. In some embodiments, the method further includes structurally modifying the mutein to increase its half-life. In other embodiments, the method further includes that the modification includes one or more modifications selected from the group consisting of fusion to a human antibody Fe fragment, fusion to albumin, and PEGylation.

[0016] This document also provides, in some aspects, methods for preventing the proliferation of potentially inflammatory T cells and / or preventing the secretion of IFNγ from CD8+ T cells or other subsets of inflammatory immune cells. The methods include contacting a cell expressing an interleukin-2 receptor γ (IL-2Rγ) with any of the IL-2 mutains disclosed herein. In other modalities, the potentially inflammatory T cells are either CD8+CD44+IFNγ+ T cells or CD8+CD44+IFNγ+ T cells. In some modalities, the method is carried out in vitro, in vivo, or ex vivo. In some modalities of any of the methods disclosed herein, the IL-2 mutain is structurally modified to increase its half-life.In some forms of any of the forms disclosed in this document, the modification is one or more modifications selected from the group consisting of fusion to a human antibody Fe fragment, fusion to albumin, and PEGylation.

[0017] In another respect, methods for decreasing the proliferation of regulatory T cells (Tregs) are provided herein, including contacting a Treg cell with an interleukin 2 (IL-2) mutan that has: (i) reduced binding affinity for the interleukin 2 receptor γ (IL-2Ry) compared to the polypeptide encoded by the ID of SEC NO: 2; and (ii) 0-50% of Emax compared to the polypeptide encoded by the ID of SEC NO: 2.In some embodiments, the IL-2 mutein includes: (i) one or more amino acid substitutions that increase the binding affinity of IL-2Rp compared to the polypeptide encoded by ID of SEC NO: 1, selected from L80F, R81D, L85V, I86V, and I92F, numbered according to the amino acid sequence of ID of SEC NO: 1; and (ii) one or more amino acid substitutions that reduce the binding affinity of the IL-2Ry receptor and result in 0-50% of Emax compared to the polypeptide encoded by ID of SEC NO: 2, selected from (A) L18R and Q22E; and (B) amino acid position 126, numbered according to the amino acid sequence of ID of SEC NO: 2.

[0018] In another aspect, methods for decreasing regulatory T cell (Treg) proliferation are provided herein, including contacting a Treg cell with an IL-2 mutan having: (i) reduced binding affinity for IL-2Ry compared to the polypeptide encoded by ID of SEC NO: 1; and (ii) 0-50% of Emax compared to the polypeptide encoded by ID of SEC NO: 1. In some embodiments, the IL-2 mutan includes one or more amino acid substitutions that reduce the binding affinity of the IL-2Ry receptor and result in 0-50% Emax compared to a polypeptide encoded by ID of SEC NO: 1, selected from (A) L18R and Q22E; and (B) amino acid position 126, numbered according to the amino acid sequence of ID of SEC NO: 1.In some embodiments, the amino acid substitution at position 126 of either SEC NO: 1 or SEC NO: 2 is selected from the group consisting of Q126A, Q126C, Q126D, Q126E, Q126G, Q126H, Q126I, Q126K, Q126M, Q126R, Q126S, or Q126T. In some embodiments, the IL-2 mutein includes the Q126H, Q126K, or Q126M amino acid substitution. In some embodiments, the IL-2 mutein includes a Q126H amino acid substitution. In some embodiments of any of the embodiments disclosed herein, the method further includes administering the IL-2 muteins with an antibody that directs the mutein to a Treg cell. In some variations of any of the methods disclosed herein, the antibody is covalently or non-covalently bound to the mutein. In some variations of any of the methods disclosed herein, the method is carried out in vitro, in vivo, or ex vivo.

[0019] Each of the aspects and modalities described in this document has the ability to be used together, unless explicitly or clearly excluded from the context of the modality or aspect.

[0020] The brief description of the foregoing invention is illustrative only and is not intended to be limiting in any way. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects, and features of the disclosure will become fully apparent from the drawings, the detailed description, and the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 represents the amino acid sequences of natural-type IL-2 and muteins constructed using the H9 antecedent.

[0022] Figure 2 represents the amino acid sequences of natural-type IL-2 and muteins constructed using the natural-type antecedent.

[0023] Figure 3 represents the results of a dose-response phospho-STAT5 signaling assay of IL-2 variants. Human YT-like NK cells were stimulated for 15 min with different human IL-2 variants fused with mouse serum albumin (MSA) at different concentrations as shown in the graph (from 1 μM to 0 μM, 10-fold dilution). The Y-axis shows the ratio of the pSTAT5 signal of each IL-2 variant normalized to the pSTAT5 signal of WT IL-2 for each concentration.

[0024] Figures 4A-4B represent the time course for the phosphoSTAT5 signaling assay. Figure 4A shows the results of a p-STAT5 signaling assay in YT cells stimulated for 15 min with the indicated concentration of IL-2 partial agonists. The Y-axis shows the ratio of the p-STAT5 signal of each IL-2 variant normalized to the p-STAT5 signal of WT IL-2 for each concentration. Figure 4B shows that YT cells were stimulated at different time points with 1 μM of different IL-2 variants as indicated in the graph. The Y-axis indicates the median fluorescence intensity (MFI) for the p-STAT5 signal.

[0025] Figure 5A represents the experimental protocol for administering IL-2 agonists to Lcn / nznz / q / Yi mice. Figure 5B, Figure 5C, and Figure 5D represent the frequency of the indicated immune cell subset for each condition, normalized to the respective frequency in PBS-treated mice. B cells were defined as cells activated to CD3 CD19+. NK cells were activated to CD3 NK1.11. Cells activated to Ly6g (Gr1)+CD3+CD11b+ were defined as granulocytes.

[0026] Figures 6A-6B represent the results of a phosphoSTAT5 signaling assay with dose response of IL-2 variants. Figure 6A represents NK-like YT cells, while Figure 6B represents starved mouse blast T cells (B) stimulated for 15 min with different human IL-2 variants fused with mouse serum albumin (MSA) at different concentrations as indicated in the graph (from 5 μM to 0 μM). The Y-axis shows the ratio of the p-STAT5 signal of each IL-2 variant normalized to the p-STAT5 signal of WT IL-2 for each concentration.

[0027] Figures 7A-7E represent the results of IL-2 variant administration in a B16 melanoma mouse model. Figure 7A represents changes in humor volume. Figure 7B, Figure 7C, Figure 7D, and Figure 7E represent the frequency of the indicated immune cell subset for each condition normalized to the respective frequency in WT mice.

[0028] Figures 8A-8G graphically summarize the results of experiments conducted to demonstrate that several exemplary IL-2R partial agonists can elicit cell-type-specific responses in vivo.

[0029] Figures 9A-9C graphically summarize the experimental results of the experiments performed to illustrate that the IL-2R partial agonist REH increases the frequency of FoxP3+ regulatory T cells.

[0030] Figure 10 is a graphical summary of the results of experiments conducted to demonstrate that Tregs from mice treated with partial IL-2R REH agonist suppress the proliferation of conventional CD4+ T cells.

[0031] Figure 11 is a graphical summary of the results of experiments conducted to illustrate that the partial IL-2R agonist REH supports CD8+ T cell proliferation but not IFNy production.

[0032] Figure 12 is a graphical summary of the results of experiments conducted to demonstrate that pre-treatment and co-treatment with the IL-2R partial agonist REH are protective against various autoimmune symptoms in the EAE rodent model (an animal model of brain inflammation). Disease scores were as follows: 0 - healthy; 1 - floppy tail; 2 - partial hind limb paralysis; 3 - complete hind limb paralysis; 4 - whole-body paralysis; 5 - death. DETAILED DESCRIPTION OF THE INVENTION

[0033] This disclosure generally relates to the field of immunology and medicine, including compositions and methods for modulating the interleukin-2 (IL-2) mediated signal transduction pathway in a subject in need. More particularly, in some embodiments, the disclosure provides novel IL-2 muteins with modulated affinity for at least one of the IL-2 receptors, e.g., interleukin-2 receptor alpha (IL-2Ra), interleukin-2 receptor beta (IL-2RP), interleukin-2 receptor gamma (IL-2Ry), thereby antagonizing either completely or partially the downstream signal transduction mediated by the respective IL-2Ra, IL-2Rp, and / or IL-2Ry receptors. Some disclosure modalities provide partial IL-2 agonists that do not promote the activation of immune cells responsible for unwanted adverse autoimmune events, such as inflammation.Some aspects of the disclosure relate to compositions and methods useful for producing such IL-2 muteins, as well as methods for treating health conditions and disorders associated with signal transduction disturbances mediated by the IL-2 signaling pathway.

[0034] IL-2 exerts a broad spectrum of effects on the immune system and plays crucial roles in regulating both immune activation and homeostasis. As an immune system stimulator, IL-2 has found use in the treatment of cancer and chronic viral infections. However, the stimulatory effects of IL-2 can also wreak havoc, mediating autoimmunity and transplant rejection. Due to its instrumental role in immune regulation and disease, the identification of novel IL-2 molecules, such as IL-2 partial agonists, remains an active area of ​​research.

[0035] In most circumstances, IL-2 works through three different receptors: IL-2Ra, IL-2RP, and IL-2Ry. Most cells, such as resting T cells, are not sensitive to IL-2 because they only express IL-2R3 and IL-2Ry, which have low affinity for IL-2. Upon stimulation, resting T cells express the relatively high-affinity IL-2Ra. The binding of IL-2 to IL-2Ra causes this receptor to sequentially bind to IL-2Rp and IL-2Ry, resulting in T cell activation.

[0036] The disclosure described herein provides, among other things, novel IL-2 compositions based on new insights into how IL-2 interacts with its cognate receptors, particularly IL-2Ry. Remarkably, the inventors have discovered that mutations in the IL-2 binding site for IL-2Ry result in partial agonists capable of activating regulatory T cells (Tregs). Furthermore, these IL-2 partial agonists do not activate CD8+ T cells and other potentially inflammatory immune cell subsets, nor do they induce inflammatory cells to secrete interferon-gamma (IFN-γ). As such, these molecules may be used to treat autoimmune disorders and conditions. I. General Techniques

[0037] The practice described herein will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those experienced in the field. Such techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, Fourth Edition (Sambrook et al., 2012) and Molecular Cloning: A Laboratory Manual, Third Edition (Sambrook and Russell, 2001), (collectively referred to herein as Sambrook); Current Protocol in Molecular Biology (F.M. Ausubel et al., eds., 1987, including supplements up to 2014); PCR: The Polymerase Chain Reaction (Mullís et al., eds., 1994); Beaucage et al. eds., Current Protocol in Nucleic Acid Chemistry, John Wiley & Sons, Inc., New York, 2000, (including supplements up to 2014), Gene Transfer and.Expression in Mammalian Cells (Makrides, ed., Elsevier Sciences BV, Amsterdam, 2003), and Current Protocol in Immunology (Horgan K and S. Shaw (1994) (including supplements up to 2014). As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out according to the protocols and / or parameters defined by the manufacturer unless otherwise indicated. II. Definition

[0038] Unless otherwise defined, all subject terms, annotations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those experienced in the subject matter to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from what is generally understood in the field. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those experienced in the field.

[0039] The singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B”.

[0040] The term “approximately”, as used herein, has its ordinary meaning of plus or minus. If the degree of approximation is not clear from the context, “approximately” means either plus or minus 10% of the value given, or rounded to the nearest significant figure, in all cases inclusive of the value given. Where ranges are given, these are inclusive of the boundary values.

[0041] As used herein, the term “IL-2” means natural-type IL-2, either native or recombinant. As such, an IL-2 polypeptide refers to any IL-2 polypeptide, including but not limited to recombinantly produced IL-2 polypeptides, synthetically produced IL-2 polypeptides, and IL-2 extracted from cells or tissues. Mature human IL-2 is presented as a 133-amino-acid sequence (less the signal peptide, which consists of an additional 20 N-terminal amino acids), as described in Fujita et al., Proc. Nati. Acad. Sel. USA, 80, 7437–7441 (1983). The human IL-2 amino acid sequence (SEQ ID NO: 17) is located in GenBank under accession number NP_000577.2. The amino acid sequence of mature human IL-2 is represented in SEC ID NO: 1. The amino acid sequence of murine IL-2 (Mus musculus) is located in Genbank under the accession locator (SEC ID NO: 18).The amino acid sequence of mature murine IL-2 is represented in the ñor Lcn / nznz / q / Yi. IDdeSEC NO: 19.

[0042] As used herein, “IL-2 mutein” refers to an IL-2 polypeptide in which specific substitutions have been made to the interleukin-2 protein. IL-2 muteins are characterized by insertions, deletions, substitutions, and modifications of amino acids at one or more sites on the other residues of the native IL-2 polypeptide chain. According to this disclosure, any such insertions, deletions, substitutions, and modifications result in an IL-2 mutein that retains IL-2R-binding activity. For example, the muteins disclosed herein may have high or low affinity for IL-2Ro and / or IL-2Rp or may have an affinity for these receptors identical or similar to that of natural-type IL-2. Exemplary muteins may include substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.Muteins may also include conservative modifications and substitutions at other IL-2 positions (i.e., those that have minimal effect on the secondary or tertiary structure of the mutein). Such conservative substitutions include those described by Dayhoff in The Atlas of Protein Sequence and Structure 5 (1978) and by Argos in EMBO J, 8:779-785 (1989). For example, amino acids belonging to one of the following groups represent conservative changes: Group I: Ala, Pro, Gly, Gln, Asn, Ser, Thr; Group II: Cys, Ser, Tyr, Thr; Group III: Val, Ie, Leu, Met, Ala, Phe; Group IV: Lys, Arg, His; Group V: Phe, Tyr, Trp, His; and Group VI: Asp, Glu.

