IL-2 variants and uses thereof

Abstract

The present disclosure relates to variants of the cytokine IL-2 with altered functional properties, including increased expression titer and selective binding to different IL-2 receptor protein complexes, pharmaceutical compositions containing these variant IL-2 polypeptides, and the use of these compositions as therapeutic agents, for example, in the treatment of cancer and autoimmune disorders. This Summary is intended to introduce the subject matter of the present disclosure, but does not exhaustively describe each and every embodiment, combination, or variation contemplated and described within the disclosure. Further embodiments are contemplated and described by the disclosure of the detailed description, drawings, and claims.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 385,610, filed November 30, 2022, which is incorporated herein by reference in its entirety for all purposes. (Technical field)

[0002] The present disclosure relates to mutant IL-2 polypeptides, and fusions of these polypeptides with half-life extending proteins such as Fc monomers or Fc dimers, serum albumin polypeptides, and / or specific binding biomolecules such as VHH polypeptides, and antibodies, pharmaceutical compositions comprising these polypeptides, and the use of these compositions as therapeutic agents, for example, in the treatment of cancer or autoimmune disorders.

[0003] Sequence Listing Reference An official copy of the Sequence Listing is being submitted contemporaneously herewith through the USPTO Patent Center as an XML file in WIPO standard ST.26 format with the file name "17195-001PV1.xml," created on November 30, 2022, and 163,978 bytes in size. This Sequence Listing submitted through the USPTO Patent Center is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] Interleukin-2 (IL-2), also known as T cell growth factor (TCGF), is a pluripotent cytokine produced primarily by activated T cells, particularly CD4+ helper T cells. IL-2 signaling is mediated through binding to three distinct receptor proteins: IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132). Immune cells express dimeric or trimeric complexes of IL-2 receptor proteins. The dimeric receptor (IL-2Rβγ) is expressed on cytotoxic CD8+ T cells and natural killer (NK) cells, whereas the trimeric receptor (IL-2Rαβγ) is primarily expressed on activated lymphocytes and CD4+CD25+FoxP3+ inhibitory regulatory T cells (Treg). Treg cells express high levels of IL-2Rα (CD25), and their proliferation is stimulated by IL-2. However, resting effector T cells and NK cells do not have CD25 on their cell surface and are relatively insensitive to IL-2.

[0005] IL-2 binding to the three different receptor proteins differs significantly. IL-2 binds to the trimeric receptor via the K D It has a high affinity of approximately 10 pM, and a K D It has a medium affinity of approximately 1 nM and a K D The IL-2 receptor complexes have low affinity, approximately 10 nM. The IL-2 signaling activities mediated by the different receptor complexes also vary greatly. In general, IL-2Rβ and IL-2Rγ are essential for IL-2 signaling, whereas IL-2Rα (CD25) has been found to be non-essential.

[0006] IL-2 binding to IL-2 receptor proteins 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 natural killer (NK) cell production, proliferation, and activation. IL-2 has been approved as an immunotherapeutic agent for the treatment of cancer and chronic viral infections. However, IL-2 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)). Furthermore, IL-2 treatment has been associated with vascular leak syndrome (VLS) and pulmonary edema in patients, which is thought to result from direct binding of IL-2 to a trimeric receptor (IL-2Rαβγ) on pulmonary endothelial cells (Krieg et al., Proc Nat Acad Sci USA 107, 11906-11 (2010)).

[0007] Manipulation of IL-2 with mutations has been proposed to reduce these toxic side effects by altering the selectivity or preference of IL-2 for different IL-2 receptor subunits, thereby improving its therapeutic efficacy. For example, it has been proposed that targeting IL-2 to cells expressing IL-2Rβ but not IL-2Rα can induce expansion of a cell population enriched in IL-2Rβ, thereby improving the therapeutic efficacy of IL-2 therapy (Boyman et al., Science 311, 1924-1927 (2006)). U.S. Patent Publication 2018 / 0142037A1 describes the introduction of mutations at amino acid positions 42, 45, and 72 of IL-2, again with the aim of reducing the affinity of IL-2 for the IL-2Rα receptor. Another mutant IL-2, called "IL-2H9," contains five mutations: L80F, R81D, L85V, I86V, and I92F, and exhibits enhanced binding to IL-2Rβ, resulting in stimulation of CD25 cells (see Levin et al., Nature, Vol. 484, p. 529-533, DOI: 10.1038 / nature10975). The mutant IL-2 protein, "IL-23x," contains three mutations: R38D, K43E, and E61R, which result in significantly lower binding affinity to 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, making it unsuitable for subsequent large-scale pharmaceutical manufacturing. There remains a need for variant forms of IL-2 that exhibit improved functional properties for therapeutic use, such as in pharmaceutical compositions for the treatment of diseases such as infections, cancer, and autoimmune disorders. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Fontenot et al., Nature Immunol. 6, 1142-51 (2005) [Non-patent document 2] D'Cruz and Klein, Nature Immunol. 6, 1152-59 (2005) [Non-patent document 3] Maloy and Powrie, Nature Immunol. 6, 1171-72 (2005) [Non-patent document 4] Krieg et al.,Proc Nat Acad Sci USA 107,11906-11(2010) [Non-Patent Document 5] Boyman et al., Science 311, 1924-1927 (2006) [Non-patent document 6] Levin et al., Nature, Vol 484, p 529-533 [Non-Patent Document 7] Rodrigo Vazquez-Lombardi et al., Nature Communications, 8:15373 Summary of the Invention

[0009] The present disclosure relates generally to variants of the cytokine IL-2, pharmaceutical compositions, and the use of these compositions as therapeutic agents, for example, in the treatment of cancer. This Summary is intended to introduce the subject matter of the present disclosure, but does not exhaustively describe each and every embodiment, combination, or variation contemplated and described within the 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 to an IL-2 receptor protein, the polypeptide comprising 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 the following: [Table 6]

[0011] In at least one embodiment, the polypeptide further comprises one or more amino acid differences relative to SEQ ID NO: 10 selected from K35N, F42A, Y45R, N88D, D109N, and Q126T.

[0012] In at least one embodiment of a variant IL-2 polypeptide of the present disclosure, the polypeptide comprises a combination of amino acid differences relative to SEQ ID NO: 10 selected from the following: [Table 7-1] [Table 7-2]

[0013] In at least one embodiment of a variant IL-2 polypeptide of the present disclosure, the polypeptide comprises a combination of amino acid differences selected from the following: [Table 8-1] [Table 8-2]

[0014] In at least one embodiment of a variant IL-2 polypeptide of the disclosure, the polypeptide is selected from the group consisting of SEQ ID NOs: 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, 1 18, 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 a variant IL-2 polypeptide of the present disclosure, the polypeptide has the following properties compared to the C125SIL-2 polypeptide of SEQ ID NO: 10: (i) increased titer when expressed in mammalian cell culture systems; (ii) increased solubility; (iii) a reduced binding affinity to the IL-2Rα receptor; and / or (iv) reduced binding affinity to the IL-2Rβ and IL-2Rγ receptors.

[0016] In at least one embodiment of a variant IL-2 polypeptide of the present disclosure, the polypeptide is fused to a monomeric or dimeric Fc polypeptide via a linker; optionally, the Fc polypeptide is a monomeric Fc polypeptide comprising the amino acid sequence of SEQ ID NO: 146, 147, or 148. In at least one embodiment, the linker comprises 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 variant 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 variant 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 variant 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, the mammalian cell is selected from Chinese hamster ovary (CHO) cells, myeloma cells (e.g., Y0, NS0, Sp2 / 0), monkey kidney cells (COS-7), human embryonic kidney line (293), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells, human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor cells, TR1 cells, Medical Research Council 5 (MRC5) cells, and FS4 cells.

[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 conditions suitable for expression of 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 subject (e.g., an IL-2-mediated disease) 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, the cancer is selected from colorectal cancer, pancreatic cancer, ovarian cancer, liver cancer, renal cancer, breast cancer, lung cancer, esophageal cancer 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 administered mutant IL-2 polypeptide exhibits selectively reduced IL-2Rα binding affinity compared 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 the following: [Table 9]

[0023] In at least one embodiment of the method of treating a disease, the disease is an autoimmune disease; optionally, 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.

