WO2024118780A2

WO2024118780A2 - Il-2 mutants and uses thereof

Info

Publication number: WO2024118780A2
Application number: PCT/US2023/081609
Authority: WO
Inventors: Shouhua XIAO; Weijun Feng; Yumin Dai; Jie Xue; Halei ZHAI
Original assignee: Binacea Pharma, Inc.
Priority date: 2022-11-30
Filing date: 2023-11-29
Publication date: 2024-06-06
Also published as: WO2024118780A3

Abstract

The present disclosure relates to mutants of the cytokine IL-2 with altered functional characteristics including increased expression titer, and selective binding to different IL-2 receptor protein complexes, pharmaceutical compositions comprising these mutant IL-2 polypeptides, and uses of these compositions as therapeutics, for example in the treatment of cancer and autoimmune disorders.

Description

IL-2 MUTANTS AND USES THEREOF CROSS REFERENCE TO RELATED APPLICATIONS [0001] The present application claims priority to US Prov. Pat. Appl. Ser. No.63/385,610, filed November 30, 2022, which is hereby incorporated by reference in its entirety for all purposes. FIELD [0002] The present disclosure relates to mutant IL-2 polypeptides, and fusions of these polypeptides with half-life extending proteins, such Fc monomer or Fc dimer, serum albumin polypeptides, and/or specific binding biomolecules, such as VHH polypeptides, and antibodies, pharmaceutical compositions that include these polypeptides, and uses of these composition as therapeutics, for example in the treatment of cancer or autoimmune diseases. REFERENCE TO SEQUENCE LISTING [0003] The official copy of the Sequence Listing is submitted concurrently with the specification via USPTO Patent Center as an WIPO Standard ST.26 formatted XML file with file name “17195-001PV1.xml”, a creation date of November 30, 2022, and a size of 163,978 bytes. This Sequence Listing filed via USPTO Patent Center is part of the specification and is incorporated in its entirety by reference herein. BACKGROUND [0004] Interleukin-2 (IL-2), also known as T-cell growth factor (TCGF), is a pluripotent cytokine produced mainly by activated T cells, in particular CD4+ T helper cells. IL-2 signaling is mediated through binding to three different receptor proteins: IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132). Immune cells express dimeric or trimeric complexes of the IL-2 receptor proteins. The dimeric receptor (IL-2Rβγ) is expressed on cytotoxic CD8+ T cells and natural killer cells (NK), whereas the trimeric receptor (IL-2Rαβγ) is expressed predominantly on activated lymphocytes and CD4+ CD25+ FoxP3+ suppressive regulatory T cells (Treg). Treg cells express high levels of IL-2Rα (CD25) and Treg proliferation is stimulated by IL-2. Effector T cells and NK cells in a resting state, however, do not have CD25 on the cell surface and are relatively insensitive to IL-2. [0005] IL-2 binding to the three different receptor proteins varies significantly. IL-2 high affinity for the trimeric receptor with a K_D of about 10 pM, an intermediate affinity for the dimeric receptor with a K_D of about 1 nM, and low affinity for the monomeric IL-2Rα receptor, with a K_D of about 10 nM. IL-2 signaling activity mediated by the different receptor complexes also varies significantly. Generally, it has been found that IL-2Rβ and IL-2Rγ are critical for IL-2 signaling, while IL-2Rα (CD25) is not essential. [0006] IL-2 binding to the IL-2 receptor protein expressed on different cells mediates different immune responses. IL-2 can stimulate immune responses, such as: T cell proliferation and differentiation, cytotoxic T lymphocyte (CTL) production, B cell proliferation and differentiation, immunoglobulin synthesis, and production, proliferation, and activation of natural killer (NK) cells. IL-2 has been approved as an immunotherapeutic agent for the treatment of cancer and chronic viral infection. IL-2, however, can also promote the activation and proliferation of immunosuppressive CD4+ CD25+ Treg cells resulting in immunosuppression (Fontenot et al., Nature Immunol.6, 1142-51 (2005); D'Cruz and Klein, Nature Immunol.6, 1152-59 (2005); Maloy and Powrie, Nature Immunol.6, 1171-72 (2005)). In addition, IL-2 treatment is associated with vascular leak syndrome (VLS), and pulmonary edema in patients. It is believed that the pulmonary edema is due to the direct binding of IL-2 to trimeric receptors (IL-2Rαβγ) on lung endothelial cells (Krieg et al., Proc Nat Acad Sci USA 107, 11906-11 (2010)). [0007] The engineering of IL-2 with mutations has been proposed to reduce these toxic side effects by altering the selectivity or preference of IL-2 for the different IL-2 receptor subunits and thereby improve its therapeutic effect. For example, it has been proposed that targeting IL- 2 to cells expressing IL-2Rβ but not IL-2Rα, can induce amplification of cell populations high in IL-2Rβ, which improves the therapeutic effect of IL-2 therapy (Boyman et al., Science 311, 1924-1927 (2006)). US Patent Publ.2018/0142037 A1 describes introducing mutations at IL-2 amino acid positions 42, 45, and 72, also for the purpose of reducing affinity of IL-2 for the IL- 2Rα receptor. Another mutant IL-2 called “IL-2H9” which includes the five mutations L80F, R81D, L85V, I86V, and I92F, exhibits enhanced binding to IL-2Rβ, resulting in the stimulation of CD25− cells (see, Levin et al., Nature, Vol 484, p 529-533, DOI: 10.1038/nature10975). A mutant IL-2 protein “IL-23x,” has three mutations, R38D, K43E, and E61R that result in very low binding affinity for IL-2Rα (see, Rodrigo Vazquez-Lombardi et al., Nature Communications, 8:15373, DOI: 10.1038/ncomms15373). However, the activation preference of IL-23X for CD25+ cells still exists, and the expression level of the mutant polypeptide is low, which is not conducive to subsequent large-scale drug production. [0008] There remains a need for variant forms of IL-2 that exhibit improved functional properties for therapeutics uses, such as in pharmaceutical compositions for the treatment of diseases, such as infections, cancers, and autoimmune disorders. SUMMARY [0009] The present disclosure relates generally to mutants of the cytokine IL-2, pharmaceutical compositions, and uses of these composition as therapeutics, for example in the treatment of cancer. This summary is intended to introduce the subject matter of the present disclosure, but does not cover each and every embodiment, combination, or variation that is contemplated and described within the present disclosure. Further embodiments are contemplated and described by the disclosure of the detailed description, drawings, and claims. [0010] In at least one embodiment, the present disclosure provides a mutant IL-2 polypeptide that specifically binds an IL-2 receptor protein, wherein the polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 10 (wild-type IL-2) and one or more amino acid differences relative to SEQ ID NO: 10 selected from: E61N, and L63T; E61N, and L63S; P65N and E67T; [0011 ino

differences relative to SEQ ID NO: 10 selected from: K35N, F42A, Y45R, N88D, D109N, and Q126T. [0012] In at least one embodiment of the mutant IL-2 polypeptide of the present disclosure, the polypeptide comprises a combination of amino acid differences relative to SEQ ID NO: 10 selected from: K35N, P65N, E67T; K35N, P65N, E67S;

Y45R, R81N, P82A, R83T, E95N, K97T; Y45R, R81N, P82A, R83S, E95N, K97S; Y45R R81N P82A R83T E95N K97S; [0013

ure, the polypeptide comprises a combination of amino acid differences selected from: R81N, P82X, and R83T, wherein X is any amino acid other than P; R81N P82X and R83S wherein X is an amino acid other than P

R81N, P82A, R83T, P65N, E67T, E61N, L63T, E95N, K97T; R81N, P82A, R83T, P65N, E67S, E61N, L63T, E95N, K97T; R81N P82A R83T P65N E67T E61N L63S E95N K97T;

[0014] In at least one embodiment of the mutant IL-2 polypeptide of the present disclosure, the polypeptide comprises a an amino acid sequence selected from SEQ ID NO: 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, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, and 145. [0015] In at least one embodiment of the mutant IL-2 polypeptide of the present disclosure, the polypeptide is characterized by one or more of the following properties relative to the C125S IL- 2 polypeptide of SEQ ID NO: 10: (i) increased titer when expressed in a mammalian cell culture system; (ii) increased solubility; (iii) reduced binding affinity for IL-2Rα receptor; and/or (iv) reduced binding affinity for IL-2Rβ and IL-2Rγ receptor. [0016] In at least one embodiment of the mutant IL-2 polypeptide of the present disclosure, the polypeptide is fused through a linker to a monomeric or dimeric Fc polypeptide; optionally, wherein the Fc polypeptide is a monomeric Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 146, 147, or 148. In at least one embodiment, the linker consists of a polypeptide having a length of at least 1-50 amino acids. In at least one embodiment, the linker comprises a polypeptide having an amino acid sequence selected from (GGGGS)₁ (SEQ ID NO: 149), (GGGGS)2 (SEQ ID NO: 150), (GGGGS)3 (SEQ ID NO: 151), (GGGGS)4 (SEQ ID NO: 152), (GRPGS)₂ (SEQ ID NO: 153), (GRPGS)₄ (SEQ ID NO: 154), and (GGGGS)₃GGG (SEQ ID NO: 155). In at least one embodiment, the linker is conjugated to the C-terminus of the Fc polypeptide and the N-terminus of the mutant IL-2 polypeptide. In at least one embodiment, the linker is conjugated to the N-terminus of the Fc polypeptide and the C-terminus of the mutant IL-2 polypeptide. [0017] In at least one embodiment, the present disclosure provides a polynucleotide encoding a mutant IL-2 polypeptide of the present disclosure. In at least one embodiment, the present disclosure provides an expression vector comprising a polynucleotide encoding a mutant IL-2 polypeptide of the present disclosure. [0018] In at least one embodiment, the present disclosure also provides an isolated host cell comprising a polynucleotide encoding a mutant IL-2 polypeptide of the present disclosure or an expression vector comprising such a polynucleotide. In at least one embodiment, the host cell is a mammalian cell or a yeast cell; optionally, a mammalian cell selected from a Chinese hamster ovary (CHO) cell, a myeloma cell (e.g.,Y0, NS0, Sp2/0), a monkey kidney cell (COS- 7), a human embryonic kidney line (293), a baby hamster kidney cell (BHK), a mouse Sertoli cell (e.g., TM4), an African green monkey kidney cell (VERO-76), a human cervical carcinoma cell (HELA), a canine kidney cell, a human lung cell (W138), a human liver cell (Hep G2), a mouse mammary tumor cell, a TR1 cell, a Medical Research Council 5 (MRC 5) cell, and a FS4 cell. [0019] In at least one embodiment, the present disclosure also provides a method for producing a mutant IL-2 polypeptide of the present disclosure, the method comprising culturing a host cell comprising a polynucleotide or expression vector encoding a mutant IL-2 polypeptide of the present disclosure under a condition suitable for expressing the polypeptide. [0020] In at least one embodiment, the present disclosure also provides a pharmaceutical composition comprising a mutant IL-2 polypeptide of the present disclosure and a pharmaceutically acceptable carrier. [0021] In at least one embodiment, the present disclosure also provides a method for treating a in a subject (e.g., an IL-2 mediated disease), the method comprising administering to the subject a therapeutically effective amount of a mutant IL-2 polypeptide of the present disclosure or administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a mutant IL-2 polypeptide of the present disclosure and a pharmaceutically acceptable carrier. In at least one embodiment of the method, the disease is cancer; optionally, wherein the cancer is selected from colorectal cancer, pancreatic cancer, ovarian cancer, liver cancer, renal cancer, breast cancer, lung cancer, esophageal and gastric cancer, head and neck cancer, cervical cancer, prostate cancer, melanoma, bladder cancer, or oral cancer. [0022] In at least one embodiment of the method of treating cancer, the mutant IL-2 polypeptide administered exhibits selectively reduced IL-2Rα binding affinity relative to the IL- 2Rα binding affinity of the C125 IL-2 polypeptide of SEQ ID NO: 10. In at least one embodiment of the method, the polypeptide comprises a combination of amino acid differences relative to SEQ ID NO: 10 selected from: P65N, E67T; P65N E67S; [002