[0043] The phrase “numbered according to” means to identify a chosen amino acid by reference to the position at which that amino acid normally appears in a given amino acid sequence, such as the amino acid sequence of natural-type IL-2. For example, R81 refers to the eighty-first amino acid, arginine, which appears in ID of SEQ NO: 1.

[0044] The term “identity,” as used herein in reference to polypeptide or DNA sequences, refers to the subunit sequence identity between two molecules. When a subunit position in both molecules is occupied by the same monomeric subunit (i.e., the same amino acid or nucleotide residue), then the molecules are identical at that position. The similarity between sequences of two amino acids or two nucleotides is a direct function of the number of identical positions. In general, sequences are aligned in such a way as to obtain the highest-order match. If necessary, identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molecular BioL 215:403, 1990).Sequence can be measured using sequence analysis software such as the Analysis Software Package of the Genetics Computer Group at the Biotechnology Center of the University of Wisconsin, (1710 University Avenue, Madison, Wis. 53705), with its default parameters.

[0045] As used herein, the terms “protein” and “polypeptide” refer to a polymer of amino acid residues and are not limited to a minimum product length. Therefore, polypeptides, peptides, polypeptide fragments, fusion polypeptides, oligopeptides, and the like are encompassed within the definition. ñor icn / nznz / q / YiAi Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like. Furthermore, for the purposes of this disclosure, a “polypeptide” refers to a protein that includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, provided that the protein retains its desired activity. These modifications may be deliberate, such as through site-directed mutagenesis, or through host mutations that produce the proteins, or due to errors in PCR amplification.Therefore, the term “IL-2 polypeptide” refers to native IL-2 sequences as well as IL-2 analogues, mutains, and IL-2 fragments, unless explicitly or clearly excluded from the context of modality or appearance.

[0046] As used herein, “potentially inflammatory T cells” or “inflammatory immune cell subsets” refers to one or more T cells that produce, secrete, or are capable of producing or secreting interferon gamma. In some modalities, potentially inflammatory T cells express CD44 on their cell surface and produce or secrete interferon gamma. In other modalities, potentially inflammatory T cells express both CD44 and CD4 on their cell surface and produce or secrete interferon gamma. In further modalities, potentially inflammatory T cells express both CD44 and CD8 on their cell surface and produce or secrete interferon gamma. In still other modalities, potentially inflammatory T cells express both CD4 and CD8 on their cell surface and produce or secrete interferon gamma.

[0047] As used herein, “regulatory T cells” or “Treg cells” refers to T cells (T lymphocytes) that regulate the activity of other T cells and / or other immune cells, generally by suppressing their activity. In some modalities, Treg cells are CD4+ and FoxP3+ cells (but it will be appreciated by those experienced in the subject that Tregno cells are restricted entirely to this phenotype).

[0048] “Emax”, as referred to herein, is the maximum p-STAT5 signal that can be generated by IL-2 muteins measured at a higher concentration. WT IL-2 Emax is used as a reference for calculating a ratio of normalized IL-2 mutein p-STAT5 signal to WT IL-2 p-STAT5 signal at the higher concentration.

[0049] An “agonist” is a compound that interacts with a target to cause or promote an increase in activation of the target.

[0050] A “partial agonist” is a compound that interacts with the same target as an agonist but does not produce as large a magnitude of a biomechanical and / or physiological effect as the agonist, even by increasing the dosage of the partial agonist.

[0051] A “super agonist” is a type of agonist that is capable of producing a higher maximum response than the endogenous agonist for the target receptor, and therefore has an efficacy of more than 100%.

[0052] An “antagonist” is a compound that opposes the actions of an agonist, for example, by preventing, reducing, inhibiting, or neutralizing the activity of an agonist. An “antagonist” may also prevent, inhibit, or reduce the constitutive activity of a target, for example, a target receptor, even when no agonist has been identified.

[0053] “Operationally linked” is intended to mean that the nucleotide sequence of interest (i.e., a sequence encoding an IL-2 protein) is linked to the regulatory sequence(s) in a manner that permits expression of the nucleotide sequence (e.g., in an in vitro transcription / conversion system in a host cell when the vector is introduced into the host cell). “Regulatory sequences” include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). See, for example, Goeddel (1990) in Gene Expression Technology: Methods in Enzymology, 185 (Academic Press, San Diego, Calif).Regulatory sequences include those that direct the constitutive expression of a nucleotide sequence in many host cell types and those that direct the expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Those experienced in the field will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the desired level of protein expression, and the like. The expression constructs described herein can be introduced into host cells to produce the human IL-2 mutains disclosed herein or to produce biologically active variants thereof.

[0054] The terms “host cell” and “recombinant host cell” are used interchangeably in this document. It is understood that such terms refer not only to the cell in question but also to the progeny or potential progeny of that cell. Because certain modifications may occur in successive generations due to mutation or environmental influences, such progeny may, in fact, not be identical to the original cell but still be included within the scope of the term as used herein.

[0055] The term “vector” is used in this document to refer to a nucleic acid molecule or sequence capable of transferring or carrying another nucleic acid molecule. The transferred nucleic acid is usually linked to, or inserted into, the nucleic acid molecule of the vector. A vector may include sequences that direct direct autonomous replication in a cell, or it may include sequences sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-defective retroviruses and lentiviruses. In one respect, a vector is a gene delivery vehicle. In one respect, a vector is used as a gene delivery vehicle to transfer a gene into a cell.

[0056] As used herein, the terms “transformation” and “transfection” refer to a variety of techniques recognized in the field for inducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAEdextran-mediated transfection, lipofection, particle gun, or electroporation.

[0057] As used herein, a “subject” or an “individual” or a “patient” includes animals, such as human animals (e.g., human subjects) and non-human animals. The term “non-human animals” includes all vertebrates, such as mammals, such as rodents, such as mice, and non-mammals, such as non-human primates, such as sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0058] As used herein, the term “pharmaceutically acceptable vehicle” includes, but is not limited to, saline solution, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption retardants, and the like, compatible with pharmaceutical administration. Complementary active compounds (e.g., antibiotics) may also be incorporated into the compositions.

[0059] It is understood that the aspects and modalities of disclosure described in this document include “comprising”, “consisting”, and “essentially consisting of” aspects and modalities.

[0060] As will be understood by anyone experienced in the subject, for any and all purposes, such as in terms of providing a written description, all intervals disclosed herein also encompass any and all possible sub-intervals and combinations thereof. Any interval listed may be readily recognized as describing and sufficiently enabling the interval itself by breaking it down into at least halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each interval mentioned herein may be readily broken down into a lower third, middle third, and upper third, etc.As anyone experienced in this area will understand, all language such as “up to,” “at least,” “greater than,” “less than,” and the like includes the number mentioned and refers to intervals that can be further broken down into sub-intervals as previously discussed. Finally, as anyone experienced in this area will understand, an interval includes each individual member. Therefore, for example, a group having 1–3 items refers to groups having 1, 2, or 3 items. Similarly, a group having 1–5 items refers to groups having 1, 2, 3, 4, or 5 items, and so on.

[0061] The subheadings, for example, (a), (b), (i), etc., are included only for ease of reading of the specification and the claims. The use of subheadings in the specification or claims does not require that the steps or elements be carried out in alphabetical or numerical order or in the order in which they are presented. III. Compositions of Dissemination

[0062] Some aspects of the disclosure relate to novel IL-2 muteins with modulated affinity, e.g., increased or reduced binding affinity for one or more IL-2 receptors (e.g., IL-2Ro, IL-2Rp, and / or IL-2Ry) compared to a natural-type IL-2 polypeptide. In particular, some modalities of the disclosure relate to IL-2 muteins in which one or more molecular alterations confer reduced binding affinity for IL-2Ra, IL-2Rp, and / or IL-2Ry. In other words, some of the novel IL-2 muteins disclosed herein are partial agonists of the IL-2-mediated signaling pathway. In some modalities, the IL-2 partial agonists disclosed in this document do not promote the activation of immune cells responsible for unwanted adverse autoimmune events, such as inflammation. Mr. Lcn / nznz / q / Yi A. Partial IL-2 agonists

[0063] In one respect, some disclosure modalities provide IL-2 muteins that are partial agonists. In some modalities, IL-2 muteins are provided herein that contain one or more mutations that reduce the binding affinity of the IL-2 mutein to the IL-2R receptor compared with Natural type IL-2 (e.g., human IL-2, SEO ID NO: 2). As used in this document, all of the terms “common gamma chain,” “γ0,” IL-2Ryc, “I,” “IL-2Ry,” “IL-2 receptor gamma unit,” and “IL-2RG” (GenBank accession numbers: NM_000206 and NP_000197 (human) and NM_013563 and NP_038591 (mouse)) refer to a type I cytosine receptor family member that is a cytosine receptor subunit in receptor complexes for at least six different interleukin receptors, including, but not limited to, IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptors. IL-2Ryc interacts with IL-2Rp to form an intermediate-affinity IL-2 receptor primarily on memory T cells and natural killer (NK) cells and interacts with IL-2Ra and IL2Rp to form a high-affinity IL-2 receptor on activated T cells and Tregs.

[0064] In some embodiments, IL-2 muteins as disclosed herein are man-made recombinant IL-2 muteins, and may be, for example, any recombinant IL-2 polypeptide, engineered IL-2 polypeptide, or naturally occurring IL-2 polypeptide having modulated binding affinity to an IL-2 receptor (e.g., IL-2Ra, IL-2Rp, and / or IL-2Ry).

[0065] Muteins of The IL-2 specimens in question are at least approximately the the the the 65%, 85%, 91%, 94%, at least less less less less approximately approximately approximately approximately the the the the 70%, 87%, 92%, 95%, at least less less less less approximately approximately approximately approximately approximately the the the the the 50%, 80%, 90%, 93%, 96%, at least at least at least at least at least at least at least approximately 97%, at least approximately 98%, at least approximately 99% identical to the corresponding wild-type IL-2 (WT IL-2). The mutation may consist of a change in the number or content of amino acid residues. For example, the mutant IL-2 may have a larger or smaller number of amino acid residues than the corresponding wild-type IL-2. Alternatively, or additionally, a exemplary mutant polypeptide may contain a substitution of one or more amino acid residues that are present in wild-type IL-2. In different ways, the mutant IL-2 polypeptide may differ from wild-type IL-2 by the addition, deletion, or substitution of a single amino acid residue.

[0066] By way of non-limiting illustration, an IL-2 mutein that includes an amino acid sequence that is at least 95% identical to the reference amino acid sequence of SEO ID NO: 1 is a polypeptide that includes a sequence that is identical to the reference sequence except for the inclusion of up to five alterations of the reference amino acid sequence of SEO ID NO: 2. Accordingly, in some embodiments, the IL-2 mutein of the disclosure that includes an amino acid sequence that is at least 95% identical to the reference amino acid sequence of SEO ID NO: 1 is a polypeptide that includes a sequence that is identical to the reference sequence except for the inclusion of 1, 2, 3, 4, or 5 alterations of the reference amino acid sequence.For example, up to 5% of the amino acid residues in the reference sequence may be deleted or replaced with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations to the reference sequence may occur at the amino (N-) or carboxy (C-) terminal positions of the reference amino acid sequence or anywhere between these terminal positions, interspersed either individually between residues in the reference sequence or in one or more contiguous groups within the reference sequence.

[0067] In some forms, the disclosure IL-2 mutein binds to IL-2Ry with an affinity that is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% lower than natural-type IL-2, inclusive of any value falling between these percentages. The binding affinity of IL-2 mutein can also be expressed as 1.2, 1.4, 1.5, 2, 5, 10, 15, 20, 25, 50, 100, 200, 250 or more times the lower affinity for IL-2α than that of natural-type IL-2. The binding affinity of a given IL-2 mutein for IL-2αRβ can be measured using any suitable method known in the field.Suitable methods for measuring IL-2Ry binding include, but are not limited to, radioactive ligand binding assays (e.g., saturation binding, Scatchard plot, nonlinear curve fitting programs, and competitive binding assays); non-radioactive ligand binding assays (e.g., fluorescence polarization (FP), fluorescence resonance energy transfer (FRET) (see, e.g., Drescher et al., Methods Mol Biol 493:323-343 (2009)); liquid-phase ligand binding assays (e.g., real-time polymerase chain reaction (RT-qPCR), and immunoprecipitation); and solid-phase ligand binding assays (e.g., multi-well plate assays, bead ligand binding assays, column ligand binding assays, and filter assays).Not being limited by theory, it is believed that partial agonists constructed by means of mutations in the IL-2Ry receptor binding site would be less dose-dependent compared to natural or other IL-2R variants.

[0068] In some forms, IL-2 mutein disrupts the association of IL-2Rp with IL-2Ry such that this IL-2Rp / IL-2Ry interaction is reduced by approximately 2%, approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 50%, approximately 75%, approximately 90%, approximately 95% or more (inclusive of any value falling between these percentages) relative to natural-type IL-2.

[0069] In some embodiments, one or more mutations that reduce the binding affinity of the IL-2 mutein for the IL-2Ry receptor constitute an amino acid substitution. In some embodiments, the IL-2 mutein in question consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions compared to a natural-type IL-2 (SEC ID NO: 1). The substituted amino acid residue(s) may be, but are not necessarily, conservative substitutions, generally including substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; Urine, arginine; and phenylalanine, tyrosine. In particular forms, the substitutions are in amino acid residues of IL-2 that make contact with the IL-2Ry binding interface.