[0024] In at least one embodiment of the method of treating an autoimmune disease, the polypeptide exhibits selectively reduced IL-2Rβγ binding affinity compared 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 the following: [Table 10-1] [Table 10-2]

[0025] A better understanding of the novel features and advantages of the present disclosure will be obtained by reference to the following detailed description and accompanying drawings (also referred to herein as "Figure" and "FIG.") that set forth illustrative embodiments, in which the principles of the present disclosure are utilized: [Brief explanation of the drawings]

[0026] [Figure 1A] 1 shows the results of biolayer interferometry (BLI) measurements performed as described in Example 1 to measure binding to IL-2Rα by exemplary mutant IL-2 polypeptide Fc fusion constructs p132 and p115 compared to the control p123 (C125S IL2) construct. [Figure 1B] 1 shows the results of biolayer interferometry (BLI) measurements performed as described in Example 1 to measure binding to IL-2Rα by the exemplary mutant IL-2 polypeptide Fc fusion construct p132 compared to the control p123 (C125S IL2) construct. [Figure 1C] 1 shows the results of biolayer interferometry (BLI) measurements performed as described in Example 1 to measure binding to IL-2Rα by the exemplary mutant IL-2 polypeptide Fc fusion construct p115 compared to the control p123(C125S IL2) construct. [Figure 1D] 1 shows the results of biolayer interferometry (BLI) measurements performed as described in Example 1 to measure binding to IL-2Rβγ by exemplary mutant IL-2 polypeptide Fc fusion constructs p132 and p115 compared to the control p123 (C125S IL2) construct. [Figure 1E] 1 shows the results of biolayer interferometry (BLI) measurements performed as described in Example 1 to measure binding to IL-2Rβγ by the exemplary mutant IL-2 polypeptide Fc fusion construct p132 compared to the control p123 (C125S IL2) construct. [Figure 1F] 1 shows the results of biolayer interferometry (BLI) measurements performed as described in Example 1 to measure binding to IL-2Rβγ by the exemplary mutant IL-2 polypeptide Fc fusion construct p115 compared to the control p123(C125S IL2) construct. [Figure 1G]1 shows the results of biolayer interferometry (BLI) measurements performed as described in Example 1 to measure binding to IL-2Rα by the exemplary mutant IL-2 polypeptide Fc fusion construct p151 compared to the control p123(C125S IL2) construct. [Figure 1H] 1 shows the results of biolayer interferometry (BLI) measurements performed as described in Example 1 to measure binding to IL-2Rα by the exemplary mutant IL-2 polypeptide Fc fusion construct p151 compared to the control p123(C125S IL2) construct. [Figure 1I] 1 shows the 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 construct p151 compared to the control p123(C125S IL2) construct. [Figure 1J] 1 shows the results of biolayer interferometry (BLI) measurements performed as described in Example 1 to measure binding to IL-2Rβγ by the exemplary mutant IL-2 polypeptide Fc fusion construct p151 compared to the control p123(C125S IL2) construct.

[0027] [Figure 2A] The SEC profile obtained as described in Example 1 for the exemplary mutant IL-2 polypeptide Fc fusion construct p123 C125S IL2 fusion control is shown as follows: Control p123(C125S IL2) construct. [Figure 2B] The SEC profile obtained as described in Example 1 for the exemplary mutant IL-2 polypeptide Fc fusion construct p123 C125S IL2 fusion control is shown as follows: Control p123(C125S IL2) construct. [Figure 2C]The SEC profile obtained as described in Example 1 for an exemplary mutant IL-2 polypeptide Fc fusion construct p296 is shown below: p296. [Figure 2D] The SEC profile obtained as described in Example 1 for an exemplary mutant IL-2 polypeptide Fc fusion construct, p307, is shown below: p307. [Figure 2E] The SEC profile obtained as described in Example 1 for an exemplary mutant IL-2 polypeptide Fc fusion construct, p406, is shown below: p406. [Figure 2F] The SEC profile obtained as described in Example 1 for an exemplary mutant IL-2 polypeptide Fc fusion construct p297 is shown below: p297. [Figure 2G] The SEC profile obtained as described in Example 1 for an exemplary mutant IL-2 polypeptide Fc fusion construct, p300, is shown below: p300. [Figure 2H] The SEC profile obtained as described in Example 1 for an exemplary mutant IL-2 polypeptide Fc fusion construct p308 is shown below: p308. [Figure 2I] The SEC profile obtained as described in Example 1 for an exemplary mutant IL-2 polypeptide Fc fusion construct p214 is shown below: p214. [Figure 2J] The SEC profile obtained as described in Example 1 for an exemplary mutant IL-2 polypeptide Fc fusion construct, p310, is shown below: p310. [Figure 2K] The SEC profile obtained as described in Example 1 for an exemplary mutant IL-2 polypeptide Fc fusion construct p411 is shown below: p411. [Figure 2L]The SEC profile obtained as described in Example 1 for an exemplary mutant IL-2 polypeptide Fc fusion construct p298 is shown below: p298.

[0028] [Figure 3A] Plots of binding curves and EC50 values ​​measured in the HEK Blue IL-2 reporter assay described in Example 1 are shown for exemplary mutant IL-2 polypeptide Fc fusion constructs, p124, p132, and p167, as follows: p124, p132, and p167. [Figure 3B] Plots of binding curves and EC50 values ​​measured in the HEK Blue IL-2 reporter assay described in Example 1 are shown for exemplary mutant IL-2 polypeptide Fc fusion constructs, p124, p132, and p167, as follows: p124, p132, and p167. [Figure 3C] Plots of binding curves and EC50 values ​​measured in the HEK Blue IL-2 reporter assay described in Example 1 are shown for exemplary mutant IL-2 polypeptide Fc fusion constructs, p296, p300, and p214, as follows: p296, p300, and p214. [Figure 3D] Plots of binding curves and EC50 values ​​measured in the HEK Blue IL-2 reporter assay described in Example 1 are shown for exemplary mutant IL-2 polypeptide Fc fusion constructs, p296, p300, and p214, as follows: p296, p300, and p214. [Figure 3E] Plots of binding curves and EC50 values ​​measured in the HEK Blue IL-2 reporter assay described in Example 1 are shown for exemplary mutant IL-2 polypeptide Fc fusion constructs, p307, p214, and p123 C125S IL2 fusion control, as follows: p123, p307, and p214. [Figure 3F]Plots of binding curves and EC50 values ​​measured in the HEK Blue IL-2 reporter assay described in Example 1 are shown for exemplary mutant IL-2 polypeptide Fc fusion constructs, p307, p214, and p123 C125S IL2 fusion control, as follows: p123, p307, and p214. [Figure 3G] Plots of binding curves and EC50 values ​​measured in the HEK Blue IL-2 reporter assay described in Example 1 are shown for exemplary mutant IL-2 polypeptide Fc fusion constructs, p296, p308, and p123 C125S IL2 fusion control, as follows: p123, p296, and p308. [Figure 3H] Plots of binding curves and EC50 values ​​measured in the HEK Blue IL-2 reporter assay described in Example 1 are shown for exemplary mutant IL-2 polypeptide Fc fusion constructs, p296, p308, and p123 C125S IL2 fusion control, as follows: p123, p296, and p308. [Figure 3I] Plots of binding curves and EC50 values ​​measured in the HEK Blue IL-2 reporter assay described in Example 1 are shown for exemplary mutant IL-2 polypeptide Fc fusion constructs, p214, p310, and p123 C125S IL2 fusion control, as follows: p123, p310, and p214. [Figure 3J] Plots of binding curves and EC50 values ​​measured in the HEK Blue IL-2 reporter assay described in Example 1 are shown for exemplary mutant IL-2 polypeptide Fc fusion constructs, p214, p310, and p123 C125S IL2 fusion control, as follows: p123, p310, and p214. [Figure 3K] Plots of binding curves and EC50 values ​​measured in the HEK Blue IL-2 reporter assay described in Example 1 are shown for exemplary mutant IL-2 polypeptide Fc fusion constructs p296, p214, p297, and p298, as follows: p296, p297, p298, and p214. [Figure 3L] Plots of binding curves and EC50 values ​​measured in the HEK Blue IL-2 reporter assay described in Example 1 are shown for exemplary mutant IL-2 polypeptide Fc fusion constructs p296, p214, p297, and p298, as follows: p296, p297, p298, and p214. [Figure 3M] Plots of binding curves and EC50 values ​​measured in the HEK Blue IL-2 reporter assay described in Example 1 are shown for exemplary mutant IL-2 polypeptide Fc fusion constructs, p214, p411, and p123 C125S IL2 fusion control, as follows: p411, p214, and p123. [Figure 3N] Plots of binding curves and EC50 values ​​measured in the HEK Blue IL-2 reporter assay described in Example 1 are shown for exemplary mutant IL-2 polypeptide Fc fusion constructs, p406 and p411, as follows: p406, and p214. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present disclosure provides mutant IL-2 polypeptides with mutations that alter the glycosylation of the polypeptide and affect various physicochemical and functional properties of IL-2, including recombinant expression potency, solubility, and binding affinity to the monomeric, dimeric, and trimeric forms of the IL-2R chains, i.e., IL-2Rα, IL-2Rβ, and IL-2Rγ. The altered binding characteristics of the mutant IL-2 polypeptides with different IL-2R chains can inhibit, reduce, and / or completely block the function of the IL-2R receptor, particularly its function as a cell surface receptor that mediates immunoregulation. Therefore, it is contemplated that any compositions or formulations comprising the mutant IL-2 polypeptides of the present disclosure can be used as therapeutic agents for treating diseases mediated by the function of IL-2R or its cognate ligand, IL-2, such as cancer and autoimmune disorders. Furthermore, it is contemplated that the mutant IL-2 polypeptides of the present disclosure can be used as therapeutic agents in combination with other therapeutic agents, such as antibodies that target immune checkpoint molecules.