3] n at east one embodment o t e met od o treatng a dsease, t e dsease s an autoimmune disease; optionally, wherein the autoimmune disease is selected from Crohn’s disease, Ulcerative colitis, celiac disease, systemic lupus erythematosus, psoriatic arthritis, rheumatoid arthritis, Sjogren’s syndrome, type 1 diabetes, atopic dermatitis, psoriasis, multiple sclerosis. [0024] In at least one embodiment of the method of treating an autoimmune disease, the polypeptide exhibits selectively reduced IL-2Rβγ binding affinity relative to the IL-2Rβγ binding affinity of the C125 IL-2 polypeptide of SEQ ID NO: 10. In at least one embodiment of the method, the polypeptide comprises a combination of amino acid differences relative to SEQ ID NO: 10 selected from: E61N, L63T; E61N, L63S; E N K 7T

R81N, P82A, R83S, E61N, L63S, E95N, K97T, N90S; and R81N, P82A, R83S, E61N, L63S, E95N, K97S, N90S.

BRIEF DESCRIPTION OF THE DRAWINGS [0025] A better understanding of the novel features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which: [0026] FIGS.1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1I, and 1J depict results of biolayer interferometry (BLI) measurements performed as described in Example 1 to measure binding to IL-2Rα and IL-2Rβγ by the exemplary mutant IL-2 polypeptide Fc fusion constructs p132, p115, and p151 relative to the control p123 (C125S IL2) construct. [0027] FIGS.2A, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I, 2J, 2K, and 2L depict SEC profiles obtained as described in Example 1 for the exemplary mutant IL-2 polypeptide Fc fusion constructs p296, p307, p406, p297, p300, p308, p214, p310, p411, p298, and the p123 C125S IL2 fusion control, as follows: control p123 (C125S IL2) construct (FIG.2A and FIG.2B), p296 (FIG.2C), p307 (FIG.2D), p406 (FIG.2E), p297 (FIG.2F), p300 (FIG.2G), p308 (FIG.2H), p214 (FIG. 2I), p310 (FIG.2J), p411 (FIG.2K), and p298 (FIG.2L). [0028] FIGS.3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K, 3L, 3M, and 3N depict plots of binding curves and EC₅₀ values measured in the HEK Blue IL-2 reporter assay as described in Example 1 for the exemplary mutant IL-2 polypeptide Fc fusion constructs, p124, p132, p167, p296, p300, p214, p307, p308, p310, p297, p298, and p411, and the p123 C125S IL2 fusion control as follows: p124, p132, and p167 (FIG.3A, FIG.3B); p296, p300, and p214 (FIG.3C, FIG.3D); p123, p307, and p214 (FIG.3E, FIG.3F); p123, p296, and p308 (FIG.3G, FIG.3H); p123, p310, and p214 (FIG.3I, FIG.3J); p296, p297, p298, and p214 (FIG.3K, FIG.3L); p411, p214, p123, p406, and p214 (FIG.3M, FIG.3N). DETAILED DESCRIPTION [0029] The present disclosure provides mutant IL-2 polypeptides with mutations that alter glycosylation of the polypeptide and affect various physicochemical and functional characteristics of the IL-2 including recombinant expression titer, solubility, and binding affinity to the IL-2R chains IL-2Rα, IL-2Rβ, and IL-2Rγ, in the monomeric, dimeric, and trimeric forms. The altered binding characteristics of the mutant IL-2 polypeptides with the different IL-2R chains allows for the inhibit, decrease, and/or fully block the function of the IL-2R receptor, particularly its function as a cell surface receptor in mediating immune regulation. Accordingly, it is contemplated that any of the compositions or formulations comprising a mutation IL-2 polypeptide of the present disclosure can be used as therapeutics for treatment of diseases mediated by the function of IL-2R or its cognate ligand, IL-2, such as treatment of cancers and autoimmune disorders. Further, it is contemplated that the mutant IL-2 polypeptides of the present disclosure can be used as a therapeutic in combination with other therapeutics, such as antibodies that target immune checkpoint molecules. [0030] Overview of Terminology and Techniques [0031] For the descriptions herein and the appended claims, the singular forms “a”, and “an” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “a protein” includes more than one protein, and reference to “a compound” refers to more than one compound. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. The use of “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.” [0032] Where a range of values is provided, unless the context clearly dictates otherwise, it is understood that each intervening integer of the value, and each tenth of each intervening integer of the value, unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of these limits, ranges excluding (i) either or (ii) both of those included limits are also included in the invention. For example, “1 to 50,” includes “2 to 25,” “5 to 20,” “25 to 50,” “1 to 10,” etc. [0033] Generally, the nomenclature used herein and the techniques and procedures described herein include those that are well understood and commonly employed by those of ordinary skill in the art, such as the common techniques and methodologies described in e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Vols.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2012 (hereinafter “Sambrook”); and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., originally published in 1987 in book form by Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., and regularly supplemented through 2011, and now available in journal format online as Current Protocols in Molecular Biology, Vols.00 - 130, (1987-2020), published by Wiley & Sons, Inc. in the Wiley Online Library (hereinafter “Ausubel”). [0034] All publications, patents, patent applications, and other documents referenced in this disclosure are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference herein for all purposes. [0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. It is to be understood that the terminology used herein is for describing particular embodiments only and is not intended to be limiting. For purposes of interpreting this disclosure, the following description of terms will apply and, where appropriate, a term used in the singular form will also include the plural form and vice versa. [0036] “IL-2” or “IL-2 polypeptide” as used herein refers to the cytokine, interleukin-2, and includes naturally occurring and recombinant forms of the interleukin-2 polypeptide from human, mouse, rat, or non-human primate, and in its unprocessed (with signal peptide) and processed forms (without signal peptide). In addition, this term includes naturally occurring IL-2 variants, such as allelic and splice variants, isotypes, homologs, and species homologs, and recombinant (i.e., man-made) IL-2 variants or mutants, including mutant IL-2 polypeptides having from 1-15 amino acid substitutions relative to the amino acid sequence of the naturally occurring IL-2. For example, the term encompasses the recombinant human IL-2 amino acid sequence of UniProt P60568 with an amino acid substitution at position C125, such as C125S, or C125A. This term is also intended to encompass IL-2 polypeptides that are covalently conjugated (or fused) to another polypeptide or protein. Exemplary IL-2 fusions of the present disclosure include a mutant IL-2 polypeptide fused to other cytokines (e.g., IL-15), or fused to a half-life extending polypeptide (e.g., monomeric Fc, dimeric Fc, or human serum albumin). [0037] “IL-2 receptor” or “IL-2R,” as used herein refers to the heterotrimeric protein expressed on the surface of certain immune cells and endothelial cells and also encompasses each of the polypeptide subunits, IL-2Rα, IL-2Rβ, and IL-2Rγ (also known as cytokine receptor common subunit gamma), in their monomeric form, and in the dimeric form, such as IL-2Rβγ. [0038] “Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an IL-2 polypeptide) and its binding partner (e.g., an IL-2 receptor). “Binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (K_D). Affinity can be measured by common methods known in the art, including those described herein. “Binds specifically” or “specific binding” refers to binding of IL-2 polypeptide to its receptor with an affinity value of no more than about 1 x 10^-7 M. Illustrative and exemplary embodiments for measuring binding affinity and/or specific binding are described elsewhere herein including the Examples. [0039] “Host cell” as used herein refers to a cell capable of being functionally modified with recombinant nucleic acids and functioning to express recombinant products, including polypeptides and compounds produced by activity of the polypeptides. [0040] “Nucleic acid,” or “polynucleotide” as used herein interchangeably to refer to two or more nucleosides that are covalently linked together. The nucleic acid may be wholly comprised ribonucleosides (e.g., RNA), wholly comprised of 2'-deoxyribonucleotides (e.g., DNA) or mixtures of ribo- and 2'-deoxyribonucleosides. The nucleoside units of the nucleic acid can be linked together via phosphodiester linkages (e.g., as in naturally occurring nucleic acids), or the nucleic acid can include one or more non-natural linkages (e.g., phosphorothioester linkage). Nucleic acid or polynucleotide is intended to include single- stranded or double-stranded molecules, or molecules having both single-stranded regions and double-stranded regions. Nucleic acid or polynucleotide is intended to include molecules composed of the naturally occurring nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), or molecules comprising that include one or more modified and/or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. [0041] “Protein,” “polypeptide,” and “peptide” are used herein interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristilation, ubiquitination, etc.). As used herein “protein” or “polypeptide” or “peptide” polymer can include D- and L-amino acids, and mixtures of D- and L-amino acids. [0042] “Naturally-occurring” or “wild-type” as used herein refers to the form as found in nature. For example, a naturally occurring nucleic acid sequence is the sequence present in an organism that can be isolated from a source in nature, and which has not been intentionally modified by human manipulation. [0043] “Recombinant,” “engineered,” or “non-naturally occurring” when used herein with reference to, e.g., a cell, nucleic acid, or polypeptide, refers to a material, or a material corresponding to the natural or native form of the material, that has been modified in a manner that would not otherwise exist in nature, or is identical thereto but is produced or derived from synthetic materials and/or by manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant cells expressing genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise expressed at a different level. [0044] “Nucleic acid derived from” as used herein refers to a nucleic acid having a sequence at least substantially identical to a sequence of found in naturally in an organism. For example, cDNA molecules prepared by reverse transcription of mRNA isolated from an organism, or nucleic acid molecules prepared synthetically to have a sequence at least substantially identical to, or which hybridizes to a sequence at least substantially identical to a nucleic sequence found in an organism. [0045] “Coding sequence” refers to that portion