[0070] In some embodiments, the amino acid substitutions are substitutions at one or more natural-type IL-2 amino acid positions selected from positions 18, 22, and 126, numbered according to natural-type hlL2 (e.g., SEC ID NO: 1). In some embodiments, the amino acid substitutions that decrease IL-2Ry receptor binding affinity include Leu-to-Arg (L18R), Gln-to-Glu (Q22E), and / or one of Gln-to-His (Q126H), Gln-to-Met (Q126M), or Gln-to-Lys (Q126K), or combinations thereof.

[0071] In additional embodiments, the amino acid substitution that decreases the binding affinity of the IL-2Ry receptor includes L18Ry Q22E and any of Q126A, Q126C, Q126D, Q126E, Q126G, Q126H, Q126I, Q126K, Q126M, Q126R, Q126S, or Q126T. In still other embodiments, the IL-2 mutein may have additional amino acid substitutions at one or more natural-type IL-2 amino acid positions selected from positions 80, 81, 85, 86, and 192, numbered according to natural-type hlL-2 (e.g., SEC ID NO: 1). In another form, IL-2 mutein may have additional amino acid substitutions selected from one or more of L80F, R81D, L85V, I86V, and / or I92F.

[0072] In some forms, the IL-2 mutain may further have an increased binding affinity for the IL-2Rp receptor and may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mutations that increase the IL-2Rp binding affinity. As used herein, both of the terms “IL-2Rp” and “CD122” (GenBank accession numbers NM_000878 and NP_000869 (human)) refer to a member of the type I cytokine receptor family that interacts with IL-2Ry to form an intermediate-affinity IL-2 receptor primarily on memory T cells and natural killer (NK) cells and interacts with IL-2Ro and IL-2Ry to form a high-affinity IL-2 receptor on activated T cells and regulatory T cells (Tregs).In some forms, the IL-2 mutain in question includes at least one mutation (e.g., a deletion, addition, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) relative to a natural-type IL-2 (e.g., SEC ID NO: 1), and binds to IL-2Rp with higher affinity than a natural-type IL-2. In some forms, IL-2 mutein binds to IL-2Rp with an affinity that is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% greater than natural-type IL-2 (including any value falling between these percentages). The binding affinity of IL-2 mutein can also be expressed as 1.2, 1.4, 1.5, 2, 5, 10, 15, 20, 25, 50, 100, 200, 250 or more times greater affinity for IL-2R3 than natural-type IL-2.The binding of the IL-2 mutain in question to IL-2Rp can be assessed by any suitable method known to those experienced in the field, including, but not limited to, the methods described above. In some forms, at least one mutation that increases the binding affinity of IL-2Rp is present. 2Rβ is an amino acid substitution. In some embodiments, the amino acid substitutions that increase the binding affinity of IL-2Rp include substitutions at amino acid positions 124, P65, Q74, L80, R81, L85, I86, I89, I92, and / or V93 numbered according to the natural-type hlL-2 (SEC ID NO: 1): In some embodiments, the substitutions include I24V, P65H, Q74R, Q74H, Q74N, Q74S, L80F, L80V, R81I, R81T, R81D, L85V, I86V, I89V, I92F, and / or V93I or combinations thereof. In some versions, the replacements include Q74N, Q74H, Q74S, L80F, L80V, R81D, R81T, L85V, I86V, I89V, and / or I93V or combinations thereof.

[0073] In some forms, the IL-2 mutein may further have decreased binding affinity for the IL-2Rp receptor and may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mutations that decrease the IL-2Rp binding affinity. In some forms, the IL-2 mutein in question includes at least one mutation (e.g., a deletion, addition, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) relative to a natural-type IL-2 (e.g., SEC ID NO: 1), and binds to IL-2R3 with lower affinity compared to a natural-type IL-2. In some forms, IL-2 mutein binds to IL-2RP with an affinity that is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% lower than natural-type IL-2 (including any value falling between these percentages).The IL-2 mutein binding affinity can also be expressed as 1.2, 1.4, 1.5, 2, 5, 10, 15, 20, 25, 50, 100, 200, 250 or more times lower affinity for IL-2RP than natural-type IL-2.

[0074] In additional modalities, any of the IL-2 muteins disclosed herein may bind to the IL-2RP receptor with similar (e.g., varying by less than one percent) or identical affinity to a natural-type IL-2 (e.g., SEC ID NO: 1).

[0075] In additional modalities, the IL-2 mutan may further have an increased binding affinity for the IL-2Ro receptor and may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mutations that increase the IL-2Ra binding affinity. As used herein, both of the terms “IL-2Ra” and “CD25” (GenBank accession numbers NM_000417 and NP_000408 (human)) refer to a member of the type I cytokine receptor family that interacts with IL-2R3 and IL-2Ry to form a high-affinity IL-2 receptor on activated T cells and regulatory T cells (Tregs). In some forms, the IL-2 mutein in question includes at least one mutation (e.g., a deletion, addition, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) relative to a natural-type IL-2 (e.g., SEC ID NO: 1), and binds to IL-2Ra with greater affinity than a natural-type IL-2.In some forms, IL-2 mutein binds to IL-2Ra with an affinity that is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% greater than natural-type IL-2 (including any value falling between these percentages). The binding affinity of IL-2 mutein can also be expressed as 1.2, 1.4, 1.5, 2, 5, 10, 15, 20, 25, 50, 100, 200, 250 or more times greater affinity for IL-2RP than natural-type IL-2. The binding of the IL-2 mutein in question to IL-2RP can be assessed by any suitable method known to those experienced in the field, including, but not limited to, the methods described above. In some forms, at least one mutation that increases the binding affinity of IL-2RP is an amino acid substitution.

[0076] In still other forms, the IL-2 mutan may further have a decreased binding affinity for the IL-2Ra receptor and may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mutations that decrease the binding affinity of IL-2Ra. In some forms, the IL-2 mutan in question includes at least one mutation (e.g., a deletion, addition, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) relative to a natural-type IL-2 (e.g., SEC ID NO: 1), and binds to IL-2Ra with lower affinity than a natural-type IL-2. In some forms, IL-2 mutein binds to IL-2Ro with an affinity that is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% lower than natural-type IL-2 (including any value falling between these percentages).The binding affinity of IL-2 mutein can also be expressed as 1.2, 1.4, 1.5, 2, 5, 10, 15, 20, 25, 50, 100, 200, 250 or more times lower affinity for IL-2Ra than natural type IL-2.

[0077] In additional modalities, any of the IL-2 muteins disclosed herein may bind to the IL-2Ro receptor with similar (e.g., varying by less than one percent) or identical affinity to a natural-type IL-2 (e.g., SEC ID NO: 1).

[0078] In some embodiments, the IL-2 mutein includes the amino acid substitutions L80F, R81D, L85V, I86V, I92F, L18R, Q22E, and Q126H. In some embodiments, the IL-2 mutein has the following amino acid sequence: APTSSSTKKTQLQLEHLRLDLEMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSK NFHFDPRDWSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFCCHSIISTLT (SEQ ID NO: 8)

[0079] In other embodiments, the IL-2 mutein includes the amino acid substitutions L80F, R81D, L85V, I86V, I92F, L18R, Q22E, and Q126K. In some embodiments, the IL-2 mutein has the following amino acid sequence: APTSSSTKKTQLQLEHLRLDLEMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSK NFHFDPRDWSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFCKSIISTLT (SEQ ID NO: 10)

[0080] In additional embodiments, the IL-2 mutein includes the amino acid substitutions L80F, R81D, L85V, I86V, I92F, L18R, Q22E, and Q126M. In some embodiments, the IL-2 mutein has the following amino acid sequence: APTSSSTKKTQLQLEHLRLDLEMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSK NFHFDPRDWSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFCMSIISTLT (SEQ ID NO: 11).

[0081] In another embodiment, the IL-2 mutein includes amino acid substitutions at L18R, Q22E, and Q126H. In some embodiments, the IL-2 mutein has the following amino acid sequence: APTSSSTKKTQLQLEHLRLDLEMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSK ñor Lcn / nznz / q / Yi NFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCHSIISTLT (SEQ ID NO: 15)

[0082] In another embodiment, the IL-2 mutein includes amino acid substitutions at L18R, Q22E, and Q126M. In some embodiments, the IL-2 mutein has the following amino acid sequence: APTSSSTKKTQLQLEHLRLDLEMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSK NFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCMSIISTLT (SEQ ID NO: 16).

[0083] In different modalities, the IL-2 mutein in question has an amino acid sequence according to the formula: APTSSSTKKTQLQLEHL-(X1)nLDL-(X2)nMlLNGlNNYKNPKLTRMLTF-KFYM-PK-KATELKHLQCLEEELKPLEEV-LNLAQSKNF H-(X3)n-(X4)nPRD-(X5)n-(X6)nSNlNV-(X7)nV-LE-LKGSETTFMCEYADETATlVE-FLN-RW-lTFC-(X13)nSllSTLT, where: each n if you individually select 0 or 1; X1es L (natural type) or R; X2esQ (natural type) or E; X3es L (natural type), FoV; X4es R (natural type), I, T or D; X5es L (natural type) or V; X6esl (natural type) or V; X7es I (natural type) or F; X13es Q (natural type) or Η, Μ, K, C, D, E, G, I, R, S, or T (ID de SEC NO: 20).

[0084] In some embodiments of IL-2 mutein according to SEC ID NO: 20, at least one amino acid in X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, or X13 is not a naturally occurring amino acid. In some embodiments, at least one amino acid in X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, or X13 is not a naturally occurring amino acid. In some modalities, the IL-2 mutein has at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or approximately 100% homology with the IL-2 mutein of ID SEC NO: 20.

[0085] In some modalities, the IL-2 mutains in question that are partial agonists have one or more reduced functions, compared to natural-type IL-2, such as (i) reduced Emax, (ii) reduced ability to stimulate signaling pathways that depend on IL-2RB / IL-2Ry heterodimerization, (iii) reduced production and / or secretion of interferon gamma (IFNy) from potentially inflammatory immune cells, (iv) reduced proliferation of potentially inflammatory T cells.

[0086] In some forms, IL-2 mutein has reduced capabilities to stimulate one or more signaling pathways that are dependent on IL-2Rp / IL-2Ry heterodimerization. In some forms, the IL-2 mutan in question has a reduced capacity to stimulate STAT5 phosphorylation in a T cell compared to natural-type IL-2 (see, e.g., Example 1 and Figures 4A-4B). In some forms, the IL-2 mutan stimulates STAT5 phosphorylation in a T cell at a level that is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% (inclusive of values ​​in between) of the level at which natural-type IL-2 stimulates STAT5 phosphorylation in the same cell. In some forms, the T cell is a natural killer (NK) cell.STAT5 signaling can be measured, for example, by STAT5 phosphorylation using any suitable method known to the field. For instance, STAT5 phosphorylation can be measured using antibodies specific for the phosphorylated version of this molecule in combination with flow cytometry analysis.

[0087] In additional embodiments, the IL-2 muteins disclosed herein have a reduced Emax compared to the Emax of natural-type IL-2. In some embodiments, the muteins have any of approximately 15-95%, such as approximately 20-95%, approximately 30-95%, approximately 40-95%, approximately 50-95%, approximately 60-95%, approximately 70-95%, approximately 80-95%, or approximately 90-95% of Emax compared to natural-type IL-2 (such as the polypeptide encoded by SEC ID NO: 1). In some embodiments, the IL-2 muteins disclosed herein have 15-95% Emax compared to the Emax of natural-type IL-2. In some forms, the IL-2 muteins disclosed in this document have 70-95% of the Emax compared to the Emax of natural-type IL-2.In another modality, the IL-2 muteins disclosed in this document have any of approximately 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%. 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% of Emax compared to natural-type IL-2 (such as the polypeptide encoded by SEC ID NO: 1). In a non-limiting modality, Emax is calculated based on the ratio of the maximum level of STAT5 phosphorylation (pSTAT5, STAT5 phosphorylation) induced in an immune cell by the IL-2 mutain to the maximum pSTAT5 signal generated by natural-type IL-2.

[0088] In still other modalities, the IL-2 mutains disclosed herein may result in lower production and / or secretion of interferon gamma (IFNy) from potentially inflammatory immune cells compared to the amount of IFNy production and / or secretion induced by natural-type IL-2 (such as, the polypeptide encoded by the ID of SEC NO: 1) (see, for example, Example 2 and Figures 5A-5D). The reduction in IFNγ production and / or secretion may be at a level that is approximately 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (inclusive of values ​​in between) of the level that stimulates natural-type IL-2 in potentially inflammatory immune cells at comparable concentrations and under similar conditions. In some modalities, the IL-2 mutains disclosed herein do not induce IFNγ secretion from potentially inflammatory immune cells.In one non-limiting modality, the potentially inflammatory immune cell is a CD8+ T cell and / or another subset of inflammatory immune cells. In another non-limiting modality, the potentially inflammatory immune cell is a CD4+IFNγ+ T cell or a CD8+IFNγ+ T cell.

[0089] In additional modalities, the IL-2 mutains in this document may result in reduced proliferation of potentially inflammatory T cells compared to the proliferation of potentially inflammatory T cells induced by natural-type IL-2 (such as the polypeptide encoded by the ID of SEC NO: 1). Non-limiting examples of potentially inflammatory T cells include CD4+IFNγ+ T cells or CD8+IFNγ+ T cells. In some modalities, the reduction in the proliferation of potentially inflammatory T cells may be at a level that is 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (inclusive of values ​​between these percentages) or less than the level that natural-type IL-2 stimulates at comparable concentrations and under similar conditions.In some cases, the IL-2 mutains disclosed in this document do not induce any proliferation of potentially inflammatory immune cells. In one non-limiting case, the potentially inflammatory immune cell is a CD8+ T cell and / or another subset of inflammatory immune cells. In another non-limiting case, the potentially inflammatory immune cell is a CD4+IFNγ+ T cell or a CD8+IFNγ+ T cell.