[0030] Terminology and Technology Overview

[0031] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a protein" includes a plurality of proteins, and a reference to "a compound" refers to a plurality of compounds. It is further noted that the claims may be drafted to exclude any element. Accordingly, this statement is intended to serve as a guidepost for the use of exclusive terminology such as "solely," "only," or the use of "negative" limitations in connection with the recitation of claim elements. The use of "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and not intended to be limiting. Where the description of various embodiments uses the term "comprising," those skilled in the art will further understand that in some specific instances, an embodiment may alternatively be described using the 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 and tenth of each intervening integer between the upper and lower limits of that range, as well as 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 within the smaller ranges and are also encompassed herein, subject to any specific excluded limits in the stated range. When a stated range includes one or both of those upper and lower limits, ranges excluding (i) either one or (ii) both of those included upper and lower limits are also encompassed within 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 well understood and commonly used by those of skill in the art, such as, for example, the general techniques and methodology described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 2012 (hereinafter "Sambrook"), and Current Protocols in Molecular Biology, edited by FMA Ausubel, first published in book form by Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. in 1987, supplemented periodically until 2011, and now available in journal form 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 incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated herein by reference for all purposes.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It is to be understood that the terms used herein are for the purpose of describing particular embodiments and are not intended to be limiting. For purposes of interpreting this disclosure, the following terminology explanations apply, and where appropriate, terms used in the singular also include the plural and vice versa.

[0036] As used herein, "IL-2" or "IL-2 polypeptide" refers to the cytokine interleukin-2 and includes naturally occurring and recombinant forms of interleukin-2 polypeptide from human, mouse, rat, or non-human primate, as well as unprocessed (with signal peptide) and processed (without signal peptide) forms. Furthermore, the term includes naturally occurring IL-2 variants, such as alleles and splice variants, isotypes, homologs, and interspecies homologs, as well as recombinant (i.e., artificial) IL-2 variants or mutants, including mutant IL-2 polypeptides with 1 to 15 amino acid substitutions relative to the amino acid sequence of naturally occurring IL-2. For example, the term encompasses the recombinant human IL-2 amino acid sequence of UniProt P60568, which has an amino acid substitution at position C125, such as C125S or C125A. The term is also intended to encompass IL-2 polypeptides covalently conjugated (or fused) to another polypeptide or protein. Exemplary IL-2 fusions of the present disclosure include mutant IL-2 polypeptides fused to other cytokines (e.g., IL-15) or fused to half-life extending polypeptides (e.g., monomeric Fc, dimeric Fc, or human serum albumin).

[0037] As used herein, "IL-2 receptor" or "IL-2R" refers to a heterotrimeric protein expressed on the surface of certain immune and endothelial cells and includes each of the polypeptide subunits, IL-2Rα, IL-2Rβ, and IL-2Rγ (also known as cytokine receptor common subunit gamma), in their monomeric and dimeric forms, such as IL-2Rβγ.

[0038] "Affinity" refers to the strength of the sum of non-covalent 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 the 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 is generally determined by the equilibrium dissociation constant (K D ) Affinity can be measured by common methods known in the art, including those described herein. "Specifically binds" or "specific binding" refers to the binding of an IL-2 polypeptide to its receptor at a concentration of about 1 x 10 -7 It refers to binding with an affinity value of less than or equal to M. Exemplary and exemplary embodiments for measuring binding affinity and / or specific binding are described elsewhere herein, e.g., in the Examples.

[0039] As used herein, "host cell" refers to a cell that can be functionally modified with a recombinant nucleic acid and that can function to express recombinant products, including polypeptides and compounds produced by the activity of the polypeptides.

[0040] "Nucleic acid" or "polynucleotide" are used interchangeably herein to refer to two or more nucleosides linked together by a covalent bond. A nucleic acid may be composed entirely of ribonucleosides (e.g., RNA), entirely of 2'-deoxyribonucleotides (e.g., DNA), or a mixture of ribonucleosides and 2'-deoxyribonucleosides. The nucleoside units of a nucleic acid may be linked together via phosphodiester bonds (e.g., as in naturally occurring nucleic acids), or the nucleic acid may contain one or more non-natural bonds (e.g., phosphorothioester bonds). A nucleic acid or polynucleotide is intended to include single-stranded or double-stranded molecules, or molecules having both single-stranded and double-stranded regions. Nucleic acid or polynucleotide is intended to include molecules composed of naturally occurring nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine) or molecules that contain one or more modified and / or synthetic nucleobases, e.g., inosine, xanthine, hypoxanthine, etc.

[0041] "Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristylation, ubiquitination, etc.). As used herein, a "protein" or "polypeptide" or "peptide" polymer can contain D- and L-amino acids, as well as mixtures of D- and L-amino acids.

[0042] As used herein, "naturally occurring" or "wild-type" refers to a form found in nature. For example, a naturally occurring nucleic acid sequence is one that is present in an organism, can be isolated from a source in nature, and has not been intentionally modified by human manipulation.

[0043] As used herein, "recombinant," "engineered," or "non-naturally occurring," e.g., when used with respect to a cell, nucleic acid, or polypeptide, refers to a substance that has been modified in a way that does not occur in nature, or that is identical to but produced or derived from synthetic material and / or by manipulation using recombinant technology, or a substance that corresponds to a natural or native form of a substance. Non-limiting examples include, among others, recombinant cells expressing genes not found in the native (non-recombinant) form of the cell, or recombinant cells that express native genes that are otherwise expressed at different levels.

[0044] As used herein, "nucleic acid derived from" refers to a nucleic acid having a sequence at least substantially identical to a sequence found naturally in an organism, such as a cDNA molecule prepared by reverse transcription of mRNA isolated from an organism, or a nucleic acid molecule prepared synthetically to have a sequence at least substantially identical to a nucleic acid sequence found in an organism, or a nucleic acid molecule that hybridizes to a sequence at least substantially identical to a nucleic acid sequence found in an organism.

[0045] "Coding sequence" refers to a portion of a nucleic acid (eg, a gene) that codes for the amino acid sequence of a protein.

[0046] As used herein, "heterologous nucleic acid" 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 the host cell.

[0047] "Codon optimization" refers to changing the codons of a polynucleotide encoding a protein to those preferentially used in a particular organism so that the encoded protein can be efficiently expressed in the organism of interest. While the genetic code is degenerate, with most amino acids represented by several codons called "synonyms" or "synonymous" codons, it is well known that codon usage by a particular organism is non-random and biased toward certain codon triplets. This codon usage bias can be higher for a given gene, for common functions or ancestral genes, for highly expressed proteins versus low copy number proteins, and for the entire protein-coding region of an organism's genome. In some embodiments, a polynucleotide encoding an imine reductase enzyme may be codon-optimized for optimal production from the host organism selected for expression.

[0048] A "preferred, optimal, or high codon usage bias codon" refers to a codon that is used more frequently in a protein-coding region than other codons that encode the same amino acid. Preferred codons can be determined in relation to the codon usage frequency in a single gene, a set of genes with a common function or origin, highly expressed genes, the codon frequency throughout the protein-coding region of an entire organism, the codon frequency throughout the protein-coding region of related organisms, or a combination thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. Various methods are known for determining codon frequencies (e.g., codon usage, relative synonymous codon usage) and codon preferences in a particular organism, including, for example, multivariate analysis using cluster analysis or correspondence analysis, and the effective number of codons used in a gene (see, for example, 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, e.g., 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 DC, pp. 2047-2066). Data sources for obtaining codon usage can rely on any available nucleotide sequence capable of encoding a protein.These datasets include nucleic acid sequences known to actually encode expressed proteins (e.g., complete protein-coding sequences - CDS), expressed sequence tags (ESTS), or predicted coding regions of genomic sequences (see, e.g., Mount, D., Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; Uberbacher, EC, 1996, Methods Enzymol. 266:259-281; ​​Tiwari et al., 1997, Comput. Appl. Biosci. 13:263-270).

[0049] As used herein, the term "control sequences" refers to all sequences necessary or advantageous for the expression of polynucleotides and / or polypeptides used in the present disclosure. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, promoter, polyadenylation sequence, propeptide sequence, signal peptide sequence, and transcription terminator. At a minimum, control sequences typically include a promoter, and transcriptional and translational stop signals. Control sequences may be provided with linkers for the purpose of introducing specific restriction sites to facilitate ligation of restriction sequences with the coding region of the nucleic acid sequence encoding the polypeptide.

[0050] As used herein, "operably linked" refers to a configuration in which a control sequence is suitably positioned (e.g., in a functional relationship) with a polynucleotide or polypeptide sequence of interest such that the control sequence directs or controls expression of the sequence of interest.