of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein. [0046] “Heterologous nucleic acid” as used herein refers to any polynucleotide that is introduced into a host cell by laboratory techniques and includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into a host cell. [0047] “Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the imine reductase enzymes may be codon optimized for optimal production from the host organism selected for expression. [0048] “Preferred, optimal, high codon usage bias codons” refers to codons that are used at higher frequency in the protein coding regions than other codons that code for the same amino acid. The preferred codons may be determined in relation to codon usage in a single gene, a set of genes of common function or origin, highly expressed genes, the codon frequency in the aggregate protein coding regions of the whole organism, codon frequency in the aggregate protein coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. A variety of methods are known for determining the codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in specific organisms, including multivariate analysis, for example, using cluster analysis or correspondence analysis, and the effective number of codons used in a gene (see GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, John Peden, University of Nottingham; McInerney, J. O, 1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res.222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables are available for a growing list of organisms (see for example, Wada et al., 1992, Nucleic Acids Res.20:2111-2118; Nakamura et al., 2000, Nucl. Acids Res.28:292; Duret, et al., supra; Henaut and Danchin, "Escherichia coli and Salmonella," 1996, Neidhardt, et al. Eds., ASM Press, Washington D.C., p.2047-2066. The data source for obtaining codon usage may rely on any available nucleotide sequence capable of coding for a protein. These data sets include nucleic acid sequences actually known to encode expressed proteins (e.g., complete protein coding sequences-CDS), expressed sequence tags (ESTS), or predicted coding regions of genomic sequences (see for example, Mount, D., Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Uberbacher, E. C., 1996, Methods Enzymol.266:259-281; Tiwari et al., 1997, Comput. Appl. Biosci.13:263-270). [0049] “Control sequence” as used herein refers to all sequences, which are necessary or advantageous for the expression of a polynucleotide and/or polypeptide as used in the present disclosure. Each control sequence may be native or foreign to the nucleic acid sequence encoding a polypeptide. Such control sequences include, but are not limited to, a leader, a promoter, a polyadenylation sequence, a pro-peptide sequence, a signal peptide sequence, and a transcription terminator. At a minimum, control sequences typically include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide. [0050] “Operably linked” as used herein refers to a configuration in which a control sequence is appropriately placed (e.g., in a functional relationship) at a position relative to a polynucleotide sequence or polypeptide sequence of interest such that the control sequence directs or regulates the expression of the sequence of interest. [0051] “Promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of a polynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. [0052] “Percentage of sequence identity,” “percent sequence identity,” “percentage homology,” or “percent homology” are used interchangeably herein to refer to values quantifying comparisons of the sequences of polynucleotides or polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (or gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage values may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math.2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol.48:443, by the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, J. Mol. Biol.215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison Wis.), using default parameters provided. [0053] “Reference sequence” refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length nucleic acid or polypeptide sequence. A reference sequence typically is at least 20 nucleotide or amino acid residue units in length but can also be the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity. “Comparison window” refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (or gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. [0054] “Substantial identity” or “substantially identical” refers to a polynucleotide or polypeptide sequence that has at least 70% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95 % sequence identity, or at least 99% sequence identity, as compared to a reference sequence over a comparison window of at least 20 nucleoside or amino acid residue positions, frequently over a window of at least 30-50 positions, wherein the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that includes deletions or additions which total 20 percent or less of the reference sequence over the window of comparison. [0055] “Corresponding to,” “reference to,” or “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of an engineered imine reductase, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned. [0056] “Isolated” as used herein in reference to a molecule means that the molecule (e.g., cannabinoid, polynucleotide, polypeptide) is substantially separated from other compounds that naturally accompany it, e.g., protein, lipids, and polynucleotides. The term embraces nucleic acids which have been removed or purified from their naturally occurring environment or expression system (e.g., host cell or in vitro synthesis). [0057] “Substantially pure” refers to a composition in which a desired molecule is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition) and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. [0058] “Immunoconjugate” refers to an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytokine, such as IL-2. [0059] “Treatment,” “treat” or “treating” refers to clinical intervention in an attempt to alter the natural course of a disorder in the individual being treated and can be performed either for prophylaxis or during the course of clinical pathology. Desired results of treatment can include, but are not limited to, preventing occurrence or recurrence of the disorder, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disorder, preventing metastasis, decreasing the rate of progression, amelioration or palliation of a disease state, and remission or improved prognosis. For example, treatment can include administration of a therapeutically effective amount of pharmaceutical formulation comprising an IL-2 mutant polypeptide to a subject to delay development or slow progression of a disease or condition mediated by IL-2R or a disease or condition in which IL-2R may play a role in the pathogenesis and/or progression. [0060] “Pharmaceutical formulation” refers to a preparation in a form that allows the biological activity of the active ingredient(s) to be effective, and which contain no additional components which are toxic to the subjects to which the formulation is administered. A pharmaceutical formulation may include one or more active agents. For example, a pharmaceutical formulation may include a mutation IL-2 polypeptide as the sole active agent of the formulation or may include a mutant IL-2 polypeptide and one or more additional active agents, such as e.g., an immune checkpoint inhibitor. [0061] “Pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to the subject to whom it is administered. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. [0062] “Therapeutically effective amount” refers to the amount of an active ingredient or agent (e.g., a pharmaceutical formulation) to achieve a desired therapeutic or prophylactic result, e.g., to treat or prevent a disease, disorder, or condition in a subject. In the case of an IL-2 mediated disease or condition, the therapeutically effective amount of the therapeutic agent is an amount that reduces, prevents, inhibits, and/or relieves to some extent one or more of the symptoms associated with the disease, disorder, or condition. For cancer therapy, efficacy in vivo can, for example, be measured by assessing the growth of a primary tumor, occurrence and/or growth of secondary tumor(s), occurrence and/or number of metastases, duration, severity, and/or recurrence of symptoms, the response rate (RR), duration of response, and/or quality of life. [0063] “Individual” or “subject” refers to a mammal, including but not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). [0064] IL-2 Receptors [0065] IL-2 signaling is mediated through binding to three different IL-2 receptor protein subunits: IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132). Immune cells express dimeric or trimeric IL-2 receptors. The dimeric receptor is expressed on cytotoxic CD8+ T cells and natural killer cells (NK), whereas the trimeric receptor is expressed predominantly on activated lymphocytes and CD4+ CD25+ FoxP3+ suppressive regulatory T cells (Treg) (see, Byman et al., J. Nat. Rev. Immunol.12, 180-190 (2012)). Effector T cells and NK cells in a resting state do not have CD25 on the cell surface and thus, are relatively insensitive to IL-2. Treg cells, however, express high levels of CD25, and thus, Treg proliferation is stimulated by IL-2. [0066] The trimeric receptor, IL-2Rαβγ, formed by the combination of IL-2Ra, IL-2Rβ, and IL- 2Rγ, is an IL-2 high affinity receptor with a K_D of about 10 pM. The dimeric receptor (IL-2Rβγ) is an intermediate affinity receptor with a K_D of about 1 nM. The monomeric IL-2Rα receptor is a low affinity IL-2 receptor. IL-2 signaling activity mediated by the receptors and their complexes also varies significantly. Generally, it has been found that IL-2Rβ and IL-2Rγ are critical for IL-2 signaling, while IL-2Rα (CD25) is not essential for signaling, but the presence of IL-2Rα enables high affinity IL-2 binding to the receptor complex (see e.g., Krieg et al., Proc Natl Acad Sci 107, 11906-11 (2010)). [0067] The amino acid sequence of the IL-2Rα subunit can be found at UniProt P01589 and is set forth herein as SEQ ID NO: 2 (the polynucleotide sequence encoding SEQ ID NO: 2 is included in the Sequence Listing as SEQ ID NO: 1). The amino acid sequence of the IL-2Rβ subunit can be found at UniProt P14784 and is set forth herein as SEQ ID NO: 4 (the polynucleotide sequence encoding SEQ ID NO: 4 is included in the Sequence Listing as SEQ ID NO: 3). The amino acid sequence of the IL-2Rγ subunit can be found at UniProt 31785 and is set forth herein as SEQ ID NO: 6 (the polynucleotide sequence encoding SEQ ID NO: 6 is included in the Sequence Listing as SEQ ID NO: 5). Table 1 below provides a summary description of the amino acid sequences of the various IL-2R polypeptides of the present disclosure, and their sequence identifiers. The sequences also are included in the accompanying Sequence Listing. [0068] TABLE 1: IL-2R protein subunits SEQ ID :

FPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQ VAVAGCVFLLISVLLLSGLTWQRRQRKSRRTI