[0090] In still other modalities, the IL-2 mutains disclosed herein may have enhanced functions such as, for example, increased proliferation of Treg cells. In some modalities, the IL-2 mutains disclosed herein may increase Treg cell proliferation by any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times compared to the amount of Tregin cell proliferation induced by natural-type IL-2 (such as the polypeptide encoded by SEC ID NO: 1). In some modalities, the IL-2 mutains disclosed in this document may increase the proliferation of any Treg cell by approximately 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% compared to the amount of Tregin cell proliferation induced by natural-type IL-2 (such as the polypeptide encoded by the SEC NO: 1 ID). In some modalities, the Treg cell is a CD4+Foxp3+ cell.

[0091] As described in greater detail below, in some forms of disclosure, the IL-2 mutains disclosed herein and the nucleic acids encoding such IL-2 mutains may be incorporated into compositions, including pharmaceutical compositions. Generally, such compositions include the polypeptide or nucleic acid molecule and a pharmaceutically acceptable vehicle. β. Nucleic acids

[0092] In one aspect, some of the modalities disclosed in this document refer to nucleic acid molecules encoding the IL-2 mutain, including expression cassettes, and expression vectors containing these nucleic acid molecules operatively linked to heterologous nucleic acid sequences such as, for example, regulatory sequences that allow in vivo expression of the IL-2 mutain in a host cell or cell-free ex vivo expression system.

[0093] In various embodiments, the polypeptides used in the practice of the present invention are synthetic or produced by the expression of a recombinant nucleic acid molecule. In the event that the polypeptide is a chimera (for example, a fusion protein containing at least one mutant IL-2 polypeptide and a heterologous polypeptide), it may be encoded by a hybrid nucleic acid molecule containing a sequence encoding all or part of the mutant IL-2 and a second sequence encoding all or part of the heterologous polypeptide. For example, the IL-2 mutains described herein may be fused with a hexahistidine marker to facilitate the purification of bacterially expressed protein, or with a hemagglutinin marker to facilitate the purification of eukaryotically expressed protein.

[0094] The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably in this document and refer to both RNA and DNA molecules, including nucleic acid molecules comprising cDNA, genomic DNA, synthetic DNA, and DNA or RNA molecules containing nucleic acid analogues. A nucleic acid molecule may be double-stranded or single-stranded (e.g., a sense strand or an antisense strand). A nucleic acid molecule may contain unconventional or modified nucleotides. The terms “polynucleotide sequence” and “nucleic acid sequence,” as used interchangeably in this document, refer to the sequence of a polynucleotide molecule. The nomenclature for nucleotide bases used herein is that set forth in 37 CFR §1.822.

[0095] The nucleic acid molecules of the present disclosure may be nucleic acid molecules of any length, including nucleic acid molecules that are preferably between approximately 0.5 Kb and 50 Kb, for example between approximately 0.5 Kb and 10 Kb, between approximately 1 Kb and 8 Kb, between approximately 2 Kb and 7 Kb, or between approximately 2 Kb and 20 Kb, for example between approximately 2 Kb and 10 Kb, between approximately 3 Kb and 15 Kb, between approximately 4 Kb and 10 Kb, between approximately 5 Kb and 15 Kb, or between approximately 3 Kb and 9 Kb.In some forms, the nucleic acid molecules in this disclosure may be between 5 Kb and 50 Kb, for example between approximately 5 Kb and 40 Kb, between approximately 5 Kb and 30 Kb, between approximately 5 Kb and 20 Kb, or between approximately 10 Kb and 50 Kb, for example between approximately 15 Kb and 30 Kb, between approximately 20 Kb and 50 Kb, between approximately 20 Kb and 40 Kb, between approximately 5 Kb and 25 Kb, or between approximately 30 Kb and 50 Kb.

[0096] Methods for constructing a DNA sequence encoding IL-2 mutains and expressing those sequences in a suitably transformed host include, but are not limited to, using a PCR-assisted mutagenesis technique. Mutations consisting of deletions or additions of amino acid residues to an IL-2 polypeptide can also be made using standard recombinant techniques. In the event of a deletion or addition, the nucleic acid molecule encoding IL-2 is optionally digested with an appropriate restriction endonuclease. The resulting fragment can be expressed directly or further manipulated, for example, by ligating it to a second fragment. Ligation can be facilitated if the two ends of the nucleic acid molecules contain complementary nucleotides that overlap, but truncated-end fragments can also be ligated.The nucleic acids generated by POR can also be used to generate different mutant sequences.

[0097] The complete amino acid sequence can be used to construct a retro-converted gene. A DNA oligomer containing a nucleotide sequence encoding the IL-2 mutain can be synthesized. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. Typically, the individual oligonucleotides contain 5' or 3' overhangs for complementary assembly.

[0098] In addition to generating mutant polypeptides through the expression of nucleic acid molecules that have been altered by recombinant molecular biological techniques, the IL-2 mutains in question can be chemically synthesized. Chemically synthesized polypeptides are routinely generated by those experienced in the field.

[0099] Once assembled (by synthesis, site-directed mutagenesis, or another method), the DNA sequences encoding an IL-2 mutain will be inserted into an expression vector and operatively linked to an appropriate expression control sequence for IL-2 mutain expression in the desired transformed host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the field, in order to obtain high levels of expression of a transfected gene in a host, the gene must be operatively linked to transcription and conversion expression control sequences that are functional in the chosen expression host.

[00100] The DNA sequence encoding the IL-2 mutain, whether prepared by site-directed mutagenesis, chemical synthesis, or other methods, may also include DNA sequences encoding a signal sequence. Such a signal sequence, if present, must be recognized by the cell chosen for IL-2 mutain expression. It may be prokaryotic, eukaryotic, or a combination of both. It may also be the signal sequence of native IL-2. The inclusion of a signal sequence depends on whether the IL-2 mutain is to be secreted from the recombinant cells in which it is made. If the cells chosen are prokaryotic, it is generally preferred that the DNA sequence not encode a signal sequence. If the cells chosen are eukaryotic, it is generally preferred that a signal sequence be encoded, and more preferably that the signal sequence of native IL-2 be used.

[00101] In some embodiments, the IL-2 mutein in question, either alone or as part of a chimeric polypeptide, such as those described above, can be obtained by expression of a nucleic acid molecule. Just as IL-2 muteins can be described in terms of their identity with natural-type IL-2 polypeptides, the nucleic acid molecules encoding them will necessarily have a certain degree of identity with those encoding natural-type IL-2. For example, the nucleic acid molecule encoding a particular IL-2 mutein may be at least 50%, at least 65%, preferably at least 75%, more preferably at least 85%, and still more preferably at least 95% (e.g., 99%) identical to the nucleic acid encoding natural-type IL-2.Therefore, in some embodiments, the nucleic acid molecule encoding a questioned IL-2 mutein disclosed herein is at least 50%, at least 65%, preferably at least 75%, more preferably at least 85%, and still more preferably at least 95% (e.g., 96%, 97%, 98%, or 99%) identical to the nucleic acid encoding a natural-type IL-2 having the amino acid set forth in SEC ID NO: 1. In some embodiments, the nucleic acid molecule encoding a questioned IL-2 mutein disclosed herein is at least 50%, at least 65%, preferably at least 75%, more preferably at least 85%, and still more preferably at least 95% (e.g., 96%, 97%, 98%, or 99%) identical to the nucleic acid encoding a natural-type IL-2 that has the amino acid established in the ID of SEC NO: 2.

[00102] The nucleic acid molecules provided may contain naturally occurring sequences, or sequences that differ from those of natural origin but, due to the degeneracy of the genetic code, encode the same polypeptide. These nucleic acid molecules may consist of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis), or combinations or modifications of nucleotides within these types of nucleic acids. Additionally, the nucleic acid molecules may be double-stranded or single-stranded (i.e., either a sense strand or an antisense strand).

[00103] Nucleic acid molecules are not limited to sequences that code for polypeptides; they can also include some or all of the non-coding sequences that are upstream or downstream of a coding sequence (e.g., the coding sequence for IL-2). Those experienced in molecular biology are familiar with routine procedures for isolating nucleic acid molecules. They can be generated, for example, by treating genomic DNA with restriction endonucleases, or by performing the polymerase chain reaction (PCR). If the nucleic acid molecule is a ribonucleic acid (RNA), the molecules can be produced, for example, by in vitro transcription.

[00104] Isolated nucleic acid molecules exemplary in this disclosure may include fragments not found as such in nature. Therefore, this disclosure covers recombinant molecules, such as those in which a nucleic acid sequence (for example, a sequence encoding a mutated IL-2) is incorporated into a vector (for example, a plasmid or viral vector) or into the genome of a heterologous cell (or the genome of a homologous cell, at a position other than the natural chromosomal location).

[00105] As described above, the IL-2 mutan in question may exist as part of a chimeric polypeptide. In addition to, or instead of, the heterologous polypeptides described above, a nucleic acid molecule in question may contain sequences encoding a “marker” or “reporter.” Examples of marker or reporter genes include β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neoR, G418), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK) (which encodes β-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT).Someone experienced in the subject will be aware of additional useful reagents, for example, additional sequences that can serve as a marker or reporter.

[00106] The nucleic acid molecules in question can be obtained by introducing a mutation into the DNA encoding IL-2 obtained from any biological cell, such as a mammalian cell. Therefore, the nucleic acids in question (and the polypeptides they encode) can be those from a mouse, rat, guinea pig, cow, sheep, horse, pig, rabbit, monkey, baboon, dog, or cat. In some modalities, the nucleic acid molecules will be those from a human. C. Vectors and host cells

[00107] This document also provides vectors, plasmids, or viruses containing one or more of the nucleic acid molecules encoding any of the IL-2 mutan polypeptides disclosed herein. The nucleic acid molecules described above may be contained within a vector capable of directing their expression in, for example, a cell transduced with the vector. Accordingly, in addition to the IL-2 mutans in question, expression vectors containing a nucleic acid molecule encoding an IL-2 mutan in question and cells transfected with these vectors are among the preferred modalities. Vectors suitable for use in eukaryotic and prokaryotic cells are known in the field and are commercially available or readily prepared by a skilled craftsman. Additional vectors can also be found, for example, in Ausubel, FM, et al., Current Protocols ín Molecular Bíology, (Current Protocol, 1994) y Sambrook etal.,Molecular Cloning: A Laboratory Manual, 2aEd. (1989).

[00108] It should certainly be understood that not all vectors and expression control sequences will work equally well for expressing the DNA sequences described herein. Nor will all hosts work equally well with the same expression system. However, someone experienced in the field can make a selection among these vectors, expression control sequences, and hosts without undue experimentation. For example, when selecting a vector, the host must be considered because the vector must replicate in it. The number of copies of the vector, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, must also be considered. For example, vectors that can be used include those that allow the DNA encoding IL-2 mutains to be amplified in copy number.Such amplified vectors are well known in the field. They include, for example, vectors capable of being amplified by means of DHFR amplification (see, for example, Kaufman, U.S. Patent No. 4,470,461, Kaufman and Sharp, “Construction of a Modular Dihydrafolate Reductase cDNA Gene: Analysis of Signals Utilized for Efficient Expression”, Mol. Cell. Blol., 2, pp. 1304-19 (1982)) or glutamine synthetase (GS) amplification (see, for example, U.S. Patent No. 5,122,464 and European Published Application 338,841).

[00109] In some embodiments, the human IL-2 mutains of this disclosure will be expressed from vectors, preferably expression vectors. Vectors are useful for autonomous replication in a host cell or can be integrated into the host cell genome after introduction into the host cell, and thus replicate along with the host genome (e.g., non-episomal mammalian vectors). Expression vectors have the ability to direct the expression of coding sequences to which they operatively bind. In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids (vectors). However, other forms of expression vectors are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[00110] Exemplary recombinant expression vectors may include one or more regulatory sequences, selected based on the host cells to be used for expression, operationally linked to the nucleic acid sequence to be expressed.

[00111] Expression connector constructs may be designed for expression of an IL-2 mutain or variant thereof in prokaryotic or eukaryotic host cells.

[00112] Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Coid Spring Harbor Laboratory Press, Plainview, NY) and other standard molecular biology laboratory manuals.

[00113] Protein expression in prokaryotes is most often carried out in Escherichia coli using vectors containing constitutive or inducible promoters. Strategies for maximizing recombinant protein expression in E. coli can be found, for example, in Gottesman (1990) in Gene Expression Technology: Methods in Enzymology Vol. 185 (Academic Press, San Diego, Calif.), pp. 119-128 and Wada et al. (1992) Nucleic Acids Res. 20:2111-2118. Processes for culturing, harvesting, disrupting, or extracting the IL-2 covariant mutain from cells are substantially described in, for example, U.S. Patents Nos. 4,604,377; 4,738,927; 4,656,132; 4,569,790; 4,748,234; 4,530,787; 4,572,798; 4,748,234; and 4,931,543, incorporated in this document by reference in their entirety.