[0051] A "promoter sequence" refers to a nucleic acid sequence recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence comprises transcriptional control sequences that mediate expression of the polynucleotide of interest. The promoter may be any nucleic acid sequence that shows transcriptional activity in the host cell of choice, including mutant promoters, truncated promoters, and hybrid promoters, and may be derived from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0052] "Percentage of sequence identity," "percent sequence identity," "percent homology," or "percent homology" are used interchangeably herein to refer to a value that quantifies the comparison of polynucleotide or polypeptide sequences and is determined by comparing two optimally aligned sequences over a comparison window, where a portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (or gaps) compared to the reference sequence in order to optimally align the two sequences. The percentage value can be calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Alternatively, the percentage can be calculated by determining the number of positions in both sequences where either the same nucleic acid base or amino acid residue appears or where the nucleic acid bases or amino acid residues are aligned with gaps to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Those skilled in the art will appreciate that there are many established algorithms available for aligning two sequences.Optimal alignment of sequences for comparison can be achieved, for example, 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 computer 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 collaboration between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1995 Supplement) (Ausubel)). Examples of algorithms suitable for determining percent sequence identity and percent sequence similarity include 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 website of the National Center for Biotechnology Information. This algorithm identifies high-scoring sequence pairs (HSPs) by first identifying short words of length W in the query sequence that, when aligned with words of the same length in the database sequences, either match or meet some positive threshold score T.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. For nucleotide sequences, cumulative scores are calculated using 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 word hits in each direction is stopped when: the cumulative alignment score decreases by an amount X from its maximum performed value; the cumulative score falls below zero 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 by default a word length (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 by default a word length (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). For exemplary sequence alignment and determination of percent sequence identity, the BESTFIT or GAP programs from the GCG Wisconsin Software package (Accelrys, Madison Wis.) can be used using the default parameters provided.

[0053] A "reference sequence" refers to a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of a larger sequence, such as a segment of a full-length nucleic acid or polypeptide sequence. A reference sequence is typically at least 20 nucleotides or amino acid residues in length, but can be the entire length of a nucleic acid or polypeptide. Because two polynucleotides or polypeptides may each contain (1) similar sequences (i.e., a portion of the complete sequence) between the two sequences and (2) additional sequences that differ between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues, where a sequence can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and the portion of the sequence within the comparison window may contain no more than 20 percent additions or deletions (or gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences.

[0054] "Substantial identity" or "substantially identical" refers to a polynucleotide or polypeptide sequence having 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 compared to a reference sequence over a comparison window of at least 20 nucleoside or amino acid residue positions, frequently a window of at least 30-50 positions, where the percentage of sequence identity is calculated by comparing the reference sequence to a sequence that contains deletions or additions totaling no more than 20% of the reference sequence over the entire comparison window.

[0055] "Corresponding to," "referring to," or "relative to," when used in reference to the numbering of a given amino acid sequence or polynucleotide sequence, refers to the numbering of residues in a specified reference sequence when comparing a given amino acid sequence or polynucleotide sequence to a reference sequence. In other words, residue numbers or residue positions in a given polymer are specified relative to the reference sequence, rather than the actual numerical positions of the residues in the given amino acid sequence or polynucleotide sequence. For example, a given amino acid sequence, such as the amino acid sequence of an engineered imine reductase, can be aligned to a reference sequence by introducing gaps to optimize residue matching between the two sequences. In these cases, despite the presence of gaps, the numbering of residues in a given amino acid sequence or polynucleotide sequence is relative to the reference sequence to which it is aligned.

[0056] "Isolated," as used herein with respect to a molecule, means that the molecule (e.g., cannabinoid, polynucleotide, polypeptide) has been substantially separated from other compounds that naturally accompany it, e.g., proteins, lipids, and polynucleotides. The term encompasses nucleic acids that have been removed or purified from their naturally occurring environment or expression system (e.g., a host cell or in vitro synthesis).

[0057] "Substantially pure" refers to a composition in which the desired molecule is the predominant species present (i.e., the desired molecule is more abundant than other individual macromolecular species in the composition, on a molar or weight basis); generally, a composition is substantially purified when the desired species constitutes at least about 50% of the macromolecular species present, on a molar or weight basis.

[0058] An "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 a clinical intervention aimed at altering the natural history of a disorder in the individual being treated and can be carried out prophylactically or during the course of clinical pathology. Desired results of treatment can include, but are not limited to, preventing the onset or recurrence of the disorder, alleviating symptoms, reducing any direct or indirect pathological consequences of the disorder, preventing metastasis, reducing the rate of progression, ameliorating or alleviating the disease state, and achieving remission or improving prognosis. For example, treatment can include administering to a subject a therapeutically effective amount of a pharmaceutical formulation comprising an IL-2 variant polypeptide to delay the onset or slow the progression of an IL-2R-mediated disease or condition, or a disease or condition in which IL-2R may play a role in the pathogenesis and / or progression.

[0060] A "pharmaceutical formulation" refers to a preparation in which the biological activity of the active ingredient(s) can be effective and which does not contain additional ingredients that are toxic to the subject to which the formulation is administered. A pharmaceutical formulation may include one or more active agents. For example, a pharmaceutical formulation may include a mutant IL-2 polypeptide as the only active agent in the formulation, or may include a mutant IL-2 polypeptide and one or more additional active agents, such as, for example, an immune checkpoint inhibitor.

[0061] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject to which it is administered. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0062] A "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, a therapeutically effective amount of a therapeutic agent is that amount that reduces, prevents, inhibits, and / or alleviates to some extent one or more of the symptoms associated with the disease, disorder, or condition. With respect to cancer therapy, in vivo efficacy can be measured, for example, by assessing primary tumor growth, the occurrence and / or growth of secondary tumor(s), the occurrence and / or number of metastases, the duration, severity, and / or recurrence of symptoms, response rate (RR), duration of response, and / or quality of life.

[0063] "Individual" or "subject" refers to mammals, including, but not limited to, domestic 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 receptor

[0065] IL-2 signaling is mediated through binding to three distinct 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 (NK) cells, whereas the trimeric receptor is expressed primarily on activated lymphocytes and CD4+CD25+FoxP3+ inhibitory regulatory T cells (Tregs) (see Byman et al., J. Nat. Rev. Immunol. 12, 180-190 (2012)). Resting effector T cells and NK cells lack CD25 on their cell surface and are therefore relatively insensitive to IL-2. However, Treg cells express high levels of CD25, and therefore, Treg proliferation is stimulated by IL-2.

[0066] IL-2Rαβγ, a trimeric receptor formed by the combination of IL-2Ra, IL-2Rβ, and IL-2Rγ, is a K D The dimeric receptor (IL-2Rβγ) has a K D The IL-2 receptor is a medium-affinity receptor with an affinity of approximately 1 nM. The monomeric IL-2Rα receptor is a low-affinity IL-2 receptor. The IL-2 signaling activity mediated by the receptors and their complexes also differs significantly. In general, IL-2Rβ and IL-2Rγ are essential for IL-2 signaling, whereas IL-2Rα (CD25) is not essential for signaling, although the presence of IL-2Rα has been found to allow high-affinity binding of IL-2 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-2R α subunit can be found in 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-2R β subunit can be found in 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-2R γ subunit can be found in 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 summarizes the amino acid sequences of various IL-2R polypeptides of the present disclosure, and their sequence identifiers. These sequences are also included in the attached Sequence Listing.

[0068] [Table 1]

[0069] Mutant IL-2 polypeptides

[0070] The present disclosure provides IL-2 polypeptides having mutations that alter glycosylation (compared to wild-type or C125S IL-2) and thereby affect the functional properties of the polypeptide, such as increased expression potency, increased solubility, and / or altered binding affinity to the IL-2 receptor complex. The altered glycosylation and functional properties resulting from mutations in amino acid sequence (also referred to as amino acid substitutions or amino acid differences) relative to the parent IL-2 polypeptide result in IL-2 polypeptides with improved properties for use in pharmaceutical compositions for the treatment of IL-2-mediated diseases, such as cancer and autoimmune diseases.

[0071] Naturally occurring human IL-2 is a 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), which includes a 20 amino acid N-terminal signal peptide: MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 8)

[0072] The structure of the IL-2 polypeptide contains 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 secreted IL-2 polypeptide of 133 amino acids of SEQ ID NO:9, shown below: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (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 disclosed herein as SEQ ID NO:10 is the active component of aldesleukin, a drug approved for human use in the treatment of cancer. APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTT (SEQ ID NO: 10)

[0074] 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, mutant IL-2 polypeptides have been engineered with amino acid substitutions relative to the C125S IL-2 polypeptide of SEQ ID NO: 10, which provide new N-glycosylation sites in the expressed mutant IL-2 polypeptide. Engineering N-glycosylation motifs in proteins is well known in the art. Generally, the three amino acid sequence motifs asparagine-X-serine (NXS) and asparagine-X-threonine (NXT), where X can be any amino acid except proline, provide potential N-glycosylation sites on a polypeptide. Depending on the sequence of the polypeptide, N-glycosylation sites can be introduced into the polypeptide by engineering amino acid substitutions at one, two, or three positions in the amino acid sequence.