[0069] Mutant IL-2 Polypeptides [0070] The present disclosure provides IL-2 polypeptides with mutations (relative to wild-type or C125S IL-2) that alter glycosylation, and thereby affect the functional properties of the polypeptides, including increasing expression titer, increasing solubility, and/or altering binding affinity to the IL-2 receptor complexes. The altered glycosylation and functional properties resulting from the mutations in the amino acid sequence relative to the parent IL-2 polypeptide (also referred to as amino acid substitutions or differences) result in IL-2 polypeptides with improved properties for use in pharmaceutical compositions for treatment of IL-2 mediated diseases, such as cancer and autoimmune disorders. [0071] The naturally occurring human IL-2 is the 153 amino acid polypeptide sequence (Uniprot: P60568; disclosed herein as SEQ ID NO: 8; the corresponding polynucleotide sequence encoding SEQ ID NO: 8 is included in the Sequence Listing as SEQ ID NO: 7) that includes a 20 amino acid N-terminal signal peptide: MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMP KKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI VEFLNRWITFCQSIISTLT (SEQ ID NO: 8) [0072] The structure of the IL-2 polypeptide includes four antiparallel and amphipathic α- helices, which form a quaternary structure essential for its function (see e.g., Smith, Science 240, 1169-76 (1988); Bazan, Science 257, 410-413 (1992)). The 153 amino acid precursor IL- 2 of SEQ ID NO: 8 is processed to remove the signal peptide resulting in the mature secretory IL-2 polypeptide of 133 amino acids of SEQ ID NO: 9 shown below: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 9) [0073] The mature IL-2 polypeptide of SEQ ID NO: 9 has been engineered for human pharmaceutical use by removing the cysteine residue at position C125, thereby reducing aggregation of the polypeptide. The C125S IL-2 mutant polypeptide, which is disclosed herein as SEQ ID NO: 10, is the active ingredient in aldesleukin, a drug which has been approved for use in humans in the treatment of cancers. APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLE EVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 10) [0074] The exemplary mutant IL-2 polypeptides with improved properties of the present disclosure are derived from this parent C125S IL-2 polypeptide of SEQ ID NO: 10. As described elsewhere herein, the mutant IL-2 polypeptides have been engineered with amino acid substitutions relative to C125S IL-2 of SEQ ID NO: 10 that provide new N-glycosylation sites on the expressed mutant IL-2 polypeptide. The engineering of N-glycosylation motifs in proteins is well-known in the art. Generally, the three amino acid sequence motifs, aspargine- X-serine (N-X-S) and asparagine-X-threonine (NXT), where X can be any amino acid except proline, provide result in a potential N-glycosylation site on the polypeptide. Depending on the sequence of the polypeptide, an N-glycosylation site can be introduced into a polypeptide by engineering amino acid substitutions at one, two, or three positions of an amino acid sequence. [0075] Table 2 below provides a summary of the sequences of exemplary mutant IL-2 polypeptides of the present disclosure that have been engineered with one or more N- glycosylation sites that are not present in the naturally occurring mature IL-2 polypeptide of SEQ ID NO: 9 or the C125S IL-2 polypeptide of SEQ ID NO: 10. The complete amino acid sequences and polynucleotide sequences encoding the polypeptides are provided in the accompanying Sequence Listing. [0076] TABLE 2: Exemplary mutant IL-2 polypeptides Amino acid SEQ differences relative ID :

_{P65N, E67S;} ^{APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFY} 14 MPKKATELKHLQCLEEELKNLSEVLNLAQSKNFHLRPRDLISNIN VIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT

K35N, Y45R, E95N, APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPNLTRMLTFKFR 32 K97T; MPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNIN VIVLNLTGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT

Y45R, R81N, P82A, APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFR 50 R83T, E95N, K97T; MPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLNATDLISNIN VIVLNLTGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT

Y45R, R81N, P82A, APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFR 68 R83S, E61N, L63S, MPKKATELKHLQCLENESKPLEEVLNLAQSKNFHLNASDLISNIS N90S, E95N, K97S; VIVLNLSGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT

R81N, P82A, R83S, APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFY 86 P65N, E67T; MPKKATELKHLQCLEEELKNLTEVLNLAQSKNFHLNASDLISNIN VIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT

R81N, P82A, R83T, APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFY 104 P65N, E67T, E61N, MPKKATELKHLQCLENESKNLTEVLNLAQSKNFHLNATDLISNIN L63S, E95N, K97S; VIVLNLSGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT

R81N, P82A, R83T, APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFY 122 E95N, K97T; MPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLNATDLISNIN VIVLNLTGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT

R81N, P82A, R83T, APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFY 140 E61N, L63S, E95N, MPKKATELKHLQCLENESKPLEEVLNLAQSKNFHLNATDLISNIS K97T, N90S; VIVLNLTGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT

[0078] As described elsewhere herein, including the Examples, the mutant IL-2 polypeptides of the present disclosure engineered with N-glycosylation sites exhibit surprising and advantageous technical effects relative to the parent C125S IL-2 polypeptide of SEQ ID NO: 10. Among the technical effects exhibited are increased IL-2 polypeptide expression titer during preparation in mammalian cell culture, increased solubility, and altered binding affinity for the trimeric and dimeric IL-2R complexes formed by the IL-2Rα subunit of SEQ ID NO: 2, IL- 2Rβ subunit of SEQ ID NO: 4, and the IL-2Rγ subunit of SEQ ID NO: 6. For example, certain glycosylation mutations disclosed herein reduce affinity of the mutant IL-2 for binding to IL-2Rα subunit but not for binding to dimeric IL-2Rβγ. [0079] It has been observed that engineering N-glycosylation mutations in a recombinant gene encoding a polypeptide can result in increased titer of the expressed polypeptide in mammalian cell culture systems. Additionally, it has been observed that engineering N-glycosylation mutations in a recombinant gene encoding a polypeptide can result in decreased aggregation and increased solubility of the resulting polypeptide as shown by an increased amount of monomeric form of polypeptide. In the mutant IL-2 polypeptides of the present disclosure, it has been found that the presence of certain N-glycosylation mutations alone or in combination result in an IL-2 polypeptide that exhibits increased expression titer in a mammalian cell culture system and increased solubility (measured in terms of % monomeric polypeptide). Exemplary sets of amino acid differences of the present disclosure that exhibit these functional improvements of increased expression titer and increased solubility are provided in Table 3 below. [0080] TABLE 3 Amino acid differences relative to C125S IL-2 Relative AA ID

(monomer %) R81N P82A R83T; ++ + 15

Y45R, R81N, P82A, R83S, E61N, L63S, N90S, +++ +++ 67 E95N, K97T; Y45R R81N P82A R83S E61N L63S N90S +++ +++ 68

- g y - yp p [0082] It also has been observed that engineering N-glycosylation mutations in a recombinant gene encoding an IL-2 polypeptide can result in a mutant IL-2 polypeptide that exhibits altered binding affinity for the different IL-2R receptors subunits, IL-2Rα, IL-2Rβ, and IL-2Rγ, in the monomeric, dimeric, and trimeric complex forms. As described elsewhere herein, in at least one embodiment, the mutant IL-2 polypeptide exhibits reduced affinity for IL-2Rα without little or no loss in binding affinity for IL-2β, IL-2γ, or the IL-2βγ dimer complex. It is believed that such mutant IL-2 polypeptide that exhibit reduced binding affinity for IL-2Rα can provide an improved therapeutic compound for treatment of cancers, e.g., due to reduced or no stimulation of immunosuppressive CD25+ cells. Additionally, is believed that mutant IL-2 polypeptides that exhibit reduced binding affinity for IL-2Rβγ with little or no reduction in binding affinity for IL-2Rα can provide an improved therapeutic compound for treatment of autoimmune disorders, e.g., due to the preferential stimulation of immunosuppressive CD25+ cells. Exemplary sets of amino acid differences of the present disclosure that exhibit selectively reduced binding affinity for IL- 2Rα, and/or IL-2Rβγ, are provided in Table 4 below. [0083] TABLE 4 Reduced Reduced AA Amino acid differences relative to C125S IL-2 IL-2Rα IL-2Rβγ SEQ O:

F42A, R81N, P82A, R83S; +++ 0 43 Y45R, R81N, P82A, R83T; +++ 0 44 Y45R R81N P82A R83S; +++ 0 45

R81N, P82A, R83T, E95N, K97S; 0 ++ 123 R81N, P82A, R83S, E95N, K97T; 0 ++ 124 R81N P82A R83S E95N K97S; 0 ++ 125

[0085] As described elsewhere herein, it is contemplated that the mutant IL-2 polypeptides of the present disclosure can be conjugated (or fused) to other polypeptides or proteins, including but not limited to, immunoglobulin molecules, antibody fragments, and/or other cytokines (e.g., IL-15). Such fusions can result in improved properties of the IL-2 polypeptide. For example, a mutant IL-2 polypeptide of the present disclosure (e.g., a polypeptide of Table 2) can be fused to an immunoglobulin Fc region polypeptide. Conjugation to an Fc region polypeptide can result in improved pharmacokinetic properties, such as half-life, for the whole fused molecule allowing for better pharmaceutical compositions for therapeutic uses. In at least one embodiment, the present disclosure provides a mutant IL-2 polypeptide fused to a polypeptide linker to a Fc polypeptide, such as the wild-type monomeric human IgG1 Fc lower hinge region polypeptide of SEQ ID NO: 146 as shown below: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEA LHNHYTQKSLSLSPGK (SEQ ID NO: 146). [0086] Certain variants of the wild-type human IgG1 Fc polypeptide of SEQ ID NO: 146 can be used in fusions with the mutant IL-2 polypeptides, including the “KK” variant of SEQ ID NO: 147 and the “DSDL” variant of SEQ ID NO: 148, both of which are shown below: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYG STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQEGNVFSCSVLHEA LHNHYTQKSLSLSPGK (SEQ ID NO: 147) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYG STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLSSDLTVDKSRWQEGNVFSCSVLHEA LHNHYTQKSLSLSPGK (SEQ ID NO: 148). [0087] Although only three human IgG1 Fc lower hinge region polypeptides are described above, it is contemplated that mutant IL-2 polypeptide fusions of the present disclosure can be prepared using other Fc region fragments and variants that are known to provide improved properties when conjugated to polypeptides, such as cytokines, for therapeutic use. For example, an Fc polypeptide variant can be used that removes effector function, such as an Fc region with the amino acid substitutions L234A/L235A (“LALA”) (Woodle, E. Steve et al., Transplantation, 68(5): 608-616 (1999)). Other effectorless Fc region mutations are well known in the art, such as L234A/L235A/P329G (“LALAPG”) (see e.g., Schlothauer, T. et al., “Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector functions”, Protein Eng. Des. Sel., 29(10): 457– 466 (2016)), or when the Fc is of isotype IgG2 or IgG4, the amino acid substitutions S228P and/or L235E. [0088] In at least one embodiment, it is contemplated that the mutant IL-2 polypeptides of the present disclosure can be conjugated to other polypeptides or proteins (e.g., Fc polypeptide) via a linker. Any of the wide range of synthetic chain-like molecules useful as linkers between biomolecules that are known in the art can be used to fuse the mutant IL-2 polypeptides to other polypeptides. In at least one embodiment, a polypeptide linker may be used. Such polypeptide linkers comprise a chain of amino acids with each end of the chain covalently attached to one of the two different polypeptides, and thereby functioning to conjugate or fuse them. Typically, such polypeptide linkers comprise a chain of 5 to 30 amino acids. A wide range of polypeptide linkers are known in the art and can be used in the mutant IL-2 polypeptide fusions of the present disclosure. Exemplary polypeptide linkers useful in the IL-2 fusions of the present disclosure include, but are not limited to: (GGGGS)₁ (SEQ ID NO: 149), (GGGGS)2 (SEQ ID NO: 150), (GGGGS)3 (SEQ ID NO: 151), (GGGGS)4 (SEQ ID NO: 152), (GRPGS)₂ (SEQ ID NO: 153), (GRPGS)₄ (SEQ ID NO: 154), (GGGGS)₃GGG (SEQ ID NO: 155). Additional polypeptides are known in the art that may be used include: (GGGGS)_n (n is 1- 10), (SSSSG)_n (n is 1-10), (GGGG)(SGGGG)_n (n is 1-10), (EAAAK)_n (n is 1-10), (XP)_n (n is 1- 10), and ENLYFQ(-G/S). [0089] The present disclosure exemplifies fusions mutant IL-10 polypeptides (e.g., SEQ ID NO: 11-145) fused to a monomeric Fc polypeptide (e.g., SEQ ID NO: 146, 147, or 148) wherein the monomeric Fc polypeptide is conjugated from its C-terminus via a polypeptide linker (e.g., SEQ ID NO: 149-155) to the N-terminus of the mutant IL-10 polypeptide. However, it is contemplated and will be understood by one of ordinary skill in the art that other orientations of the mutant IL-2 polypeptide fusions of the present disclosure can be used. For example, in at least one embodiment, a mutant IL-2 polypeptide of the present disclosure can be conjugated to another polypeptide or protein (such as a monomeric Fc polypeptide) either via its N- terminus or its C-terminus, optionally, via a linker (e.g., a polypeptide linker). [0090] In addition to mutant IL-2 polypeptides fused to a monomeric Fc polypeptide, the present disclosure also contemplates a mutant IL-2 fused to an immunoglobulin molecule with specific antigen-binding capability, such as an antibody, or an antibody fragment (e.g., a Fab molecule, scFv, or VHH). In at least one embodiment, the fused antibody or antibody fragment provides a specific antigen binding affinity that targets the fusion to a cancer cell or other cell in a tumor environment. In at least one embodiment, the specific antigen binding affinity targets the cytokine molecule CD8, and the antibody is a VHH antibody. Exemplary anti-CD8 VHH antibodies are disclosed in e.g., US Provisional Patent Application No.63/477,529, filed December 28, 2022, which is hereby incorporated by reference herein. In at least one embodiment, the specific antigen binding affinity targets the immune checkpoint inhibitor molecule, PD-1, and the antibody is a VHH antibody. Exemplary anti-PD-1 VHH antibodies are disclosed in e.g., US Provisional Patent Application No.63/488,176, filed March 3, 2023, which is hereby incorporated by reference herein. [0091] Recombinant Methods and Compositions [0092] The mutant IL-2 polypeptide of the present disclosure can be produced using recombinant methods and materials well-known in the art of polypeptide and protein production. In some embodiments, the present disclosure provides an isolated nucleic acid encoding a mutant IL-2 polypeptide. The nucleic acid can encode an amino acid sequence comprising the IL-2 polypeptide alone or as a fusion with another polypeptide, such as a monomeric Fc polypeptide. In some embodiments, one or more vectors (e.g., expression vectors) comprising nucleic acid sequences encoding a mutant IL-2 polypeptide of the present disclosure are provided. In some embodiments, a host cell comprising nucleic acid sequences encoding a mutant IL-2 polypeptide of the present disclosure are provided. In one embodiment, the host cell has been transformed with a vector comprising a nucleic acid that encodes an amino acid sequence comprising the mutant IL-2 polypeptide. In some embodiments, the host cell used is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell, or a lymphoid cell (e.g., Y0, NS0, Sp20). [0093] In at least one embodiment, a method of making a mutant IL-2 polypeptide is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the polypeptide, as provided above, under conditions suitable for expression of the polypeptide, and optionally recovering the polypeptide from the host cell (or host cell culture medium). Briefly, recombinant production of a mutant IL-2 polypeptide is carried out by synthesizing or isolating a nucleic acid encoding the mutant IL-2 polypeptide (e.g., as described herein) and inserting this nucleic acid into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids are readily isolated and sequenced using conventional procedures well-known in the art (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding IL-2. Suitable host cells and culturing methods for cloning or expressing the IL-2 polypeptide-encoding vectors are well-known in the art and include prokaryotic or eukaryotic cells. Typically, after expression, the mutant IL-2 polypeptide may be isolated from cell paste in a soluble fraction and further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for the vectors. [0094] Examples of suitable mammalian host cell lines useful for the production of the mutant IL-2 polypeptides of the present disclosure include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (see e.g., Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); myeloma cell lines such as Y0, NS0 and Sp2/0; monkey kidney CVl line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CVl); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TR1 cells (see e.g., in Mather et al., Annals N Y. Acad. Sci.383:44-68 (1982) and US 6,235,498); Medical Research Council 5 (MRC 5) cells (such as e.g., those available from ATCC and also referred to as CCL-171); and Foreskin 4 (FS-4) cells (see e.g., in Vilcek et al. Ann. N. Y. Acad. Sci.284:703-710 (1977), Gardner & Vilcek. J. Gen. Virol.44:161-168 (1979), and Pang et al. Proc. Natl. Acad. Sci. U.S.A.77:5341- 5345 (1980)). [0095] Pharmaceutical Compositions and Formulations of Mutant IL-2 Polypeptides [0096] The present disclosure also provides pharmaceutical compositions and pharmaceutical formulations comprising a mutant IL-2 polypeptide. In some embodiments, the present disclosure provides a pharmaceutical formulation comprising a mutant IL-2 polypeptide as described herein and a pharmaceutically acceptable carrier. In some embodiments, the mutant IL-2 polypeptide is the sole active agent of the pharmaceutical composition. Such pharmaceutical formulations can be prepared by mixing a mutant IL-2 polypeptide, having the desired degree of purity, with one or more pharmaceutically acceptable carriers. Typically, such mutant IL-2 polypeptide formulations can be prepared as an aqueous solution or as a lyophilized formulation. [0097] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed. A wide range of such pharmaceutically acceptable carriers are well-known in the art (see e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Exemplary pharmaceutically acceptable carriers useful in the formulations of the present disclosure can include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m- cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn- protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). [0098] Pharmaceutically acceptable carriers useful in the formulations of the present disclosure can also include interstitial drug dispersion agents, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP) (see e.g., US Pat. Publ. Nos.2005/0260186 and 2006/0104968), such as human soluble PH-20 hyaluronidase glycoproteins (e.g., rHuPH20 or HYLENEX^®, Baxter International, Inc.). [0099] It is also contemplated that the formulations disclosed herein may contain active ingredients in addition to the mutant IL-2 polypeptide, as necessary for the particular indication being treated in the subject to whom the formulation is administered. Preferably, any additional active ingredient has activity complementary to that of the IL-2 activity and the activities do not adversely affect each other. [0100] As disclosed elsewhere herein, including the Examples, it has been shown that the mutant IL-2 polypeptide of the present disclosure can be used as a fusion to a Fc polypeptide to provide improved therapeutic effect in treating autoimmune disorders and/or cancers. [0101] As described elsewhere herein, in some embodiments the present disclosure provides pharmaceutical composition or formulation for use in a therapeutic method comprising a mutant IL-2 polypeptide conjugated to another polypeptide or protein, such as a half-life extending Fc polypeptide. In some embodiments, this pharmaceutical composition or formulation can comprise a mutant IL-2 polypeptide covalently fused to a Fc polypeptide through a linker, such as a polypeptide linker of amino acid sequence of SEQ ID NO: 149-155. Examples demonstrating such mutant IL-2 polypeptide fusions to monomeric Fc polypeptides of SEQ ID NO: 146, 147, or 148, and their use in pharmaceutical compositions for reducing treatment of cancer or autoimmune disorders as described elsewhere herein. [0102] In some embodiments, the pharmaceutical composition can comprise a mutant IL-2 polypeptide of the present disclosure and an additional active agent for treatment of cancer, such as an immune checkpoint inhibitor. Checkpoint inhibitors useful in such embodiments include, but are not limited to, an antibody comprising a specificity for an antigen that is an immune checkpoint molecule, such as, PD1, LAG3, CTLA-4, A2AR, TIM-3, BTLA, CD276, CD328, VTCN1, KIR, NOX2, VISTA, OX40, CD27, CD28, CD40, CD122, CD137, GITR, or ICOS. [0103] Generally, pharmaceutical composition active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). [0104] In some embodiments, the formulation of a mutant IL-2 polypeptide can be a sustained- release preparation of the polypeptide, and/or other active ingredients. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the mutant IL-2 polypeptide, which matrices are in the form of shaped articles, e.g., films, or microcapsules. [0105] Typically, the formulations of the present disclosure to be administered to a subject are sterile. Sterile formulations may be readily prepared using well-known techniques, e.g., by filtration through sterile filtration membranes. [0106] Uses and Methods of Treatment [0107] It is contemplated that any of the compositions or formulations comprising a mutant IL-2 polypeptide of the present disclosure can be used for any methods or uses, such as in therapeutic methods, that utilize the ability of the polypeptides to specifically bind to IL-2 receptor protein. The binding of IL-2 to the IL-2 receptor protein as it is expressed on different cells mediates different immune responses. IL-2 binding can stimulate an immune response, such as T cell proliferation and differentiation, cytotoxic T lymphocyte (CTL) production, B cell proliferation and differentiation, immunoglobulin synthesis, and production, and proliferation and activation of NK cells. The IL-2 polypeptide, such as the C125S IL-2 (Proleukin), has been approved as an immunotherapeutic agent for the treatment of cancer and chronic viral infection. The IL-2 polypeptide, however, can also promote the activation and proliferation of immunosuppressive CD4+ CD25+ Treg cells resulting in immunosuppression. Accordingly, there are a range of diseases, disorders, and conditions that can potentially be treated by altering the immune regulatory and/or immune signaling activity of IL-2 binding to IL-2 receptor proteins, particularly, the effect of IL-2 on tumor progression. Diseases, disorders, and conditions include, but are not limited to, cancers, including but not limited to colon cancer, pancreatic cancer, ovarian cancer, liver cancer, renal cancer, breast cancer, lung cancer, gastric cancer, head and neck cancer, or oral cancer. It is contemplated that any of the compositions or formulations comprising a mutant IL-2 polypeptide of the present disclosure, including the mutant IL-2 polypeptide fusions with monomeric Fc polypeptide, can be used in a method or use for the treatment of any of the above-listed cancers. Thus, in at least one embodiment, the present disclosure provides a method of treating cancer in a subject, wherein the method comprises administering to the subject in need thereof a therapeutically effective amount of a mutant IL-2 polypeptide of the present disclosure or administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising a mutant IL-2 polypeptide of the present disclosure and a pharmaceutically acceptable carrier. [0108] As disclosed herein, including in the Examples below, the mutant IL-2 polypeptides of the present disclosure have the ability to specifically bind to IL-2 receptors proteins differentially, and thereby differentially alter the immune signaling pathways mediated by IL-2 binding to IL-2 receptor proteins expressed on different cells. Accordingly, in some embodiments, the present disclosure provides a method of treating an IL-2 mediated disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of a mutant IL-2 polypeptide of the present disclosure or administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a mutant IL-2 polypeptide of the present disclosure and a pharmaceutically acceptable carrier. Similarly, in some embodiments, the present disclosure provides a method of treating a disease mediated by IL-2 binding to IL-2 receptor proteins expressed on cells in a subject, the method comprising administering to the subject, the method comprising administering to the subject a therapeutically effective amount of a mutant IL-2 polypeptide of the present disclosure or administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a mutant IL-2 polypeptide of the present disclosure and a pharmaceutically acceptable carrier. [0109] Administration of the mutant IL-2 polypeptide, composition, or pharmaceutical formulation in accordance with the method of treatment provides an antibody-induced therapeutic effect that protects the subject from and/or treats the progression of an IL-2- mediated disease in a subject. In some embodiments, the method of treatment can further comprise administration of one or more additional therapeutic agents or treatments known to those of skill in the art to prevent and/or treat the IL-2-mediated disease or condition. Such methods comprising administration of one or more additional agents can encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the mutant IL-2 polypeptide composition or formulation can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent. [0110] In some embodiments of the methods of treatment of the present disclosure, the mutant IL-2 polypeptide or pharmaceutical formulation comprising a mutant IL-2 polypeptide is administered to a subject by any mode of administration that delivers the agent systemically, or to a desired target tissue. Systemic administration generally refers to any mode of administration of the antibody into a subject at a site other than directly into the desired target site, tissue, or organ, such that the antibody or formulation thereof enters the subject's circulatory system and, thus, is subject to metabolism and other like processes. Accordingly, modes of administration useful in the methods of treatment of the present disclosure can include, but are not limited to, injection, infusion, instillation, and inhalation. Administration by injection can include intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. [0111] In at least one embodiment, a pharmaceutical formulation of the mutant IL-2 polypeptide is formulated such that the IL-2 is protected from inactivation in the gut. Accordingly, the method of treatments can comprise oral administration of the formulation. [0112] In at least one embodiment, use of the compositions or formulations comprising a mutant IL-2 polypeptide of the present disclosure as a medicament are also provided. Additionally, in some embodiments, the present disclosure also provides for the use of a composition or a formulation comprising a mutant IL-2 polypeptide in the manufacture or preparation of a medicament, particularly a medicament for treating an IL-2 mediated disease. In a further embodiment, the medicament is for use in a method for treating disease comprising administering to an individual having a disease an effective amount of the medicament. In certain embodiments, the medicament further comprises an effective amount of at least one additional therapeutic agent, or treatment. [0113] As disclosed elsewhere herein, it is also contemplated that additional therapeutic agents or treatments that can be used in such medicaments with a mutant IL-2 polypeptide of the present disclosure. Generally, it is contemplated that the mutant IL-2 polypeptides of the present disclosure can be used with any therapeutic agent or treatment, such as a therapeutic antibody, that specifically targets a cell surface receptor on an immune cell, a tumor cell, or a myeloid cell. In at least one embodiment, the additional therapeutic agent can include, but is not limited to a therapeutic antibody that specifically binds to an immune checkpoint molecule, such as PD1, PD-L1, LAG3, CTLA-4, A2AR, TIM-3, BTLA, CD276, CD328, VTCN1, IDO, KIR, NOX2, VISTA, OX40, CD27, CD28, CD40, CD122, CD137, GITR, ICOS. [0114] In a further embodiment, the medicament is for use in treating an IL-2-mediated disease, such as a cancer, in a subject comprising administering to the subject an amount effective of the medicament to treat, inhibit or prevent the IL-2-mediated disease. The appropriate dosage of the mutant IL-2 polypeptide contained in the compositions and formulations of the present disclosure (when used alone or in combination with one or more other additional therapeutic agents) will depend on the specific disease or condition being treated, the severity and course of the disease, whether the dosage is administered for preventive or therapeutic purposes, the previous therapy administered to the patient, the patient's clinical history and response to the mutant IL-2 polypeptide composition, and the discretion of the attending physician. It is contemplated that the mutant IL-2 polypeptide included in the compositions and formulations described herein, can be suitably administered to the patient at one time, or over a series of treatments. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein. [0115] Depending on the type and severity of the disease, about 1 µg/kg to 30 mg/kg of a mutant IL-2 polypeptide in a formulation of the present disclosure is an initial candidate dosage for administration to a human subject, whether, for example, by one or more separate administrations, or by continuous infusion. Generally, the administered dosage of the antibody would be in the range from about 0.05 mg/kg to about 10 mg/kg. In some embodiments, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg, or 10 mg/kg (or any combination thereof) may be administered to a patient. [0116] Dosage administration can be maintained over several days or longer, depending on the condition of the subject, for example, administration can continue until the IL-2-mediated disease is sufficiently treated, as determined by methods known in the art. In some embodiments, an initial higher loading dose may be administered, followed by one or more lower doses. However, other dosage regimens may be useful. The progress of the therapeutic effect of dosage administration can be monitored by conventional techniques and assays. Accordingly, in some embodiments of the methods of the present disclosure, the administration of the mutant IL-2 polypeptide comprises a daily dosage from about 0.05 mg/kg to about 50 mg/kg, at least about 0.5 mg/kg to about 30 mg/kg, or at least about 1 mg/kg to about 25 mg/kg. In some embodiments, the dosage of mutant IL-2 polypeptide comprises a daily dosage of at least about 0.1 mg/kg, at least about 1 mg/kg, at least about 5 mg/kg, at least about 10 mg/kg, at least about 20 mg/kg, at least about 30 mg/kg, at least about 40 mg/kg, or at least about 50 mg/kg. EXAMPLES [0117] Various features and embodiments of the disclosure are illustrated in the following representative examples, which are intended to be illustrative, and not limiting. Those skilled in the art will readily appreciate that the specific examples are only illustrative of the invention as described more fully in the claims which follow thereafter. Every embodiment and feature described in the application should be understood to be interchangeable and combinable with every embodiment contained within. Example 1: Preparation of IL-2 Glycosylation Mutant Polypeptides [0118] This example illustrates the preparation and screening of mutant IL-2 polypeptides with amino acid changes that alter glycosylation and thereby alter other functional properties, including expression titer, solubility, and binding affinity for IL-2Rα and IL-2Rβγ. [0119] Materials and Methods [0120] A. Preparation of mutant IL-2 genes: Gene fragments encoding mutant IL-2 polypeptides were reverse-transcribed and then synthesized by Integrated DNA Technologies, Inc. as gene fragments (gblocks or eblocks). These gene fragments were then cloned into expression vector as described below. [0121] B. Preparation of mutant IL-2 expression constructs and generating mutant polypeptides: Expression vector pcDNA3.1(+) (Thermo Fisher Scientific) was digested with both PstI and XbaI restriction enzymes according to the protocol provided by New England Biolabs, Inc. and purified by agarose gel and dissolved in TE buffer (10 mM Tris, pH 8.0 and 1 mM EDTA). Synthesized DNA fragments encoding mutant IL-2 polypeptides were dissolved into water. Equal volume of vector and inserts were added into 2x Gibson assembly master mix (New England Biolabs), with 1:3 molar ratio and incubated at 50^oC for 1-2 hours. Subsequently, the ligated DNA were transformed into DH5α E. coli competent cells (New England Biolabs). The resulting plasmid DNAs were sequenced according to standard molecular biology techniques. Correct plasmid DNAs were prepared using the PureLink™ Hipure Maxiprep Kit from Thermo Fisher Scientific (Catalog number K210006) to recover sterile, salt-free, and supercoiled DNA. [0122] Results: Table 2 (above) lists the amino acid sequences of 144 exemplary mutant IL-2 polypeptides prepared as described above along with their specific sets of mutations (or amino acid differences) relative to the parent C125S IL-2 polypeptide of SEQ ID NO: 10. [0123] C. Expression of IL-2 fusion proteins in Chinese hamster ovary (CHO) cells: [0124] ExpiCHO cells (Thermo Fisher Scientific) were maintained at 37 °C, 5% CO₂ and 130 rpm in 60 mL of ExpiCHO expression medium in Erlenmeyer flasks. Transfection was performed according to instructions provided with the ExpiCHO™ Expression System Kit (ThermoFisher – Catalog number A29133). Briefly, when cells reached Viable Cell Density (VCD) of 6 × 10⁶ cells/mL and doubling time of 18–20 h, 1 μg plasmid DNA per 10⁶ cells and ExpiFectamine CHO was mixed by repeated inversions, diluted with cold OptiPRO serum free medium, and finally complexed to the diluted plasmid DNA at room temperature. After 5 min, the mix was added drop by drop to the cell culture at room temperature. After transfection, cells were cultured at 200 RPM for 24-well plate or 130 RPM for 25 mL or large flasks. After 7-12 days post transfection, culture media were centrifuged at 25°C and 500×g for 5 min to pellet cells and then at 4°C and 4500×g for 30 min. The clarified supernatant was filtered by 0.45 μm filtration and fusion proteins were purified with protein A affinity column chromatography. [0125] D. Assay of relative expression titer level: Measurement of expressed protein titer levels was performed on an Agilent 1100 HPLC system (Santa Clara, California) coupled with POEOSTM A 20 µm column (Catalog number: 2100100, Thermo Fisher Scientific). The equilibrium buffer was 1X PBS (Buffer A) and the elution buffer was 0.1% HCl with 150 mM NaCl (Buffer B). In a typical HPLC run, the column was washed with 6 column volume (CV) of 1X PBS after sample injection. The binding protein was eluted with a step elution with 10 CV of elution buffer and the UV signal at 280 nm wavelength was recorded. Before sample measurement, a standard curve was generated by injecting human IgG reference of known amount. For sample titer measurements, 40 µl of culture soup was injected and the titer was calculated from the area of the elution peak and calculated with the standard curve of human IgG. [0126] Results: Table 5 below summarizes expression titer levels measured for the different mutant IL-2 polypeptide Fc fusion constructs. These results demonstrate that the genes encoding the mutant IL-2 polypeptides engineered with the sets of glycosylation mutations listed below (see also relative values listed in Table 3) provide substantially increased expression titer relative to C125S IL-2 in a mammalian expression system. [0127] TABLE 5 IL-2 Fc AA differences in IL-2 polypeptide Titer Titer Construct monomer relative to C125S IL-2 (SEQ ID NO: 10) (mg/l) (fold)