[00114] In some forms, recombinant IL-2 mutains or biologically active variants thereof can also be made in eukaryotic cells or more such as yeast or human cells. Suitable eukaryotic host cells include insect cells (examples of Baculovirus vectors available for protein expression in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. Mr. Lcn / nznz / q / Yi (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39)); yeast cells (examples of vectors for expression in S. cerevisiae of yeast include pYepSed (Baldan et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and pPicZ (Invitrogen Corporation, San Diego, Calif.)); or mammalian cells (mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195)). Suitable mammalian cells include Chinese hamster ovary (CHO) cells or COS cells. In mammalian cells, the expression vector control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyomavirus, Adenovirus 2, cytomegalovirus, and Simian Virus 40.For other expression systems suitable for prokaryotic and eukaryotic cells, see Chapters 16 and 17 of Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Coid Spring Harbor Laboratory Press, Plainview, NY). See, Goeddel (1990, supra).

[00115] The human IL-2 mutain-encoding sequences of this disclosure may be optimized for expression in the host cell of interest. The GC content of the sequence may be adjusted to average levels for a given host cell, as calculated by reference to known genes expressed in the host cell. Methods for codon optimization are well known in the field. The strands within the IL-2 mutain-coding sequence may be optimized to enhance expression in the host cell, such that approximately 1%, approximately 5%, approximately 10%, approximately 25%, approximately 50%, approximately 75%, or up to 100% of the strands within the coding sequence have been optimized for expression in a particular host cell.

[00116] Suitable vectors for use include T7-based vectors for use in bacteria (see, for example, Rosenberg et al., Gene 56:125, 1987), the pMSXND expression vector for use in mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988), and baculovirus-derived vectors (for example, the pBacPAK9 expression vector from Clontech, Palo Alto, Calif.) for use in insect cells.

[00117] In some modalities, nucleic acid inserts, which encode the IL-2 mutains in question in such vectors, can be operationally linked to a promoter, which is selected based on, for example, the cell type in which expression is sought.

[00118] When selecting an expression control sequence, a variety of factors must also be considered. These include, for example, the relative intensity of the sequence, its controllability, and its compatibility with the actual DNA sequence encoding the IL-2 mutain in question, particularly with regard to potential secondary structures. Hosts may be selected based on their compatibility with the chosen vector, the toxicity of the product encoded by the DNA sequences in this disclosure, their secretion characteristics, their ability to properly lend the polypeptides, their fermentation or culture requirements, and the ease of purification of the products encoded by the DNA sequences.

[00119] Within these parameters, someone experienced in the subject can select different Lcn / nznz / q / Yi vector / expression control sequence / host combinations that will express the desired DNA sequences in fermentation or in large-scale animal culture, for example, using CHO cells or COS cells 7.

[00120] The choice of expression control sequence and expression vector, in some modalities, will depend on the choice of host. A wide variety of host / expression vector combinations can be employed. Expression vectors useful for eukaryotic hosts include, for example, vectors with expression control sequences of SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include well-known bacterial plasmids, such as E. coli plasmids, including colEl, pCRI, pER32z, pMB9, and their derivatives; broader host-range plasmids, such as RP4; phage DNAs, for example, the numerous derivatives of lambda phage, for example, NM989; and other phage DNAs, such as M13 and filamentous single-stranded DNA phages. Expression vectors useful for yeast cells include the 2μ plasmid and its derivatives.Useful vectors for insect cells include pVL 941 and pFastBac™ 1 (Gibco BRL, Gaithersburg, Md.). Cate et al., Cell, 45, pp. 685-98 (1986).

[00121] Additionally, any of a wide variety of expression control sequences can be used in these vectors. Such useful expression control sequences include those associated with structural genes of the expression vectors mentioned above. Examples of useful expression control sequences include, for instance, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the major operator and promoter regions of lambda phage, e.g., PL, the fd envelope control regions, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the acid phosphatase promoters, e.g., PhoA, the yeast a-mating system promoters, the Baculovirus polyhedron promoter, and other sequences known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

[00122] A T7 promoter can be used in bacteria, a polyhedrine promoter in insect cells, and a cytomegalovirus or metallothionein promoter in mammalian cells. Also, in the case of higher eukaryotes, tissue-specific and cell-type-specific promoters are widely available. These promoters are named for their ability to direct the expression of a nucleic acid molecule in a given tissue or cell type within the body. Experienced craftspeople are well aware of the numerous promoters and other regulatory elements that can be used to direct nucleic acid expression.

[00123] In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, vectors may contain origins of replication and other genes that encode a selectable marker. For example, the neomycin resistance gene (neoR, neomycin-resistance) imparts G418 resistance to the cells in which it is expressed, and thus allows for phenotypic selection of the transfected cells. Those experienced in the matter can readily determine whether a given regulatory element or selectable marker is suitable for use in a particular experimental setting. Mr. Lcn / nznz / q / Yi

[00124] Viral vectors that can be used in disclosure include, for example, retroviral, adenoviral, and adeno-associated vectors, herpes virus vectors, simian virus 40 (SV40, simian virus 40), and bovine papillomavirus (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectora, CSH Laboratory Press, Coid Spring Harbor, NY).

[00125] Prokaryotic or eukaryotic cells that contain and express a nucleic acid molecule encoding a mutating IL-2 mutan in question, as disclosed herein, are also subject to disclosure. A cell of disclosure is a transfected cell, that is, a cell into which a nucleic acid molecule, for example, a nucleic acid molecule encoding a mutating IL-2 polypeptide, has been introduced by means of recombinant DNA techniques. The progeny of such a cell are also considered to be within the scope of disclosure.

[00126] The precise components of the expression system are not critical. For example, an IL-2 mutein can be produced in a prokaryotic host, such as the bacterium E. coli, or in a eukaryotic host, such as an insect cell (e.g., an Sf21 cell), or mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, VA). When selecting an expression system, it is only important that the components are compatible with each other. Experienced practitioners are capable of making such a determination. In addition, if guidance in selecting an expression system is required, experienced practitioners can consult Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987).

[00127] The expressed polypeptides can be purified from the expression system using routine biochemical procedures, and can be used, for example, as therapeutic agents, as described in this document.

[00128] In some forms, the IL-2 muteins obtained will be glycosylated or non-glycosylated depending on the host organism used to produce the mutein. If bacteria are chosen as the host, then the IL-2 mutein produced will be non-glycosylated. Eukaryotic cells, on the other hand, will glycosylate the IL-2 muteins, although perhaps not in the same way that native IL-2 is glycosylated. The IL-2 mutein produced by the transformed host can be purified according to any suitable method. Several methods for purifying IL-2 are known. See, for example, Current Protocols in Protein Science, Vol 2. Eds: John E. Coligan, Ben M. Dunn, Hidde L. Ploehg, David W. Speicher, Paul T. Wingfield, Unit 6.5 (copyright 1997), John Wiley & Sons, Inc. IL-2 muteins can be isolated from inclusion bodies generated in E.Coli, or from the conditioned medium of mammalian or yeast cultures that produce a given mutein using cation exchange, gel filtration, and / or reversed-phase liquid chromatography.

[00129] Another exemplary method for constructing a DNA sequence encoding IL-2 mutains is by chemical synthesis. This includes the direct chemical synthesis of a peptide from the protein sequence encoding an IL-2 mutain exhibiting the described properties. This method can incorporate naturally occurring and non-natural amino acids that affect the interactions of IL-2 with IL-2Ra, IL-2Rp, and / or IL-2Ry. Alternatively, a gene encoding the desired IL-2 mutain can be synthesized chemically using an oligonucleotide synthesizer. Such oligonucleotides are designed based on the amino acid sequence of the desired IL-2 mutain, preferably selecting those codons that are favored in the host cell in which the recombinant protein will be produced. In this regard, it is well known that the genetic code is degenerate—that an amino acid can be encoded by more than one strand.For example, Phe (F) is encoded by two codons, TIC or TTT, Tyr (Y) is encoded by TAC or TAT, and su (H) is encoded by CAC or CAT. Trp (W) is encoded by a single codon, TGG. Consequently, it will be appreciated that for a given DNA sequence encoding a particular IL-2 mutein, there will be many degenerate DNA sequences encoding that IL-2 mutein. For example, it will be appreciated that in addition to the preferred DNA sequence for the 5-2 mutein shown in Figure 2, there will be many degenerate DNA sequences encoding the IL-2 mutein shown. These degenerate DNA sequences are considered within the scope of this disclosure. Therefore, the term “degenerate sequences” in the context of this disclosure refers to all DNA sequences that encode, and thus permit the expression of, a particular mutein.

[00130] The biological activity of IL-2 mutains can be assayed by any suitable method known to the public. Such assays include PHA blast proliferation and NK cell proliferation. D. Fusion proteins

[00131] Any of the IL-2 muteins disclosed herein may be prepared as fusion or chimeric polypeptides comprising an IL-2 mutein in question and a heterologous polypeptide (i.e., a polypeptide that is not IL-2 or a mutant thereof) (see, for example, U.S. Patent No. 6,451,308). Exemplary heterologous polypeptides may increase the circulating half-life of the chimeric polypeptide in vivo and thus further enhance the properties of the mutant IL-2 polypeptides. In various embodiments, the circulating half-life-enhancing polypeptide may be a serum albumin, such as human serum albumin, or the Fe region of the IgG subclass of antibodies lacking the IgG heavy chain variable region.Exemplary Fe regions may include a mutation that inhibits complementary attachment and Fe receptor binding, or may be lytic, i.e., capable of binding, complementing, or using cells by another mechanism, such as antibody-dependent complement lysis (ADCC; U.S. Serial No. 08 / 355,502 filed December 12, 1994).

[00132] The “Fe region” can be a naturally occurring or synthetic polypeptide homologous to the C-terminal domain of IgG produced by digestion of IgG with papain. The Fe region of IgG has a molecular weight of approximately 50 kDa. Mutant IL-2 polypeptides may include the entire Fe region or a smaller portion that retains the ability to extend the circulating half-life of a chimeric polypeptide of which it is a part. Additionally, full-length or fragmented Fe regions may be variants of the naturally occurring molecule. Mr. Lcn / nznz / q / Yi That is, they may contain mutations that may or may not affect polypeptide function; as described later, native activity is not required or desired in all cases. In some forms, the IL-2 mutein fusion protein (e.g., an IL-2 partial agonist or antagonist as described herein) includes an Fe region of lgG1, lgG2, lgG3, or lgG4.

[00133] The Fe region can be either “lytic” or “non-lytic,” but it is generally non-lytic. A non-lytic Fe region typically lacks a high-affinity Fe receptor binding site and a C'1q binding site. The high-affinity Fe receptor binding site of murine IgG Fe includes the Leu residue at position 235 of IgG Fe. Therefore, the Fe receptor binding site can be destroyed by mutating or deleting Leu 235. For example, substituting Glu for Leu 235 inhibits the ability of the Fe region to bind the high-affinity Fe receptor. The C'1q binding site of murine Fe can be functionally destroyed by mutating or deleting the Glu residues 318, Lys 320, and Lys 322 of IgG. For example, substituting Ala residues for Glu 318, Lys 320, and Lys 322 renders IgG1 Fe unable to direct complement-dependent lysis of the antibody.In contrast, the Fe region of lytic IgG has a high-affinity Fe receptor binding site and a C'1q binding site. The high-affinity Fe receptor binding site includes the Leu residue at position 235 of IgG Fe, and the C'1q binding site includes the Glu residues 318, Lys 320, and Lys 322 of IgG 1. The Fe region of lytic IgG has either naturally occurring residues or conservative amino acid substitutions at these sites. The Fe region of lytic IgG can direct cells for antibody-dependent cellular cytotoxicity or complement-directed cytolysis (CDC). Appropriate mutations are also known for human IgG (see, for example, Morrison et al., The Immunologist 2:119-124, 1994; and Brekke et al., The Immunologist 2:125, 1994).

[00134] In other embodiments, the chimeric polypeptide may include a relevant IL-2 mutan and a polypeptide that functions as an antigenic marker, such as a FLAG sequence. FLAG sequences are recognized by highly specific biotinylated anti-FLAG antibodies, as described herein (see also Blanar et al., Science 256:1014, 1992; LeClair et al., Proc. Nati. Acad. Sci. USA 89:8145, 1992). In some embodiments, the chimeric polypeptide further includes a C-terminal c-myc epitope marker.

[00135] In other embodiments, the chimeric polypeptide includes the mutant IL-2 polypeptide and a heterologous polypeptide that functions to enhance the expression or direct cellular localization of the mutant IL-2 polypeptide, such as the agglutinin subunit of Aga2p (see, for example, Bodery Wittrup, Nature Biotechnol. 15:553-7, 1997).

[00136] In other embodiments, a chimeric polypeptide can be generated that includes a mutant IL-2 and an antibody or antigen-binding portion thereof. The antibody or antigen-binding component of the chimeric protein can serve as a targeting portion. For example, it can be used to localize the chimeric protein to a particular subset of target cells or molecules. Methods for generating chimeric cytosine-antibody polypeptides are described, for example, in U.S. Patent No. 6,617,135.

[00137] In some embodiments, the mutant IL-2 can be modified with one or more molecules of polyethylene glycol (PEG) to increase its half-life. The term “PEG” as used herein refers to a molecule of polyethylene glycol. In its typical form, PEG is a linear polymer with terminal hydroxyl groups and has the formula HO-CH2CH2-(CH2CH2O)n-CH2CH2-OH, where n is from 8 to 4000.