[0075] Table 2 below summarizes the sequences of exemplary mutant IL-2 polypeptides of the present disclosure that have been engineered to have 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 and polynucleotide sequences encoding the polypeptides are provided in the accompanying sequence listing.

[0076] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9]

[0077] Changes in expression titer and solubility of mutant IL-2 polypeptides

[0078] As described elsewhere herein, including in the Examples, variant IL-2 polypeptides of the present disclosure engineered at 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 are increased IL-2 polypeptide expression titers during preparation in mammalian cell culture, increased solubility, and altered binding affinity to the trimeric and dimeric IL-2R complexes formed by the IL-2Rα subunit of SEQ ID NO: 2, the 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 the binding affinity of variant IL-2 to the IL-2Rα subunit but do not reduce its binding affinity to the dimeric IL-2Rβγ subunit.

[0079] It has been observed that engineering N-glycosylation mutations in a recombinant gene encoding a polypeptide can increase the titer of the expressed polypeptide in mammalian cell culture systems. Furthermore, it has been observed that engineering N-glycosylation mutations in a recombinant gene encoding a polypeptide can reduce aggregation and increase the solubility of the resulting polypeptide, as indicated by an increased amount of the polypeptide in monomeric form. It has been found that the presence of certain N-glycosylation mutations, alone or in combination, in the mutant IL-2 polypeptides of the present disclosure results in IL-2 polypeptides that exhibit increased expression titers and increased solubility (measured in % monomeric polypeptide) in mammalian cell culture systems. An exemplary set of amino acid differences of the present disclosure that demonstrate these functional improvements of increased expression titers and increased solubility is shown in Table 3 below.

[0080] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0081] Altered IL-2R binding affinity of mutant IL-2 polypeptides

[0082] It has also been observed that engineering N-glycosylation mutations in a recombinant gene encoding an IL-2 polypeptide can result in mutant IL-2 polypeptides that exhibit altered binding affinities for the different IL-2R receptor subunits, i.e., IL-2Rα, IL-2Rβ, and IL-2Rγ, in their 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 with little or no loss of binding affinity for IL-2β, IL-2γ, or the IL-2βγ dimeric complex. It is believed that such mutant IL-2 polypeptides that exhibit reduced binding affinity for IL-2Rα may provide improved therapeutic compounds for the treatment of cancer, e.g., by virtue of reduced or absent stimulation of immunosuppressive CD25+ cells. Mutant IL-2 polypeptides that exhibit reduced binding affinity for IL-2Rβγ with little or no reduction in binding affinity for IL-2Rα could provide improved therapeutic compounds for the treatment of autoimmune disorders, for example, by preferentially stimulating immunosuppressive CD25+ cells. An exemplary set of amino acid differences of the present disclosure that exhibit selectively reduced binding affinity for IL-2Rα and / or IL-2Rβγ is shown in Table 4 below.

[0083] [Table 4-1] [Table 4-2] [Table 4-3]

[0084] Mutant IL-2 polypeptide fusions

[0085] As described elsewhere herein, it is contemplated that the variant 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 improve the properties of the IL-2 polypeptide. For example, a variant IL-2 polypeptide of the present disclosure (e.g., a polypeptide in 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 the half-life of the overall fusion molecule, allowing for better pharmaceutical compositions for therapeutic use. In at least one embodiment, the present disclosure provides a variant IL-2 polypeptide fused to a polypeptide linker to an Fc polypeptide, such as the wild-type monomeric human IgG1 Fc lower hinge region polypeptide of SEQ ID NO: 146 shown below. APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 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. [ka] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLSSDLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSPGK (SEQ ID NO: 148).

[0087] Although only three human IgG1 Fc lower hinge region polypeptides are described above, it is contemplated that the mutant IL-2 polypeptide fusions of the present disclosure can be prepared using other Fc region fragments and variants known to provide improved properties when conjugated to polypeptides such as cytokines for therapeutic use. For example, Fc polypeptide variants that eliminate effector function can be used, such as an Fc region with the amino acid substitutions L234A / L235A ("LALA") (see Woodle, E. Steve et al., Transplantation, 68(5):608-616 (1999)). Other effector-less Fc region mutations are known in the art, e.g., 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, if 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 variant IL-2 polypeptides of the present disclosure can be conjugated to other polypeptides or proteins (e.g., Fc polypeptides) via a linker. Any of a wide variety of synthetic linear molecules known in the art as useful linkers between biomolecules can be used to fuse the variant IL-2 polypeptides to other polypeptides. In at least one embodiment, a polypeptide linker can be used. Such a polypeptide linker comprises an amino acid chain, each end of which is covalently attached to one of two different polypeptides, thereby functioning to conjugate or fuse them. Typically, such a polypeptide linker comprises a chain of 5 to 30 amino acids. A wide variety of polypeptide linkers are known in the art and can be used in variant 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 known in the art that can be used include (GGGGS) n (n is 1 to 10), (SSSSG) n (n is 1 to 10), (GGGG) (SGGGG) n (n is 1 to 10), (EAAAK) n (n is 1 to 10), (XP) n (n is 1 to 10), and ENLYFQ(-G / S).

[0089] The present disclosure exemplifies fusion mutant IL-10 polypeptides (e.g., SEQ ID NOs: 11-145) fused to a monomeric Fc polypeptide (e.g., SEQ ID NOs: 146, 147, or 148), where the monomeric Fc polypeptide is conjugated from the C-terminus to the N-terminus of the mutant IL-10 polypeptide via a polypeptide linker (e.g., SEQ ID NOs: 149-155). However, it is contemplated, and would be understood by one of 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 (e.g., a monomeric Fc polypeptide) via either its N-terminus or its C-terminus, optionally via a linker (e.g., a polypeptide linker).

[0090] In addition to variant IL-2 polypeptides fused to monomeric Fc polypeptides, the present disclosure also contemplates variant IL-2 fused to immunoglobulin molecules having specific antigen-binding ability, such as antibodies or antibody fragments (e.g., Fab molecules, scFvs, or VHHs). In at least one embodiment, the fused antibody or antibody fragment provides specific antigen-binding affinity that targets the fusion to cancer cells or other cells in the 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, for example, in U.S. Provisional Patent Application No. 63 / 477,529, filed December 28, 2022, which is incorporated herein by reference. 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, for example, in U.S. Provisional Patent Application No. 63 / 488,176, filed March 3, 2023, which is incorporated herein by reference.

[0091] Recombinant methods and compositions

[0092] The variant IL-2 polypeptides 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 isolated nucleic acids encoding the variant IL-2 polypeptides. The nucleic acids can encode an amino acid sequence comprising an IL-2 polypeptide alone or as a fusion with another polypeptide, e.g., a monomeric Fc polypeptide. In some embodiments, one or more vectors (e.g., expression vectors) are provided that comprise a nucleic acid sequence encoding a variant IL-2 polypeptide of the present disclosure. In some embodiments, host cells are provided that comprise a nucleic acid sequence encoding a variant IL-2 polypeptide of the present disclosure. In one embodiment, the host cell is transformed with a vector comprising a nucleic acid encoding an amino acid sequence comprising a variant IL-2 polypeptide. In some embodiments, the host cell used is a eukaryotic cell, e.g., a Chinese hamster ovary (CHO) cell, or a lymphoid cell (e.g., Y0, NS0, Sp20).

[0093] In at least one embodiment, a method for producing a variant IL-2 polypeptide is provided, comprising culturing a host cell containing nucleic acid encoding the polypeptide 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 variant IL-2 polypeptide is achieved by synthesizing or isolating a nucleic acid encoding the variant IL-2 polypeptide (e.g., as described herein) and inserting the 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 an oligonucleotide probe capable of specifically binding to the IL-2-encoding gene). Suitable host cells and culture methods for cloning or expressing IL-2 polypeptide-encoding vectors are well known in the art and include prokaryotic or eukaryotic cells. Typically, after expression, the variant IL-2 polypeptide may be isolated from the cell paste as a soluble fraction and may be further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors.

[0094] Examples of suitable mammalian host cell lines useful for producing the variant 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; SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (293 or 293 cells, e.g., those described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, e.g., Mather, Biol. Reprod. 23:243-251 (1980); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL3A); human lung cells (W138); human hepatocytes (HepG2); mouse mammary tumor (MMT060562); TR1 cells (see, e.g., Mather et al., Annals N Y. Acad. Sci. 383:44-68 (1982) and U.S. Pat. No. 6,235,498); Medical Research Council 5 (MRC5) cells (such as those available from ATCC, also designated CCL-171); and Foreskin 4 (FS-4) cells (see, e.g., Vilcek et al. Ann. NY Acad. Sci. 284:703-710 (1977), Gardner & Vilcek. J. Gen. Virol. 44:161-168 (1979), and Pang et al. Proc. Natl. Acad. Sci. USA 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 mutant IL-2 polypeptides. In some embodiments, the present disclosure provides pharmaceutical formulations comprising a mutant IL-2 polypeptide described herein and a pharmaceutically acceptable carrier. In some embodiments, the mutant IL-2 polypeptide is the only active agent in the pharmaceutical composition. Such pharmaceutical formulations can be prepared by mixing a mutant IL-2 polypeptide having a desired 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 variety of such pharmaceutically acceptable carriers is 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 include buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; and proteins, such as serum. These may include, but are not limited to, albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions 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 also include interstitial drug dispersing agents, such as soluble intermediate-activity hyaluronidase glycoproteins (sHASEGPs) (e.g., U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968), including human soluble PH-20 hyaluronidase glycoprotein (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, depending on the particular indication being treated in the subject to which the formulation is administered. Preferably, any additional active ingredients have activities that complement the activity of IL-2 activity, and the activities do not adversely affect each other.