p214 DSDL K35N+Y45R (control) 240.9 1.00

[0129] The association and dissociation constants for the binding kinetics of the mutant IL-2 polypeptides to its receptors were measured by Biolayer interferometry (BLI) with Gator Prime instrument (Gator Bio, Inc.). The binding kinetics was measured on at 30°C and analyzed with the GatorOne analysis software. Streptavidin (SA) sensor probes was used to capture the biotinylated IL-2Rα protein (Acro Biosystems, Inc.; cat. #ILA-H82E6) or the biotinylated IL-2Rβγ heterodimer protein (Acro Biosystems, Inc.; cat. #ILG-H82F3). The samples were prepared using purified protein (confirmed single peak by SEC) in Tris buffer. The streptavidin sensor was set up as follows: Baseline: 1xKB buffer; Load: biotinylated IL-2Rα or IL-2Rβγ from Acro Biosystems,Inc.; Association: purified protein; Dissociation: 1xKB buffer. The sensor probes were dipped into serial dilutions of the parent C125S IL-2 polypeptide or the mutant IL-2 polypeptides. A well with buffer only was set as a reference well for subtracting background during data processing. Data were fitted with a 1:1 Langmuir model for association and dissociation using Rmax linked global fitting for each cytokine-receptor binding. [0130] Results: As shown by the BLI results plotted in FIGS.1A, 1B, 1C, 1G, and 1H, the exemplary mutant IL-2 polypeptide Fc fusion constructs p132 (K35N, Y45R, C125S) (FIG.1B), p115 (K35N, F42A, C125S) (FIG.1C), and p151 (F42A, Y45A, L72G, C125S) (FIG.1H), exhibited reduced binding to IL-2Rα relative to the control p123 (C125S IL2) construct (FIG.1A, and FIG.1G). For comparison, the results for BLI measured binding to IL-2Rβγ are also shown in FIGS.1D, 1E, 1F, 1I, and 1J as follows: control p132 (C125 IL2) construct (FIG.1D and FIG. 1I) relative to the Fc fusion constructs p132 (K35N, Y45R, C125S) (FIG.1E), p115 (K35N, F42A, C125S) (FIG.1F), and p151 (F42A, Y45A, L72G, C125S) (FIG.1J). [0131] Table 4 (above) also summarizes exemplary results showing that IL-2Rα subunit binding affinity is substantially reduced for many of the mutant IL-2 polypeptides (indicated by “+++”) relative to affinity observed for the parent C125S IL-2 polypeptide. Additionally, the results in Table 4 show that many of these same mutant IL-2 polypeptides exhibit less or no reduction in IL-2Rβγ binding affinity relative to the parent C125S IL-2 polypeptide. This differential reduction in IL-2Rα binding indicates that a mutant IL-2 polypeptide with that combination of amino acid differences will exhibit higher differential binding and stimulation of cells expressing IL-2Rβγ, such as cytotoxic CD8+ T cells and natural killer cells (NK), and therefore will be better suited for treatment of cancer. [0132] Table 4 also lists results showing that IL-2Rα binding is unchanged but IL-2Rβγ binding affinity is substantially reduced for many of the mutant IL-2 polypeptides. This differential reduction in IL-2Rβγ binding indicates that a mutant IL-2 polypeptide with that combination of amino acid differences will exhibit higher differential binding and stimulation of cells expressing IL-2Rαβγ, such as activated lymphocytes and CD4+ CD25+ FoxP3+ suppressive regulatory T cells (Treg), and therefore will be immunosuppressive and better suited for treatment of autoimmune disorders. [0133] F. Preparation of monomeric Fc conjugates of IL-2 mutants: [0134] Gene fragments encoding monomeric Fc and mutant IL-2 polypeptides were reverse- transcribed and then synthesized by Integrated DNA Technologies, Inc. as gene fragments (gblocks or eblocks). These fragments were then cloned into expression vector as described below. Expression vector pcDNA3.1(+) was digested with both PstI and XbaI restriction enzymes (New England Biolabs, Inc.) and purified by agarose gel and dissolved in TE buffer (10 mM Tris, pH 8.0 and 1 mM EDTA). DNA fragments encoding monomeric Fc and mutant IL- 2 polypeptides that were synthesized were dissolved into water. Equal volume of Vector and inserts were added into 2x Gibson assembly master mix (New England Biolabs), with 1:3 molar ratio and incubated at 50^oC for 1-2 hours. Subsequently, the ligated DNA were transformed into E coli competent cells (New England Biolabs). Resulting plasmid DNAs were sequenced according to standard molecular biology techniques. Correct plasmid DNAs were prepared using the PureLink™ Hipure Maxiprep Kit from Thermo Fisher Scientific (Catalog number K210006) to recover sterile, salt-free, and supercoiled DNA. [0135] G. Expression of mutant IL-2 polypeptide fusions with monomeric Fc in Chinese hamster ovary (CHO) cells: ExpiCHO cells (Thermo Fisher Scientific) were maintained at 37 °C, 5% CO₂ and 130 rpm in 60 mL of ExpiCHO expression medium using Erlenmeyer flasks. Transfection was performed according to instructions provided with the ExpiCHO™ Expression System Kit (Thermo Fisher Scientific– Catalog number A29133). Briefly, when cells reached Viable Cell Density (VCD) of 6 × 10⁶ cells/mL and doubling time of 18–20 h, 1 μg plasmid DNA per 10⁶ cells and ExpiFectamine CHO was mixed by repeated inversions, diluted with cold OptiPRO serum free medium, and finally complexed to the diluted plasmid DNA at room temperature. After 5 min the mix was added drop by drop to the cell culture at room temperature. After transfection, cells were cultured at 200 RPM for 24-well plate or 130 RPM for 25 mL or large flasks. After 7-12 days post transfection, culture media were centrifuged at 25°C and 500×g for 5 min to pellet cells and then at 4 °C and 4500×g for 30 min. The clarified supernatant was filtered by 0.45 μm filtration and fusion proteins were purified with protein A affinity column chromatography. [0136] H. Assay for relative solubility mutant IL-2 polypeptide fusions with monomeric Fc: Protein relative solubility (turbidity) assay was performed on an Infinite M Plex multi-mode plate reader (Tecan Systems, Inc.) using a Costar half area 96-well assay plate (Corning).0.1 mL of protein stock solution at 1 mg/mL concentration was added to plate wells, and the solution was adjusted to the designed pH using 1 M Tris. The adsorption at 340 nm wavelength (OD340) of the solution was measured. The first OD₃₄₀ nm measured right after pH adjustment was set as T=0. Time-course OD₃₄₀ was determined at 15 min, 30 min, 1h, and 2h. [0137] Results: Table 3 lists the relative solubility, in terms of % monomer measured, for the different mutant IL-2 polypeptides. These results demonstrate that the mutant IL-2 polypeptides engineered with the certain sets of glycosylation mutations listed in Table 3, when fused to monomeric Fc, also provide substantially increased solubility relative to C125S IL-2. [0138] J. Size Exclusion Chromatography: Size exclusion chromatography (SEC-HPLC) analysis was performed on an Agilent 1100 HPLC system (Santa Clara, California) with 1X PBS as a running buffer. Samples were injected into a prepacked Superose 12-300 column (Cytiva) which was equilibrated with 1 X PBS buffer. The flow rate was 0.65 mL/min, and the total running time was 40 min. [0139] Results: FIGS.2A-2L shows exemplary SEC-HPLC profiles for the control p123 (C125S IL2) construct (FIG.2A and FIG.2B) and the exemplary mutant IL-2 polypeptide Fc fusions as follows: p296 (FIG.2C), p307 (FIG.2D), p406 (FIG.2E), p297 (FIG.2F), p300 (FIG. 2G), p308 (FIG.2H), p214 (FIG.2I), p310 (FIG.2J), p411 (FIG.2K), and p298 (FIG.2L). [0140] K. IL-2 reporter assay with HEK Blue cells: IL-2 activity was measured with HEK Blue IL-2 cells. In this cell system, the HEK293 cells stably expressing IL-2 receptor subunits (CD25 for IL2Rα, CD122/CD132 for IL2Rβγ and STAT5-inducible SEAP reporter. During the assay, cell suspension above 90% viability were prepared. Test articles at the concentration indicated in each experiment were prepared by serial dilution in DMEM+10% heat inactivated FBS, added to cell suspension at 50K/well in a flat bottom 96-well plate, incubated at 37C for 20-24 hours. Next day, QuantI-Blue solution was prepared following the instructions from the Invivogen.20 μl of induced HEK Blue cell supernatant per well were added to a flat-bottom 96- well plate, then 100 μl of resuspended QUANTI-Blue Solution was added to each well, and the plate was incubated at 37C for 1-3 hrs. SEAP levels were determined by reading absorbance at 630 nm (OD₆₃₀) using spectrophotometer. [0141] Results: FIGS.3A-3N show plots of exemplary CD25+ and CD25- binding curves and EC₅₀ values measured in the HEK Blue IL-2 reporter assay for the p123 C125S IL2 fusion control and exemplary mutant IL-2 polypeptide Fc fusions as follows: p124, p132, and p167 (FIG.3A, FIG.3B); p296, p300, and p214 (FIG.3C, FIG.3D); p123, p307, and p214 (FIG.3E, FIG.3F); p123, p296, and p308 (FIG.3G, FIG.3H); p123, p310, and p214 (FIG.3I, FIG.3J); p296, p297, p298, and p214 (FIG.3K, FIG.3L); p411, p214, p123, p406, and p214 (FIG.3M, FIG.3N). [0142] While the foregoing disclosure of the present invention has been described in some detail by way of example and illustration for purposes of clarity and understanding, this disclosure including the examples, descriptions, and embodiments described herein are for illustrative purposes, are intended to be exemplary, and should not be construed as limiting the present disclosure. It will be clear to one skilled in the art that various modifications or changes to the examples, descriptions, and embodiments described herein can be made and are to be included within the spirit and purview of this disclosure and the appended claims. Further, one of skill in the art will recognize a number of equivalent methods and procedure to those described herein. All such equivalents are to be understood to be within the scope of the present disclosure and are covered by the appended claims. [0143] Additional embodiments of the invention are set forth in the following claims. [0144] The disclosures of all publications, patent applications, patents, or other documents mentioned herein are expressly incorporated by reference in their entirety for all purposes to the same extent as if each such individual publication, patent, patent application or other document were individually specifically indicated to be incorporated by reference herein in its entirety for all purposes and were set forth in its entirety herein. In case of conflict, the present specification, including specified terms, will control.