[00138] Generally, n is not a discrete value but constitutes an interval with an approximately Gaussian distribution around an average value. The terminal hydrogen can be replaced with a leveling group such as an alkyl or alkanol group. PEG can have at least one hydroxyl group, more preferably a terminal hydroxyl group. This hydroxyl group can be attached to a linker portion that can react with the peptide to form a covalent bond. Numerous PEG derivatives exist in matter. (See, for example, U.S. Patent Nos. 5,445,090; 5,900,461; 5,932,462; 6,436,386; 6,448,369; 6,437,025; 6,448,369; 6,495,659; 6,515,100 and 6,514,491 and Zalipsky, S. Bioconjugate Chem. 6: 150-165, 1995). The PEG molecule covalently bound to the IL-2 muteins of the present disclosure may be approximately 10,000, 20,000, 30,000, or 40,000 average molecular weight.PEGylation reagents may be linear or branched molecules and may be present singly or in tandem. The PEGylated IL-2 mutein peptides of this disclosure may have tandem PEG molecules attached to the C-terminus and / or the N-terminus of the peptide. The term “PEGylation” as used herein refers to the covalent attachment of one or more PEG molecules, as described above, to a molecule such as the IL-2 muteins of this disclosure. E. Pharmaceutical compositions

[00139] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (where water-soluble) or sterile dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable vehicles include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and free-flowing to allow for easy administration by syringe. It must be stable under the conditions of manufacture and storage and protected against contamination by microorganisms such as bacteria and fungi.The vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants, such as sodium dodecyl sulfate. Prevention of microbial activity can be achieved through various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols like mannitol, sorbitol, and sodium chloride, in the composition.Prolonged absorption of injectable compositions can be induced by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[00140] Sterile injectable solutions may be prepared by incorporating the active compound in the required amount into a suitable solvent with one or a combination of the ingredients listed above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into the sterile vehicle, which contains a basic dispersion medium and the other required ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization, which produces a powder of the active ingredient plus any additional desired ingredients from a previously filtered sterile solution of the same.

[00141] Oral compositions, if used, generally include an inert diluent or an edible vehicle. For the purpose of oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of tablets, coated tablets, or capsules, e.g., gelatin capsules. Oral compositions may also be prepared using a fluid vehicle for use as a mouthwash. Pharmaceutically compatible binding agents and / or adjuvant materials may be included as part of the composition.Tablets, pills, capsules, dragees and the like may contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, tragacanth gum or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel™, or corn starch; a lubricant such as magnesium stearate or Sterotes™; a gluent such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as mint, methyl salicylate, or orange flavoring.

[00142] In the event of administration by inhalation, the IL-2 muteins in question, or the nucleic acids encoding them, are delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.

[00143] Systemic administration of the IL-2 mutans or nucleic acids in question may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, appropriate penetrants are used in the formulation for the barrier to be permeated. Such penetrants are generally known in the field and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration may be achieved by the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated in ointments, balms, gels, or creams, as generally known in the field.

[00144] In some modalities, the compounds (mutant IL-2 polypeptides or nucleic acids) can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[00145] In some modalities, the compounds (the IL-2 mutains in question or nucleic acids) can also be administered by means of transfection or infection using methods known in the field, including but not limited to the methods described in McCaffrey et al. (Nature 418:6893, 2002), Xia et al. (Nature Biotechnol. 20: 1006-1010, 2002), or Putnam (Am. J. Health Syst. Pharm. 53: 151-160, 1996, erratum at Am. J. Health Syst. Pharm. Mr. Lcn / nznz / q / Yi 53:325, 1996).

[00146] In some modalities, the IL-2 mutains in question or nucleic acids are prepared with vehicles that will protect the mutating IL-2 polypeptides from rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethyl vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Such formulations can be prepared using standard techniques. The materials may also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeting cells infected with monoclonal antibodies to viral antigens) may also be used as pharmaceutically acceptable vehicles.These can be prepared according to methods known to those experienced in the matter, for example, as described in United States Patent No. 4,522,811. IV. Dissemination Methods

[00147] Some aspects of the disclosure relate to methods and related materials useful for the production of IL-2 muteins, e.g., the IL-2 partial agonists described herein, as well as methods for the treatment of health conditions and disorders associated with disturbances of signal transduction mediated by the IL-2 signaling pathway. More particularly, some modalities of the disclosure relate to the modulation of the interleukin-2 (IL-2)-mediated signal transduction pathway in a subject in need. In some modalities of the disclosure, IL-2-mediated signaling is modulated by selectively reducing IL-2 binding to one, two, or three of its receptors, e.g., interleukin-2 receptor alpha (IL-2Ra), interleukin-2 receptor beta (IL-2RP), and interleukin-2 receptor gamma (IL-2Ry).

[00148] In one respect, some modalities of disclosure relate to compositions and methods useful for producing the IL-2 mutein disclosed herein, the method including culturing the host cell as described herein under conditions suitable for the production of IL-2 muteins.

[00149] In some embodiments, the method further includes the isolation of the produced mutein. In some embodiments, the method further includes the purification of the produced mutein. The techniques, systems, and related materials suitable for the isolation and purification of recombinant proteins produced in prokaryotic and eukaryotic host cells are known in the field.

[00150] In some embodiments, the produced IL-2 muteins can be further modified to prolong their in vivo and / or ex vivo half-life. Non-limiting examples of known strategies and methodologies suitable for modifying the IL-2 muteins of the disclosure include (1) chemical modification of an IL-2 mutein polypeptide described herein with highly soluble macromolecules such as polyethylene glycol (PEG) that prevents the polypeptides from contacting proteases; (2) covalent linkage or conjugation of an IL-2 mutein described herein with an antibody or antibody fragment such as, for example, a fragment of human Fe antibody; and (3) covalent linkage or conjugation of an IL-2 mutein described herein with a stable protein such as, for example, albumin.Consequently, in some cases, the IL-2 mutations in the release can be fused to a stable protein, such as albumin. For example, human albumin is known as one of the most effective proteins for enhancing the stability of polypeptides fused to it, and many such fusion proteins have been reported.

[00151] In some embodiments, the IL-2 muteins produced from disclosure are chemically modified with one or more polyethylene glycol portions, e.g., PEGylated; or similarly modified, e.g., PASylated. In some embodiments, the PEG or PAS molecule is conjugated to one or more amino acids of the IL-2 mutein. In some embodiments, the PEGylated or PASylated IL-2 mutein contains a PEG or PAS portion on only one amino acid. In other embodiments, the PEGylated or PASylated IL-2 mutein contains a PEG or PAS portion on two or more amino acids, e.g., attached to two or more, five or more, ten or more, fifteen or more, or twenty or more different amino acid residues. In some modalities, the PEG or PAS chain is 2,000, more than 2,000, 5,000, more than 5,000, 10,000, more than 10,000, more than 10,000, 20,000, more than 20,000, and 30,000 Da.The IL-2 mutein produced can be coupled directly to PEG or PAS (e.g., without a linking group) via an amino group, a sulfhydryl group, a hydroxyl group, or a carboxyl group.

[00152] In one respect, some disclosure modalities relate to compositions and methods useful for the treatment of health conditions and disorders associated with disturbances of signal transduction mediated by the IL-2 signaling pathway. More particularly, some disclosure modalities relate to the modulation of the interleukin-2 (IL-2)-mediated signal transduction pathway in a subject in need.

[00153] In some modalities, the IL-2 mutains disclosed herein, and / or nucleic acids expressing them, may be administered to a subject to treat a disorder associated with unwanted autoimmune or immunosuppressive responses. In the treatment of such diseases, the IL-2 mutains disclosed herein may possess convenient properties, such as stimulating regulatory T cells while at the same time minimally or not at all activating potentially inflammatory immune cells.

[00154] In certain modalities, the IL-2 mutains disclosed herein may be used to treat patients who have, are suspected of having, or may be at risk of developing an autoimmune disease. The method includes administering a therapeutically effective amount of (i) any of the IL-2 mutains disclosed herein, (ii) any of the nucleic acids or vectors disclosed herein, and / or (iii) any of the pharmaceutical compositions disclosed herein to the individual. In certain modalities, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, insulin-dependent diabetes mellitus, hemolytic anemias, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, multiple sclerosis, alopecia areata, psoriasis, vitiligo, dystrophic epidermolysis bullosa, systemic lupus erythematosus, and graft-versus-host disease.In some forms, autoimmune disease is graft-versus-host disease. Mr. Lcn / nznz / q / Yi

[00155] In some modalities, IL-2 mutans, nucleic acids or vectors, and / or pharmaceutical compositions are administered to the subject as a single therapeutic agent or in combination with one or more additional therapeutic agents. In some modalities, such one or more additional therapeutic agents include an antibody that directs the mutan to a specific cell type. In some modalities, the cell type is a regulatory T cell (Treg). In some modalities, the antibody is either covalently or non-covalently bound to the IL-2 mutan.

[00156] In one aspect, this document also provides methods for preventing the proliferation of potentially inflammatory T cells and / or preventing the secretion of IFNγ from CD8+ T cells or other subsets of inflammatory immune cells. The methods include contacting a cell expressing an interleukin-2 receptor (IL2Rγ) with any of the IL-2 mutains disclosed herein. In other modalities, the potentially inflammatory T cells are CD4+CD44+IFNγ+ T cells or CD8+CD44+IFNγ+ T cells. In some modalities, the method is carried out in vitro, in vivo, or ex vivo.

[00157] As discussed above, in some embodiments, the IL-2 mutein is structurally modified to increase its half-life. In some embodiments of any of the embodiments disclosed herein, the modification is one or more modifications selected from the group consisting of fusion to a human antibody Fe fragment, fusion to albumin, and PEGylation.

[00158] In another respect, methods for decreasing the proliferation of regulatory T cells (Tregs) are provided in this document, including contacting a Treg cell with an interleukin 2 (IL-2) mutan that has: (i) reduced binding affinity for the interleukin 2 receptor (IL-2Ry) compared to the polypeptide encoded by the SEC ID NO: 2; and (ii) 0-50% of Emax compared to the polypeptide encoded by the SEO ID NO: 2.In some embodiments, the IL-2 mutein includes: (i) one or more amino acid substitutions that increase the binding affinity of IL-2Rp compared to the polypeptide encoded by ID of SEC NO: 1, selected from L80F, R81D, L85V, I86V, and I92F, numbered according to the amino acid sequence of ID of SEC NO: 1; and (ii) one or more amino acid substitutions that reduce the binding affinity of the IL-2Ry receptor and result in 0-50% Emax compared to the polypeptide encoded by ID of SEC NO: 2, selected from (A) L18R and Q22E; and (B) amino acid position 126, numbered according to the amino acid sequence of ID of SEC NO: 2.

[00159] In another aspect, methods for decreasing regulatory T cell (Treg) proliferation are provided herein, including contacting a Treg cell with an IL-2 mutan having: (i) reduced binding affinity for IL-2Ry compared to the polypeptide encoded by ID of SEC NO: 1; and (ii) 0-50% of Emax compared to the polypeptide encoded by ID of SEC NO: 1. In some embodiments, the IL-2 mutan includes one or more amino acid substitutions that reduce the binding affinity of the IL-2Ry receptor and result in 0-50% Emax compared to a polypeptide encoded by ID of SEC NO: 1, selected from (A) L18R and Q22E; and (B) amino acid position 126, numbered according to the amino acid sequence of ID of SEC NO: 1.In some embodiments, the amino acid substitution at position 126 of either SEC NO: 1 or SEC NO: 2 is selected from the group consisting of Q126A, Q126C, Q126D, Q126E, Q126G, Q126H, Q126I, Q126K, Q126M, Q126R, Q126S, or Q126T. In some embodiments, the IL-2 mutein includes the Q126H, Q126K, or Q126M amino acid substitution. In some embodiments, the IL-2 mutein includes a Q126H amino acid substitution. In some embodiments of any of the embodiments disclosed herein, the method further includes administering the IL-2 muteins with an antibody that directs the mutein to a Treg cell. In some variations of any of the methods disclosed herein, the antibody is covalently or non-covalently bound to the mutein. In some variations of any of the methods disclosed herein, the method is carried out in vitro, in vivo, or ex vivo.

[00160] A pharmaceutical composition is formulated to be compatible with its intended route of administration. The terms “administration” and “administer,” as used herein, refer to the delivery of a bioactive composition or formulation by means of a route of administration comprising, but not limited to, oral, intravenous, intra-arterial, intramuscular, intraperitoneal, subcutaneous, and topical administration, or combinations thereof. The term includes, but is not limited to, administration by a healthcare professional and self-administration. In some modalities, the IL-2 mutating polypeptides of disclosure may be given orally or by inhalation, but are more likely to be administered via a parenteral route. Examples of parenteral routes of administration include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral administration may include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and tonicity-adjusting agents such as sodium chloride or dextrose. The pH may be adjusted with acids or bases, such as mono- and / or dibasic sodium phosphate, hydrochloric acid, or sodium hydroxide (e.g., to a pH of approximately 7.2 to 7.8, e.g., 7.5). The parenteral preparation may be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[00161] The dosage, toxicity, and therapeutic efficacy of such IL-2 mutans or nucleic acid compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD50 (the lethal dose for 50% of the population) and the ED50 (the therapeutically effective dose for 50% of the population). The ratio of toxic to therapeutic effects is the therapeutic index and can be expressed as the LD50 / ED50 ratio. Compounds exhibiting high therapeutic indices are preferred. While compounds exhibiting toxic side effects may be used, care must be taken in designing a delivery system that directs such compounds to the affected tissue site in order to minimize potential damage to uninfected cells and thus reduce side effects.

[00162] Data obtained from cell culture assays and animal studies may be used to formulate a dosage range for human use. The dosage of such compounds preferably lies within a range of circulating concentrations that includes the ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form and route of administration. For any compound used in the disclosure method, the therapeutically effective dose may initially be estimated from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves maximum mean inhibition of symptoms) as determined in cell culture.This information can be used to more accurately determine effective doses for humans. Plasma levels can be measured, for example, using high-performance liquid chromatography.