[0100] As disclosed elsewhere herein, including in the Examples, it has been shown that the variant IL-2 polypeptides of the present disclosure can be used as fusions with Fc polypeptides to improve therapeutic efficacy in the treatment of autoimmune disorders and / or cancer.

[0101] As described elsewhere herein, in some embodiments, the present disclosure provides pharmaceutical compositions or formulations for use in methods of treatment comprising a mutant IL-2 polypeptide conjugated to another polypeptide or protein, e.g., a half-life-extending Fc polypeptide. In some embodiments, the pharmaceutical composition or formulation can comprise a mutant IL-2 polypeptide covalently fused to an Fc polypeptide via a linker, such as a polypeptide linker of the amino acid sequence of SEQ ID NOs: 149-155. Examples demonstrate such mutant IL-2 polypeptide fusions to the monomeric Fc polypeptide of SEQ ID NOs: 146, 147, or 148, and their use in pharmaceutical compositions for palliating the treatment of cancer or autoimmune disorders, as described elsewhere herein.

[0102] In some embodiments, pharmaceutical compositions may comprise a mutant IL-2 polypeptide of the present disclosure and an additional active agent for cancer treatment, such as an immune checkpoint inhibitor. Checkpoint inhibitors useful in such embodiments include, but are not limited to, antibodies that contain 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] In general, the active ingredients of the pharmaceutical composition may be incorporated into microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0104] In some embodiments, the formulation of the variant IL-2 polypeptide may be a sustained-release preparation of the polypeptide and / or other active ingredient. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the variant 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 administered to a subject are sterile. Sterile formulations can be readily prepared using well-known techniques, for example, by filtration through sterile filtration membranes.

[0106] Uses and Treatment Methods

[0107] It is contemplated that any composition or formulation containing a mutant IL-2 polypeptide of the present disclosure can be used in any method or use, e.g., a therapeutic method, that utilizes the polypeptide's ability to specifically bind to the IL-2 receptor protein. IL-2 binding to the IL-2 receptor protein expressed on various cells mediates various immune responses. IL-2 binding can stimulate immune responses, e.g., T cell proliferation and differentiation, cytotoxic T lymphocyte (CTL) production, B cell proliferation and differentiation, immunoglobulin synthesis, and NK cell production, proliferation, and activation. IL-2 polypeptides, such as C125S IL-2 (Proleukin), have been approved as immunotherapeutic agents for the treatment of cancer and chronic viral infections. However, IL-2 polypeptides can also promote the activation and proliferation of immunosuppressive CD4+CD25+ Treg cells, which result in immunosuppression. Thus, there are a variety of diseases, disorders, and conditions that can potentially be treated by altering the immunomodulatory and / or immune signaling activities mediated by IL-2 binding to the IL-2 receptor protein, particularly the effects of IL-2 on tumor progression. Diseases, disorders, and conditions include, but are not limited to, cancer, including, but not limited to, colorectal 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 variant IL-2 polypeptide of the present disclosure, including a variant IL-2 polypeptide fusion with a monomeric Fc polypeptide, can be used in methods or uses for treating any of the cancers listed above. Accordingly, in at least one embodiment, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to a subject in need of treatment a therapeutically effective amount of a variant IL-2 polypeptide of the present disclosure, or administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a variant IL-2 polypeptide of the present disclosure and a pharmaceutically acceptable carrier.

[0108] As disclosed herein, including in the Examples below, variant IL-2 polypeptides of the present disclosure have the ability to differentially bind specifically to IL-2 receptor proteins, thereby differentially altering 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 methods of treating an IL-2-mediated disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of a variant 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 variant IL-2 polypeptide of the present disclosure and a pharmaceutically acceptable carrier. Similarly, in some embodiments, the present disclosure provides methods of treating a disease mediated by IL-2 binding to IL-2 receptor proteins expressed on cells in a subject, comprising administering to the subject a therapeutically effective amount of a variant 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 variant IL-2 polypeptide of the present disclosure and a pharmaceutically acceptable carrier.

[0109] Administration of a variant IL-2 polypeptide, composition, or pharmaceutical formulation in accordance with the method of treatment results in an antibody-induced therapeutic effect that protects the subject from and / or treats the progression of an IL-2-mediated disease in the subject. In some embodiments, the method of treatment can further include administration of one or more additional therapeutic agents or treatments known to those of skill in the art for preventing and / or treating an IL-2-mediated disease or condition. Such methods that include administration of one or more additional agents can encompass combined administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administration, where administration of the variant IL-2 polypeptide composition or formulation can occur before, simultaneously with, and / or after administration of the additional therapeutic agent(s).

[0110] In some embodiments of the therapeutic methods of the present disclosure, a mutant IL-2 polypeptide or a 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 that administers the antibody to a site in a subject other than directly to the desired target site, tissue, or organ in the subject, such that the antibody or a formulation thereof enters the subject's circulatory system and is therefore subject to metabolic and other similar processes. Thus, modes of administration useful in the therapeutic methods of the present disclosure can include, but are not limited to, injection, infusion, instillation, and inhalation. Administration by injection includes intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion.

[0111] In at least one embodiment, the pharmaceutical formulation of the mutant IL-2 polypeptide is formulated to protect the IL-2 from inactivation in the intestinal tract, and thus, the method of treatment can include oral administration of the formulation.

[0112] In at least one embodiment, the use of a composition or formulation comprising a mutant IL-2 polypeptide of the present disclosure as a pharmaceutical is also provided. Furthermore, in some embodiments, the present disclosure also provides the use of a composition or formulation comprising a mutant IL-2 polypeptide in the manufacture or preparation of a pharmaceutical, particularly a pharmaceutical for treating an IL-2-mediated disease. In further embodiments, the pharmaceutical is for use in a method for treating a disease, the method comprising administering an effective amount of the pharmaceutical to an individual having the disease. In certain embodiments, the pharmaceutical further comprises an effective amount of at least one additional therapeutic or therapeutic agent.

[0113] As disclosed elsewhere herein, additional therapeutic or therapeutic agents that can be used in such medicaments together with the variant IL-2 polypeptides of the present disclosure are also contemplated. Generally, it is contemplated that the variant IL-2 polypeptides of the present disclosure can be used together with any therapeutic or therapeutic agent, such as a therapeutic antibody that specifically targets a cell surface receptor on immune cells, tumor cells, or myeloid cells. In at least one embodiment, the additional therapeutic agent includes, 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, or ICOS.

[0114] In a further embodiment, the medicament is for use in treating an IL-2-mediated disease, such as cancer, in a subject, comprising administering to the subject an amount of the medicament effective to treat, inhibit, or prevent the IL-2-mediated disease. The appropriate dosage of the variant 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 vary depending on the specific disease or condition being treated, the severity and course of the disease, whether the administration is for prophylactic or therapeutic purposes, previous treatments administered to the patient, the patient's medical history and response to the variant IL-2 polypeptide composition, and the discretion of the attending physician. It is contemplated that the variant IL-2 polypeptide contained in the compositions and formulations described herein can be suitably administered to a patient at one time or over a series of treatments. Various administration schedules are contemplated herein, including, but not limited to, single or multiple doses over various time points, bolus administration, and pulse infusion.

[0115] Depending on the type and severity of the disease, about 1 μg / kg to 30 mg / kg of a variant IL-2 polypeptide in a formulation of the present disclosure is an initial candidate dosage for administration to a human subject, e.g., by one or more individual administrations or by continuous infusion. Generally, the administered dosage of the antibody ranges 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) can be administered to a patient.