Claims

CLAIMS What is claimed is: 1. A polypeptide that specifically binds IL-2 receptor, wherein the polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 10 (C125 IL-2) and one or more amino acid differences relative to SEQ ID NO: 10 selected from: E61N, and L63T; E61N, and L63S; P65N d E67T

2. The polypeptide of claim 1, wherein the polypeptide further comprises one or more amino differences selected from: K35N, F42A, Y45R, N88D, D109N, and Q126T. 3. The polypeptide of claim 2, wherein the polypeptide comprises a combination of amino acid differences selected from: K35N, P65N, E67T; K35N P65N E67S;

Y45R, R81N, P82A, R83S; Y45R, R81N, P82A, R83T, E61N, L63T; Y45R R81N P82A R83S E61N L63S;

4. The polypeptide of claim 1, wherein the polypeptide comprises a combination of amino acid differences selected from: R81N, P82X, and R83T, wherein X is any amino acid other than P;

R81N, P82A, R83T, P65N, E67T, E61N, L63T; R81N, P82A, R83T, P65N, E67S, E61N, L63S; R81N P82A R83T P65N E67S E61N L63T;

R81N, P82A, R83T, E61N, L63S, E95N, K97T, N90S; R81N, P82A, R83T, E61N, L63S, E95N, K97S, N90S; R81N P82A R83S E61N L63T E95N K97T N90S;

5. The polypeptide of claim 1, wherein the polypeptide comprises a sequence selected from SEQ ID NO: 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, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, and 145. 6. The polypeptide of claim 1, wherein the polypeptide is fused to a monomeric or a dimeric Fc polypeptide; optionally, wherein the Fc polypeptide is a monomeric Fc polypeptide. 7. The polypeptide of claim 6, wherein the monomeric or dimeric Fc polypeptide comprises an amino acid sequence selected from SEQ ID NO: 146, 147, and 148. 8. The polypeptide of claim 6, wherein the polypeptide is fused via a linker; optionally, wherein the linker is a polypeptide comprising an amino acid sequence selected from (GGGGS)₁ (SEQ ID NO: 149), (GGGGS)₂ (SEQ ID NO: 150), (GGGGS)₃ (SEQ ID NO: 151), (GGGGS)₄ (SEQ ID NO: 152), (GRPGS)₂ (SEQ ID NO: 153), (GRPGS)₄ (SEQ ID NO: 154), and (GGGGS)₃GGG (SEQ ID NO: 155). 9. The polypeptide of claim 6, wherein the polypeptide is fused via a linker from the C-terminus of the monomeric or dimeric Fc polypeptide and the N-terminus of the mutant IL-2 polypeptide. 10. The polypeptide of claim 1, wherein the polypeptide is characterized in having one or more of the following properties relative to the IL-2 polypeptide of SEQ ID NO: 10: (i) increased titer when expressed in a mammalian cell culture system; (ii) increased solubility; (iii) reduced binding affinity for IL-2Rα receptor subunit of SEQ ID NO: 2; and/or (iv) reduced binding affinity for the heterodimeric IL-2Rβγ receptor protein. 11. A polynucleotide encoding the polypeptide of claim 1. 12. An expression vector comprising the polynucleotide of claim 11. 13. An isolated host cell comprising the polynucleotide of claim 11; optionally, wherein, the host cell is a mammalian cell or a yeast cell. 14. The isolated host cell of claim 13, wherein the host cell is a mammalian cell selected from a Chinese hamster ovary (CHO) cell, a myeloma cell (e.g.,Y0, NS0, Sp2/0), a monkey kidney cell (COS-7), a human embryonic kidney line (293), a baby hamster kidney cell (BHK), a mouse Sertoli cell (e.g., TM4), an African green monkey kidney cell (VERO-76), a human cervical carcinoma cell (HELA), a canine kidney cell, a human lung cell (W138), a human liver cell (Hep G2), a mouse mammary tumor cell, a TR1 cell, a Medical Research Council 5 (MRC 5) cell, and a FS4 cell. 15. A method for producing a mutant IL-2 polypeptide comprising culturing the host cell of any one of claim 13 under a condition suitable for expressing the polypeptide. 16. A pharmaceutical composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable carrier. 17. A method for treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of a mutant IL-2 polypeptide of claim 1, or administering to the subject a therapeutically effective amount of a pharmaceutical composition of claim 16. 18. The method of claim 17, wherein the disease is cancer; optionally, wherein the cancer is selected from colorectal cancer, pancreatic cancer, ovarian cancer, liver cancer, renal cancer, breast cancer, lung cancer, esophageal and gastric cancer, head and neck cancer, cervical cancer, prostate cancer, melanoma, bladder cancer, oral cancer, or hematological malignancies. 19. The method of claim 17, wherein the polypeptide exhibits selectively reduced IL-2Rα binding affinity relative to the IL-2Rα binding affinity of the C125 IL-2 polypeptide of SEQ ID NO: 10. 20. The method of claim 19, wherein the polypeptide comprises a combination of amino acid differences relative to SEQ ID NO: 10 selected from: P65N, E67T;

K35N, Y45R, P65N, E67S; K35N, Y45R, P65N, E67T; K35N F42N F44T D109N; 21.

optionally, wherein the autoimmune disease is selected from Crohn’s disease, Ulcerative colitis, celiac disease, systemic lupus erythematosus, psoriatic arthritis, rheumatoid arthritis, Sjogren’s syndrome, type 1 diabetes, atopic dermatitis, psoriasis, and multiple sclerosis. 22. The method of claim 21, wherein the polypeptide exhibits selectively reduced IL-2Rβγ binding affinity relative to the IL-2Rβγ binding affinity of the C125 IL-2 polypeptide of SEQ ID NO: 10. 23. The method of claim 22, wherein the polypeptide comprises a combination of amino acid differences relative to SEQ ID NO: 10 selected from: E61N, L63T; E61N, L63S;

R81N, P82A, R83T, E61N, L63S, E95N, K97T; R81N, P82A, R83S, E61N, L63T, E95N, K97T; R81N P82A R83S E61N L63S E95N K97S;

24. The method of claim 17, wherein the polypeptide comprises a sequence selected from SEQ ID NO: 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, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, and 145. 25. The method of claim 17, wherein the polypeptide is fused to a monomeric or a dimeric Fc polypeptide; optionally, wherein the Fc polypeptide is a monomeric Fc polypeptide. 26. The method of claim 25, wherein the monomeric or dimeric Fc polypeptide comprises an amino acid sequence selected from SEQ ID NO: 146, 147, and 148. 27. The method of claim 25, wherein the polypeptide is fused via a linker; optionally, wherein the linker is a polypeptide comprising an amino acid sequence selected from (GGGGS)₁ (SEQ ID NO: 149), (GGGGS)₂ (SEQ ID NO: 150), (GGGGS)₃ (SEQ ID NO: 151), (GGGGS)₄ (SEQ ID NO: 152), (GRPGS)₂ (SEQ ID NO: 153), (GRPGS)₄ (SEQ ID NO: 154), and (GGGGS)₃GGG (SEQ ID NO: 155). 28. The method of claim 25, wherein the polypeptide is fused via a linker from the C-terminus of the monomeric or dimeric Fc polypeptide and the N-terminus of the mutant IL-2 polypeptide.