[00163] As defined herein, a practically effective amount of a given IL-2 mutan (i.e., an effective dosage) depends on the selected polypeptide. For example, single-dose amounts may be administered in the range of approximately 0.001 to 0.1 mg / kg of the patient's body weight; in some formulations, approximately 0.005, 0.01, or 0.05 mg / kg may be administered. In some formulations, 600,000 IU / kg is administered (IU can be determined by a lymphocyte proliferation bioassay and is expressed in International Units (IU) as established by the World Health Organization's 1st International Standard for Interleukin-2 (from Human)). The dosage may be similar to, but is expected to be lower than, that prescribed for PROLEUKIN®. The formulations may be administered from one or more times daily to one or more times weekly, including a every third day.The experienced practitioner will appreciate that certain factors can influence the dosage and duration required to effectively treat a subject, including but not limited to the severity of the illness or disorder, prior treatments, the subject's general health and / or age, and other concurrent illnesses. Furthermore, treating a subject with a therapeutically effective amount of the IL-2 mutans in question may involve a single course of treatment or a series of treatments. In some formulations, the compounds are administered every 8 hours for five days, followed by a rest period of 2 to 14 days; for example, 9 days, followed by an additional five days of administration every 8 hours.

[00164] This document also provides methods for preventing, reducing, or not promoting the proliferation of potentially inflammatory T cells and / or preventing the secretion of IFNγ from CD8+ T cells or other subsets of inflammatory immune cells. The method is carried out by contacting a cell expressing an interleukin-2 receptor (IL-2Rγ) with any of the IL-2 mutains disclosed herein. The proliferation of potentially inflammatory T cells and / or prevention of IFN-γ secretion by CD8+ T cells or other subsets of inflammatory immune cells can be decreased by any of approximately 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (inclusive of values ​​in between these percentages) compared to proliferation and / or IFN-γ secretion stimulating natural-type IL-2 at comparable concentrations and under similar conditions.In some modalities, the potentially inflammatory T cells are CD4+CD44+IFNγ+ T cells or CD8+CD44+IFNγ+ T cells. Additionally, the method can be carried out in vitro, in vivo, or ex vivo. V. Systems and Kits

[00165] The systems and / or kits in this disclosure include one or more of any of the IL-2 mutans, nucleic acids, vectors, or pharmaceutical compositions disclosed herein, as well as syringes (including pre-filled syringes) and / or catheters (including pre-filled syringes) used to administer any of the IL-2 mutans, nucleic acids, vectors, or pharmaceutical compositions to an individual. The kits also include written instructions for the use of any of the IL-2 mutans, nucleic acids, vectors, or pharmaceutical compositions disclosed herein, as well as syringes and / or catheters for use with their administration.

[00166] It is intended that each maximum numerical limitation given throughout this specification shall include each lower numerical limitation, as if such lower numerical limitations were expressly stated herein. Each minimum numerical limitation given throughout this specification shall include each upper numerical limitation, as if such upper numerical limitations were expressly stated herein. Each numerical interval given throughout this specification shall include each narrower numerical interval that falls within such wider numerical interval, as if all such narrower numerical intervals were expressly stated herein.

[00167] All publications and patent applications mentioned in this disclosure are incorporated by reference in this document to the same extent as if each individual publication or patent application were specifically and individually indicated as incorporated by reference.

[00168] No admission is made that any reference cited in this document constitutes prior art. The discussion of references states what their authors claim, and the inventors reserve the right to challenge the accuracy and relevance of the cited documents. It shall be clearly understood that, although a number of sources of information, including scientific journal articles, patent documents, and textbooks, are referenced in this document, such reference does not constitute an admission that any of these documents form part of the common knowledge in the field.

[00169] The discussion of the general methods provided in this document is intended for illustrative purposes only. Other alternative methods and alternatives will become apparent to those experienced in the subject matter upon review of this disclosure, and should be included within the spirit and scope of this request. EXAMPLES Example 1

[00170] This example shows the ability of different human IL-2 variants to stimulate STAT5 phosphorylation. Human NK-like YT cells were stimulated for 15 min with different human IL-2 variants fused with mouse serum albumin (MSA) at different concentrations, as shown in the graph in Figure 3 (from 1 μM to 0 μM, 10-fold dilution). All IL-2 variants were purified from transduced HEK 293 cells except for IL-2 REK (SEC ID NO: 10), which was purified from insect cells. The Y-axis in the graph in Figure 3 shows the ratio of the p-STAT5 signal of each IL-2 variant normalized to the p-STAT5 signal of wild-type IL-2 for each concentration. As shown, substitution of residue Q126 with specific amino acids in the antecedent of IL-2 H9 (SEC ID NO: 2) results in a wide range of IL-2 efficacies.

[00171] Subsequently, a time-domain phosphoSTAT5 signaling assay was performed by stimulating YT cells for 15 minutes with the concentrations of partial IL-2 agonists indicated in the graph shown in Figure 4A. The Y-axis of the graph in Figure 4A shows the ratio of the p-STAT5 signal of each IL-2 variant normalized to the p-STAT5 signal of WT IL-2 for each concentration. As shown in Figure 4B, YT cells were stimulated at different time points with 1 μM of different IL-2 variants, as indicated in the graph. In this figure, the Y-axis indicates the mean fluorescence intensity (MFI) for the p-STAT5 signal.

[00172] These data in Figure 4B demonstrate that the p-STAT5 signal magnitude for WT (SEC NO ID: 1), H9 (SEC NO ID: 2) and novel partial agonists REE (SEC NO ID: 6), REH (SEC NO ID: 8) and REK (SEC NO ID: 10) (all in antecedent of H9) are constant over time with REE, REK and REH signaling at 25%, 50% and 75% respectively. Example 2

[00173] This example illustrates the in vivo administration of IL-2 variants in mice. On day 0, female WT C57BL / 6c mice received 30 μg of each MSA-fused IL-2 variant by intraperitoneal (IP) injection. On day 6, spleens were collected and analyzed by flow cytometry to determine cell surface marker and intracellular cytokine expression (n = 3 / group) (see Figure 5A). The graphs in Figure 5B, Figure 5C, and Figure 5D represent the frequency of the indicated cell subset for each condition, normalized to the respective frequency in PBS-treated mice. B cells were defined as CD3 CD19+ activated cells. NK cells were CD3 NK1.V activated cells. Ly6g (Gr1)+CD3+CD11b+ activated cells were defined as granulocytes.

[00174] As shown in Figures 5B-5D, variant administration of IL-2 resulted in a mild change in other immune cell types. IL-2 H9_REH (REH; SEC ID NO: 8) initiated preferential expansion of CD4 effector memory T (TEM) cells. Notably, while treatment with WT (SEQ ID NO: 1) and H9 IL-2 (SEQ ID NO: 2) resulted in a significant increase in the frequency of CD8 T cell IFNT secretion, this increase was not observed in mice treated with either REE (SEQ ID NO: 6), REH (SEQ ID NO: 8), or IL-2 H9_REH. NO: 8) and REK (SEC ID NO: 10). Example 3

[00175] This example discloses the generation and characterization of IL-2 mutans in a natural-type antecedent. The ability of these variants to stimulate STAT5 phosphorylation was measured using a phospho-STAT5 signaling assay. Human NK-like YT cells (Figure 6A) or starved mouse blast T cells (Figure 6B) were stimulated for 15 min with different human IL-2 variants fused with mouse serum albumin (MSA) at different concentrations as shown in the graph (from 5 μM to 0 μM). All IL-2 variants were purified from insect cells. The Y-axis of the graphs shown in Figure 6A and Figure 6B shows the ratio of the p-STAT5 signal of each IL-2 variant normalized to the p-STAT5 signal of wild-type IL-2 for each concentration. As shown, the new partial agonist WT_REH (SEC NO ID: 15) appears to exhibit a lower EC50 and higher Emax compared to H9_REH (SEC NO ID: 8).Both new partial agonists WT_REH (SEC ID NO: 1) and H9_REH (SEC ID NO: 8) show lower efficacies (Emax) than WT or H9 IL-2.

[00176] Naturally occurring antecedent IL-2 mutains were subsequently administered to a B16 melanoma mouse model. On day 0 (DO), 10⁶ B16F10 cells were injected subcutaneously into female C57BL / 6 mice. On days 5, 9, and 14, mice were treated with 30 μg of each of the MSA-IL2 variants by intraperitoneal (IP) injection. Tumor size was measured every three days from day 5 using forceps. Tumor volume in mm³ was calculated as follows: (length * (width)²) / 2. On day 19, spleens were collected and analyzed by flow cytometry and intracellular marker expression. (n = 5 / group).

[00177] As depicted in Figure 7A, tumor sizes were substantially smaller in mice treated with WT (SEC NO ID: 1) or H9 (SEC NO ID: 2) compared to mice treated with PBS. Administration of H9_REH (SEC NO ID: 8) and H9_REM (REM) (SEC NO ID: 11) resulted in increased tumor size compared to PBS-treated mice. Mice treated with WT_REH (SEC NO ID: 15) exhibited larger tumor size compared to mice treated with WT (SEC NO ID: 1) or H9 (SEC NO ID: 2). An increased frequency of Foxp3+ regulatory T cells (Tre8) was observed in REM-treated mice (SEC ID NO: 11) and a 3-fold increase in the number of Foxp3+ Treg cells when mice were treated with WT_REH (SEC ID NO: 15) compared to PBS mice (Figure 7B).Mice treated with WT and H9 exhibited an expansion of IFNγ-secreting CD44+CD8 memory cells, but not with the novel partial agonists WT_REH (SEC NO ID: 15), H9_REH (SEC NO ID: 8), or REM (SEC NO ID: 11). Figure 7C depicts the effect on NK cells and granulocytes. The same trend was observed with IFNγ-secreting CD4+CD44+ memory T cells (Figure 7D). Finally, while IL-2 from WT (SEC NO ID: 1) activated the proliferation of CD4+ effector T cells (KI67+ Teff cells) and Treg cells, WT_REH (SEC NO ID: 1) induced a specific expansion of Treg cells but not of Tsff cells (Figure 7E). Mr. Lcn / nznz / q / Yi Example 4

[00178] This example describes experiments conducted to demonstrate that partial agonists of IL-2R in the natural antecedent can elicit cell-type-specific responses in vivo.

[00179] In these experiments, female C57 / BL6 mice were administered 3 x 30 pg intraperitoneal doses (days 0, 3, and 6) of wild-type (WT) IL-2 (SEQ ID NO: 1), partial agonist WT_REH (SEQ ID NO: 15), partial agonist WT_RETR (SEQ ID NO: 21), partial agonist WT_REK (SEQ ID NO: 22), or PBS control. All cytokines were formatted as N-terminal fusion with mouse serum albumin (MSA). On day 7, spleens and peripheral lymph nodes were collected and analyzed by flow cytometry and expressed as secreted proteins in insect cells.

[00180] The IL-2 WT_RETR and WT_REK mutains used in these experiments included the following amino acid sequences: APTSSSTKKTQLQLEHLRLDLEMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSK NFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCTSIIRTLT (WT_ RETR; SEQ ID NO: 21). APTSSSTKKTQLQLEHLRLDLEMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSK NFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCKSIISTLT (WT_ REK; ID de SEC NO: 22).

[00181] Partial IL-2R agonists were observed to induce lymphoid organ enlargement according to their agonist activity (the spleen and inguinal lymph nodes were photographed). As shown in Figures 8A-8B, administration of the partial agonists was found to induce lymphoid organ enlargement. IL-2 and the partial agonists showed little effect on NK cell frequency (CD3-NK1.1+), while IL-2 alone, but not the partial agonists, showed a reduction in B cell frequency (CD3-CD19+). IL-2 and the partial agonists were also observed to have no effect on T cell frequency (CD3+); however, IL-2 resulted in a decrease in the CD4+ to CD8+ T cell ratio, while one partial agonist, REH, caused an increase in the CD4+ to CD8+ T cell ratio. Example 5

[00182] This example describes experiments conducted to demonstrate that treatment with an exemplary IL-2R partial agonist, REH, increased the frequency of FoxP3+ regulatory T cells with reduced activation of conventional CD4+ T cells.

[00183] In these experiments, female C57 / BL6 mice were administered 3 x 30 pg intraperitoneal doses (days 0, 3, and 6) of wild-type IL-2 (ID of SEC NO: 1), partial agonist WT_REH (ID of SEC NO: 15), partial agonist WT_RETR (ID of SEC NO: 21), partial agonist WT_REK (ID of SEC NO: 22), or PBS control. All cytokines were formatted as N-terminal fusions with mouse serum albumin (MSA) and expressed as secreted proteins in insect cells. On day 7, spleens and peripheral lymph nodes were collected and analyzed by flow cytometry to assess the frequency of TregS(CD25+FoxP3+) and T cell activation. conventional CD4+ (CD25+FoxP3-).

[00184] It was observed that the IL-2R partial agonist REH increased the frequency of CD25+FoxP3+ regulatory T cells and induced conventional CD25+ cells. Example 6

[00185] This example describes experiments conducted to demonstrate that Tregs from mice treated with partial IL-2R agonist REH suppress the proliferation of conventional CD4+ T cells.