[0116] Dosage administration can be maintained for several days or longer, depending on the subject's condition, for example, until the IL-2-mediated disorder is deemed to be sufficiently treated by methods known in the art. In some embodiments, an initial higher loading dose can be administered, followed by one or more lower doses. However, other dosing regimens may be useful. The progress of the therapeutic effect of the dosage administration can be monitored by conventional techniques and assays. Thus, in some embodiments of the disclosed methods, administration of a mutant IL-2 polypeptide comprises a daily dosage of 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 the variant 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. [Example]

[0117] Various features and embodiments of the present disclosure are illustrated in the following representative examples, which are intended to be illustrative and not limiting. As one skilled in the art will readily appreciate, the specific examples are merely illustrative of the invention, which is more fully described in the claims that follow. It should be understood that all embodiments and features described in this application are interchangeable and combinable with all embodiments contained herein. Example 1: Preparation of IL-2 glycosylation mutant polypeptides

[0118] This example demonstrates the preparation and screening of mutant IL-2 polypeptides with amino acid changes that alter glycosylation and thereby other functional properties, including expression potency, solubility, and binding affinity to 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 as gene fragments (gblocks or eblocks) by Integrated DNA Technologies, Inc. These gene fragments were then cloned into expression vectors as described below.

[0121] B. Preparation of mutant IL-2 expression constructs and mutant polypeptides: The 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., purified on an agarose gel, and dissolved in TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA). Synthetic DNA fragments encoding mutant IL-2 polypeptides were dissolved in water. Equal amounts of vector and insert were added to 2x Gibson Assembly Master Mix (New England Biolabs) at a 1:3 molar ratio and incubated at 50°C for 1-2 hours. The ligated DNA was then transformed into DH5α E. coli competent cells (New England Biolabs). The resulting plasmid DNA was sequenced using standard molecular biology techniques. Appropriate plasmid DNA was prepared using the PureLink™ Hipure Maxiprep kit from Thermo Fisher Scientific (Cat. No. K210006) to recover sterile, salt-free, 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 mutation sets (or amino acid sequence 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 in 60 mL of ExpiCHO expression medium in an Erlenmeyer flask at 37°C, 5% CO2, and 130 rpm. Transfection was performed according to the instructions provided with the ExpiCHO™ Expression System Kit (ThermoFisher - Catalog No. A29133). Briefly, cells were transfected at a cell density (VCD) of 6 x 106 When the cell density reached 10 cells / mL and the doubling time reached 18-20 hours, 6 One microgram of plasmid DNA per cell was mixed with ExpiFectamine CHO by inversion, diluted with cold OptiPRO serum-free medium, and finally complexed with the diluted plasmid DNA at room temperature. After 5 minutes, the mixture was added dropwise to the cell culture at room temperature. After transfection, cells were cultured at 200 RPM for 24-well plates or 130 RPM for 25 mL flasks or large flasks. Seven to 12 days after transfection, the culture medium was centrifuged at 500 x g for 5 minutes at 25°C to pellet the cells, followed by centrifugation at 4500 x g for 30 minutes at 4°C. The clarified supernatant was filtered through a 0.45 μm filter, and the fusion protein was purified by Protein A affinity column chromatography.

[0125] D. Assay of Relative Expression Titer Levels: Measurement of expressed protein titer levels was performed on an Agilent 1100 HPLC system (Santa Clara, California) coupled with a POEOS™ A 20 μm column (catalog number: 2100100, Thermo Fisher Scientific). The equilibration buffer was 1x PBS (buffer A), and the elution buffer was 0.1% HCl containing 150 mM NaCl (buffer B). In a typical HPLC run, after sample injection, the column was washed with 6 column volumes (CV) of 1x PBS. Bound proteins were eluted by step elution with 10 CV of elution buffer, and the UV signal at 280 nm wavelength was recorded. Prior to sample measurement, a standard curve was generated by injecting a known amount of human IgG reference. For sample titer measurement, 40 μl of culture medium was injected, and titers were calculated from the area of ​​the elution peak and calculated using a human IgG standard curve.

[0126] Results: Table 5 below summarizes the expression titer levels measured for different mutant IL-2 polypeptide Fc fusion constructs. These results demonstrate that genes encoding mutant IL-2 polypeptides engineered with the set of glycosylation mutations listed below (see also the relative values ​​listed in Table 3) result in substantially increased expression titers compared to C125S IL-2 in mammalian expression systems.

[0127] [Table 5]

[0128] E. Assays for IL-2Rα and IL-2Rβγ Binding Affinity:

[0129] The binding and dissociation constants for the mutant IL-2 polypeptides' binding kinetics to the receptor were measured by biolayer interferometry (BLI) using a Gator Prime instrument (Gator Bio, Inc.). Binding kinetics were measured at 30°C and analyzed using GatorOne analysis software. A streptavidin (SA) sensor probe was used to capture biotinylated IL-2Rα protein (Acro Biosystems, Inc.; catalog number ILA-H82E6) or biotinylated IL-2Rβγ heterodimer protein (Acro Biosystems, Inc.; catalog number ILG-H82F3). Samples were prepared using purified protein (confirmed as a single peak by SEC) in Tris buffer. The streptavidin sensor was set as follows: baseline: 1x KB buffer; loading: biotinylated IL-2Rα or IL-2Rβγ (Acro Biosystems, Inc.); association: purified protein; dissociation: 1x KB buffer. The sensor probe was immersed in serial dilutions of the parent C125S IL-2 polypeptide or mutant IL-2 polypeptide. Wells containing buffer alone were set as reference wells for background subtraction during data processing. For each cytokine-receptor binding event, data were fitted with a 1:1 Langmuir model for association and dissociation using a global fit with linked Rmax.

[0130] Results: As shown in the BLI results plotted in Figures 1A, 1B, 1C, 1G, and 1H, exemplary mutant IL-2 polypeptide Fc fusion constructs p132 (K35N, Y45R, C125S) (Figure 1B), p115 (K35N, F42A, C125S) (Figure 1C), and p151 (F42A, Y45A, L72G, C125S) (Figure 1H) showed reduced binding to IL-2Rα compared to the control p123 (C125S IL-2) construct (Figures 1A and 1G). For comparison, the results of binding to IL-2Rβγ measured by BLI are also shown in Figures 1D, 1E, 1F, 1I, and 1J: Fc-fusion constructs p132(K35N, Y45R, C125S) (Figure 1E), p115(K35N, F42A, C125S) (Figure 1F), and p151(F42A, Y45A, L72G, C125S) (Figure 1J) compared with the control p132(C125 IL-2) construct (Figures 1D and 1I).

[0131] Table 4 (above) also summarizes exemplary results showing that many of the mutant IL-2 polypeptides exhibit substantially reduced IL-2Rα subunit binding affinity (indicated by "+++") compared to the affinity observed for the parent C125S IL-2 polypeptide. Furthermore, 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 compared to the parent C125S IL-2 polypeptide. This differential reduction in IL-2Rα binding indicates that mutant IL-2 polypeptides with combinations of amino acid differences exhibit differential binding and stimulation of cells expressing IL-2Rβγ, such as cytotoxic CD8+ T cells and natural killer (NK) cells, and are therefore more suitable for the treatment of cancer.

[0132] Table 4 also lists results showing that many of the mutant IL-2 polypeptides have substantially reduced IL-2Rβγ binding affinity while IL-2Rα binding is unchanged. This differential reduction in IL-2Rβγ binding indicates that mutant IL-2 polypeptides with combinations of amino acid differences exhibit greater differential binding and stimulation of cells expressing IL-2Rαβγ, such as activated lymphocytes and CD4+CD25+FoxP3+ inhibitory regulatory T cells (Tregs), and are therefore immunosuppressive and more suitable for the treatment of autoimmune disorders.

[0133] F. Preparation of Monomeric Fc Conjugates of IL-2 Mutants:

[0134] Gene fragments encoding the monomeric Fc polypeptide and mutant IL-2 polypeptide were reverse transcribed and then synthesized as gene fragments (gblocks or eblocks) by Integrated DNA Technologies, Inc. These fragments were then cloned into expression vectors as follows: The expression vector pcDNA3.1(+) was digested with both PstI and XbaI restriction enzymes (New England Biolabs, Inc.), purified by agarose gel, and dissolved in TE buffer (10 mM Tris, pH 8.0, and 1 mM EDTA). The synthesized DNA fragments encoding the monomeric Fc polypeptide and mutant IL-2 polypeptide were dissolved in water. Equal amounts of vector and insert were added to 2x Gibson Assembly Master Mix (New England Biolabs) at a molar ratio of 1:3 and incubated at 50°C for 1-2 hours. The ligated DNA was then transformed into E. coli competent cells (New England Biolabs). The resulting plasmid DNA was sequenced using standard molecular biology techniques. Appropriate plasmid DNA was prepared using the PureLink™ Hipure Maxiprep kit from Thermo Fisher Scientific (Cat. No. K210006) to recover sterile, salt-free, supercoiled DNA.