[00186] In these experiments, female B6.FoxP3 GFP mice were administered 3 x 30 pg intraperitoneal doses (days 0, 3, and 6) of natural-type WT (ID of SEC NO: 1), partial agonist WT_REH (ID of SEC NO: 15), or PBS control. On day 7, spleens and lymph nodes were collected, and CD4+ T cells were isolated by magnetic sorting (MACS). CD4+ T cells were sorted by fluorescence-activated cell sorting (FACS) based on GFP expression to isolate Tregs (GFP+) and Tcons (GFP-). FoxP3 GFP+ cells from cytosine-treated mice were co-cultured with CelITrace Violet (CTV)-labeled Tcons from PBS-treated mice and aCD3aCD28-coated beads (1:1 bead-to-Tcon ratio). Proliferation of CTV-labeled Tcons was assessed after 72 hours.

[00187] It was observed that FoxP3+ cells from REH-treated mice were able to suppress the proliferation of FoxP3- effector cells, Example 7

[00188] This example describes experiments conducted to illustrate that the partial IL-2R agonist REH supports CD8+ T cell proliferation but not IFNy production.

[00189] In these experiments, CD8+ T cells were isolated from the spleens and lymph nodes of C57 / BL6 mice by magnetic isolation (MACS), loaded with CelITrace Violet, and co-cultured with aCD3aCD28-coated beads in the presence or absence of 20 nM WT (SEC ID NO: 1) or WT_REH (SEC ID NO: 15) for three days. Four hours before collection, GolgiStop was added to the cells to prevent further cytokine secretion. At 72 hours, the cells were fixed, permeabilized, and stained with antibodies against interferon gamma (IFNγ).

[00190] It was observed that both the partial agonist IL-2R REH and IL-2 could support proliferation but only IL-2 could induce robust IFNy production by proliferating cells. Example 8

[00191] This example describes experiments performed to demonstrate that treatment and co-treatment with the IL-2R partial agonist REH are protective against various autoimmune symptoms in the rodent model Lcn / nznz / q / Yi EAE (an animal model of brain inflammation).

[00192] In these experiments, B6 mice were either pretreated (day -7, -4, -1) or co-treated (day 0, 3, and 7) with 10 pg of WT_REH fused to MSA (SEC ID NO: 15). On day 0, mice were immunized with myelin oligodendrocyte glycoprotein (MOG) 35-55 in Freund's Complete Adjuvant 5 (CFA) and pertussis toxin (PTX), followed by a PTX boost on day 2. Disease score progression was monitored by weight loss and disease score. Disease scores were as follows: 0 - healthy; 1 - floppy tail; 2 - partial hind limb paralysis; 3- complete paralysis of the hind limb; 4- paralysis of the whole body; 5- death.

[00193] It was observed that pretreatment with the partial agonist IL-2R REH was protective, while co-treatment with REH delayed the onset of disease in a manner consistent with previous experiments examining the kinetics of REH-treated Tregsen mice.

[00194] Although particular alternatives to this disclosure have been disclosed, it should be understood that different modifications and combinations are possible and are contemplated within the true spirit and scope of the 15 appended claims. Therefore, no limitation is intended on the exact summary and disclosure presented herein.

Claims

1. An interleukin 2 (IL-2) mutan having: (a) reduced binding affinity for the interleukin 2 receptor γ (IL-2Ry) compared to an IL-2 polypeptide encoded by ID of SEC NO: 2; and (b) 15-95% of Emax compared to an IL-2 polypeptide encoded by ID of SEC NO:

2.

2. The IL-2 mutein according to claim 1, wherein the mutein comprises: (i) one or more amino acid substitutions that increase the binding affinity of IL-2Rp compared to a polypeptide encoded by ID of SEC NO: 1, selected from L80F, R81D, L85V, I86V, and I92F, numbered according to the amino acid sequence of ID of SEC NO: 1; and / or (ii) one or more amino acid substitutions that reduce the binding affinity of the IL-2Ry receptor and result in 15-95% of Emax compared to the polypeptide encoded by ID of SEC NO: 2, selected from (A) L18R and Q22E; and (B) the amino acid position 126, numbered according to the amino acid sequence of ID of SEC NO:

2.

3. The IL-2 mutein according to claim 2, wherein the amino acid substitution at position 126 of the ID of SEC NO: 2 is selected from the group consisting of Q126A, Q126C, Q126D, Q126E, Q126G, Q126H, Q126I, Q126K, Q126M, Q126R, Q126S, or Q126T.

4. The IL-2 mutein according to claim 3, wherein the mutein comprises the amino acid substitution Q126H, Q126K, or Q126M.

5. The IL-2 mutein according to claim 3, wherein the mutein comprises an amino acid substitution Q126H.

6. An interleukin 2 (IL-2) mutain having (a) reduced binding affinity for the interleukin γ receptor (IL-2Ry); and (b) 15-95% of Emax compared to a polypeptide encoded by the ID of SEC NO:

1.

7. The IL-2 mutein according to claim 6, wherein the mutein comprises one or more amino acid substitutions that reduce the binding affinity of the IL-2Ry receptor and result in 15-95% of Emax compared to a polypeptide encoded by the ID of SEC NO: 1, selected from (A) L18R and Q22E; and (B) amino acid position 126, numbered according to the amino acid sequence of the ID of SEC NO:

1.

8. The IL-2 mutein according to claim 7, wherein the mutein comprises an amino acid substitution at position 126 of the ID of SEC NO: 1 selected from the group consisting of Q126A, Q126C, Q126D, Q126E, Q126G, Q126H, Q126I, Q126K, Q126M, Q126R, Q126S, or Q126T.

9. The IL-2 mutein according to claim 8, wherein the mutein comprises an amino acid substitution Q126H, Q126K, or Q126M.

10. The IL-2 mutein according to claim 9, wherein the mutein comprises an amino acid substitution Q126H.

11. The IL-2 mutein according to any of claims 1 to 10, wherein the mutein is structurally modified to increase the half-life.

12. The IL-2 mutein according to claim 11, wherein said modification comprises one or more modifications selected from the group consisting of fusion to a human antibody Fe fragment, fusion to albumin, and PEGylation.

13. The IL-2 mutein according to any of claims 1 to 12, wherein said mutein increases the proliferation of regulatory T cells (Treg) and / or induces minimal proliferation of potentially inflammatory T cells.

14. The IL-2 mutein according to any of claims 1 to 13, wherein said mutein causes the expansion of Treg cells and does not promote the expansion of potentially inflammatory T cells and natural killer (NK) cells or granulocytes.

15. The IL-2 mutein according to claim 14, wherein the mutein induces less proliferation of potentially inflammatory T cells compared to the polypeptide encoded by SEO ID NO: 1 or SEO ID NO:

2.

16. The IL-2 mutain according to claim 15, wherein said potentially inflammatory T cells are CD4+1FNy+ T cells or CD8+IFNy+ T cells.

17. The IL-2 mutein according to any of claims 1 to 16, wherein the mutein increases Treg cell proliferation by at least 3 times and / or induces less IFNy secretion compared to the polypeptide encoded by SEO ID NO: 1 or SEO ID NO:

2.

18. The IL-2 mutain according to any of claims 1 to 17, wherein said mutain does not induce IFNy secretion from CD8+ T cells and / or other subsets of inflammatory immune cells.

19. The IL-2 mutein according to any of claims 1 to 18, wherein the mutein has 70-95% of the Emaxen compared to the polypeptide encoded by SEO ID NO:

1.

20. A nucleic acid encoding IL-2 mutain according to any of claims 1 to 19.

21. A vector comprising the nucleic acid according to claim 20.

22. A host cell comprising the nucleic acid according to claim 20 or the vector according to claim 21.

23. A sterile pharmaceutical composition comprising IL-2 mutein according to any one of claims 1 to 19 and a pharmaceutically acceptable excipient.

24. A syringe comprising (a) the IL-2 mutan according to any one of claims 1 to 19; (b) the nucleic acid according to claim 20; (c) the vector according to claim 21; (d) the host cell according to claim 22; and / or (e) the pharmaceutical composition according to claim 23.

25. A catheter comprising (a) the IL-2 mutan according to any one of claims 1 to 19; (b) the nucleic acid according to claim 20; (c) the vector according to claim 21; (d) the host cell according to claim 22; and / or (e) the pharmaceutical composition according to claim 23.

26. A kit comprising: (a) one or more of (i) the IL-2 mutein according to any one of claims 1 to 19; (ii) the nucleic acid according to claim 20; (iii) the vector according to claim 21; (iv) the host cell according to claim 22; (v) the sterile pharmaceutical composition according to claim 23; (vi) the syringe according to claim 24; and / or (vii) the catheter according to claim 25; and (b) written instructions for using (i) the IL-2 mutein according to any one of claims 1 to 19; (ii) the nucleic acid according to claim 20; (iii) the vector according to claim 21; (iv) the host cell according to claim 22; (v) the sterile pharmaceutical composition according to claim 23; (vi) the syringe according to claim 24; and / or (vil) the catheter according to claim 25.

27. A method for treating an autoimmune disease in an individual in need comprising administering (a) a therapeutically effective amount of IL-2 mutan according to any one of claims 1 to 19; (b) the nucleic acid according to claim 20; (c) the vector according to claim 21; (d) the host cell according to claim 22; and / or (e) the pharmaceutical composition according to claim 23 to the individual.

28. The method according to claim 27, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, insulin-dependent diabetes mellitus, hemolytic anemias, rheumatic fever, thyroiditis, Crohn's disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, multiple sclerosis, alopecia areata, psoriasis, vitiligo, dystrophic epidermolysis bullosa, systemic lupus erythematosus, and graft-versus-host disease.

29. The method according to claim 28, wherein the autoimmune disease is graft-versus-host disease.

30. The method according to any of claims 27 to 29 further comprises administering the IL-2 mutan according to any of claims 1 to 19 or the pharmaceutical composition according to claim 23 in combination with an antibody that directs the mutan to a specific cell type.

31. The method according to claim 30, wherein the cell type is a regulatory T cell (Treg).

32. The method according to claim 30 or 31, wherein the antibody is covalently or non-covalently linked to the IL-2 mutan.

33. A method for producing IL-2 mutein according to any of claims 1 to 19 comprising culturing the host cell according to claim 22 under conditions suitable for mutein production.

34. The method according to claim 33 further comprises isolating and / or purifying the IL2 mutein produced.

35. The method according to claim 33 or 34 further comprises structurally modifying the IL-2 mutein to increase its half-life.

36. The method according to claim 35, wherein said modification comprises one or more alterations selected from the group consisting of fusion to a human antibody Fe fragment, fusion to albumin, and PEGylation.

37. A method for preventing the proliferation of potentially inflammatory T cells and / or preventing the secretion of IFNy from CD8+ T cells or other subsets of inflammatory immune cells, said method comprising contacting a cell expressing an interleukin 2 receptor (IL-2Ry) with the IL-2 mutain according to any one of claims 1 to 19.

38. The method according to claim 37, wherein said potentially inflammatory T cells are CD4+CD44+IFNy+ T cells or CD8+CD44+IFNy+ T cells.

39. The method according to claim 37 or 38, wherein the method is carried out in vitro, in vivo, or ex vivo.

40. A method for decreasing the proliferation of regulatory T cells (Tregs) comprising contacting a Treg cell with an interleukin 2 (IL-2) mutan having: (i) reduced binding affinity for the interleukin 2 receptor (IL-2Ry) compared to the polypeptide encoded by the ID of SEC NO: 2; and (ii) 0-50% of Emax compared to the polypeptide encoded by the ID of SEC NO:

2.

41. The method according to claim 40, wherein the IL-2 mutein comprises: (i) one or more amino acid substitutions that increase the binding affinity of IL-2R3 compared to the polypeptide encoded by ID of SEC NO: 1, selected from L80F, R81D, L85V, I86V, and I92F, numbered according to the amino acid sequence of ID of SEC NO: 1; and (ii) one or more amino acid substitutions that reduce the binding affinity of the IL-2Ry receptor and result in 0-50% Emax compared to the polypeptide encoded by ID of SEC NO: 2, selected from (A) L18R and Q22E; and (B) amino acid position 126, numbered according to the amino acid sequence of ID of SEC NO:

2.

42. A method for decreasing the proliferation of regulatory T cells (Tregs) comprising contacting a Treg cell with an IL-2 mutan having: (i) reduced binding affinity for IL-2Ry compared to the polypeptide encoded by ID of SEC NO: 1; and (ii) 0-50% of Emax compared to the polypeptide encoded by ID of SEC NO:

1.

43. The method according to claim 42, wherein the mutein comprises one or more Lcn / nznz / q / Yi amino acid substitutions that reduce the binding affinity of the IL-2Ry receptor and result in 0-50% of Emax compared to a polypeptide encoded by ID of SEC NO: 1, selected from (A) L18R and Q22E; and (B) amino acid position 126, numbered according to the amino acid sequence of ID of SEC NO:

1.

44. The method according to claim 41 or 43, wherein the amino acid substitution at position 126 of ID SEC NO: 1 or ID SEC NO: 2 is selected from the group consisting of Q126A, Q126C, Q126D, Q126E, Q126G, Q126H, Q126I, Q126K, Q126M, Q126R, Q126S, or Q126T.

45. The method according to claim 44, wherein the mutein comprises the amino acid substitution Q126H, Q126K, or Q126M.

46. ​​The method according to claim 45, wherein the mutein comprises a 10 amino acid substitution Q126H.

47. The method according to any of claims 39 to 46 further comprises administering the muteins with one or more antibodies that direct the mutein to a Treg cell.

48. The method according to claim 47, wherein the antibody is covalently or non-covalently linked to the mutan. 15 49. The method according to any of claims 39 to 48, wherein the method is carried out in vitro, in vivo, or ex vivo.