[0135] G. Expression of variant IL-2 polypeptide fusions with a monomeric Fc gene in Chinese Hamster Ovary (CHO) cells: ExpiCHO cells (Thermo Fisher Scientific) were maintained in 60 mL of ExpiCHO expression medium in Erlenmeyer flasks at 37°C, 5% CO2, and 130 rpm. Transfection was performed according to the instructions provided with the ExpiCHO™ Expression System Kit (Thermo Fisher Scientific - Catalog No. A29133). Briefly, at a cell density (VCD) of 6 x 10 6 When the cell density reached 10 cells / mL and the doubling time reached 18-20 hours, 6 One microgram of plasmid DNA per cell was mixed with ExpiFectamine CHO by inversion, diluted with cold OptiPRO serum-free medium, and finally complexed with the diluted plasmid DNA at room temperature. After 5 minutes, the mixture was added dropwise to the cell culture at room temperature. After transfection, cells were cultured at 200 RPM for 24-well plates or 130 RPM for 25 mL flasks or large flasks. Seven to 12 days after transfection, the culture medium was centrifuged at 500 x g for 5 minutes at 25°C to pellet the cells, followed by centrifugation at 4500 x g for 30 minutes at 4°C. The clarified supernatant was filtered through a 0.45 μm filter, and the fusion protein was purified by Protein A affinity column chromatography.

[0136] H. Relative Solubility Assay of Variant IL-2 Polypeptide Fusions with Monomeric Fc: Protein relative solubility (turbidity) assays were performed on an Infinite M Plex multimode plate reader (Tecan Systems) using Costar half-area 96-well assay plates (Corning). 0.1 mL of protein stock solution at 1 mg / mL concentration was added to a well of the plate, and the solution was adjusted to the designated pH using 1 M Tris. The absorbance of the solution at 340 nm (OD 340 The first OD was measured immediately after the pH adjustment. 340The OD was set at T = 0. The OD was measured at 15 min, 30 min, 1 h, and 2 h. 340 The change over time was measured.

[0137] Results: Table 3 lists the measured relative solubilities in % monomer for different mutant IL-2 polypeptides. These results demonstrate that mutant IL-2 polypeptides engineered with a particular set of glycosylation mutations listed in Table 3 also confer substantially increased solubility compared to C125S IL-2 when fused to a monomeric Fc.

[0138] J. Size-Exclusion Chromatography: Size-exclusion chromatography (SEC-HPLC) analysis was performed on an Agilent 1100 HPLC system (Santa Clara, California) using 1x PBS as the running buffer. Samples were injected onto a prepacked Superose 12-300 column (Cytiva) equilibrated with 1x PBS buffer. The flow rate was 0.65 mL / min, and the total run time was 40 min.

[0139] Results: Figures 2A-2L show exemplary SEC-HPLC profiles of the control p123(C125SIL2) construct (Figures 2A and 2B) and the following exemplary mutant IL-2 polypeptide-Fc fusions: p296 (Figure 2C), p307 (Figure 2D), p406 (Figure 2E), p297 (Figure 2F), p300 (Figure 2G), p308 (Figure 2H), p214 (Figure 2I), p310 (Figure 2J), p411 (Figure 2K), and p298 (Figure 2L).

[0140] K. IL-2 reporter assay using HEK Blue cells: IL-2 activity was measured using HEK Blue IL-2 cells. In this cell line, HEK293 cells stably express IL-2 receptor subunits (CD25 for IL2Rα, CD122 / CD132 for IL2Rβγ) and a STAT5-inducible SEAP reporter. Cell suspensions with >90% viability were prepared for the assay. Test substances at the concentrations indicated in each experiment were prepared by serial dilution in DMEM + 10% heat-inactivated FBS and added to the cell suspension in a flat-bottom 96-well plate at 50K / well and incubated at 37°C for 20-24 hours. The following day, QuantI-Blue solution was prepared according to the instructions from Invivogen. 20 μl of inducible HEK Blue cell supernatant was added per well to a flat-bottom 96-well plate, followed by addition of 100 μl of resuspended QUANTI-Blue solution to each well. The plate was incubated at 37°C for 1-3 hours. SEAP levels were measured by measuring the absorbance at 630 nm (OD ) using a spectrophotometer. 630 ) was determined by reading the

[0141] Results: Figures 3A-3N show the HEK Blue staining for the p123C125S IL-2 fusion control and the following exemplary mutant IL-2 polypeptide Fc fusions: p124, p132, and p167 (Figures 3A-B); p296, p300, and p214 (Figures 3C-D); p123, p307, and p214 (Figures 3E-F); p123, p296, and p308 (Figures 3G-H); p123, p310, and p214 (Figures 3I-J); p296, p297, p298, and p214 (Figures 3K-L); and p411, p214, p123, p406, and p214 (Figures 3M-N). Exemplary CD25+ and CD25- binding curves and EC measured in an IL-2 reporter assay 50 A plot of the values ​​is shown.

[0142] Although 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, the present disclosure, including the examples, illustrations, and embodiments set forth herein, is for purposes of illustration and is intended to be illustrative and should not be construed as limiting the disclosure. It will be apparent to those skilled in the art that various modifications or variations to the examples, illustrations, and embodiments set forth herein can be made and should be included within the spirit and scope of the present disclosure and the appended claims. Moreover, those skilled in the art will recognize several methods and procedures equivalent to those described herein. All such equivalents should be understood to be within the scope of the present disclosure and encompassed 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 individual such publication, patent, patent application, or other document was individually and specifically indicated to be incorporated by reference herein in its entirety for all purposes. In case of conflict, the present specification, including specific language, will control.

Claims

1. 1. A polypeptide that specifically binds to an IL-2 receptor, said polypeptide comprising 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 the following: Table 11

2. 2. The polypeptide of claim 1, wherein the polypeptide further comprises one or more amino acid differences selected from K35N, F42A, Y45R, N88D, D109N, and Q126T.

3. 3. The polypeptide of claim 2, wherein the polypeptide comprises a combination of amino acid differences selected from: Table 12-1 Table 12-2

4. 2. The polypeptide of claim 1, wherein the polypeptide comprises a combination of amino acid differences selected from: Table 13-1 Table 13-2

5. The polypeptides are selected from the group consisting of SEQ ID NOs: 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. 2. The polypeptide of claim 1, wherein the polypeptide is fused to a monomeric or dimeric Fc polypeptide; optionally, 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 NOs: 146, 147, and 148.

8. The polypeptides are fused via a linker; optionally, the linker is (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) ３ The polypeptide of claim 6, which is a polypeptide comprising an amino acid sequence selected from the group consisting of: GGG (SEQ ID NO: 155).

9. The polypeptide of claim 6, wherein the polypeptide is fused via a linker to the C-terminus of the monomeric or dimeric Fc polypeptide and the N-terminus of the mutant IL-2 polypeptide.

10. The polypeptide has the following properties compared to the IL-2 polypeptide of SEQ ID NO: 10: (i) increased titer when expressed in mammalian cell culture systems; (ii) increased solubility; (iii) a reduced binding affinity for the IL-2Rα receptor subunit of SEQ ID NO: 2; and / or (iv) a reduced binding affinity to 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. 12. An isolated host cell comprising the polynucleotide of claim 11; optionally, the isolated host cell is a mammalian cell or a yeast cell.

14. 14. The isolated host cell of claim 13, wherein the host cell is a mammalian cell selected from Chinese hamster ovary (CHO) cells, myeloma cells (e.g., Y0, NS0, Sp2 / 0), monkey kidney cells (COS-7), human embryonic kidney line (293), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells, human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor cells, TR1 cells, Medical Research Council 5 (MRC5) cells, and FS4 cells.

15. 14. A method for producing a mutant IL-2 polypeptide, comprising culturing any one of the host cells of claim 13 under conditions suitable for expression of said polypeptide.

16. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier.

17. 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. 18. The method of claim 17, wherein the disease is cancer; optionally, 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 hematopoietic malignancies.

19. 18. The method of claim 17, wherein the polypeptide exhibits a selective reduction in IL-2Rα binding affinity compared to the IL-2Rα binding affinity of the C125 IL-2 polypeptide of SEQ ID NO:

10.

20. 20. The method of claim 19, wherein the polypeptide comprises a combination of amino acid differences relative to SEQ ID NO: 10 selected from: Table 14

21. 18. The method of claim 17, wherein the IL-2 mediated disease is an autoimmune disease; optionally, 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. 22. The method of claim 21, wherein the polypeptide exhibits a selective reduction in IL-2Rβγ binding affinity compared to the IL-2Rβγ binding affinity of the C125 IL-2 polypeptide of SEQ ID NO:

10.

23. 23. The method of claim 22, wherein the polypeptide comprises a combination of amino acid differences relative to SEQ ID NO: 10 selected from: Table 15-1 Table 15-2

24. 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, 18. The method of claim 17, comprising a sequence selected from 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. 18. The method of claim 17, wherein the polypeptide is fused to a monomeric or dimeric Fc polypeptide; and optionally, the Fc polypeptide is a monomeric Fc polypeptide.

26. 26. The method of claim 25, wherein the monomeric or dimeric Fc polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 146, 147, and 148.

27. the polypeptides are fused via a linker; optionally, the linker is (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) ３ 26. The method of claim 25, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: GGG (SEQ ID NO: 155).

28. 26. The method of claim 25, wherein the polypeptides are fused via a linker to the C-terminus of the monomeric or dimeric Fc polypeptide and the N-terminus of the mutant IL-2 polypeptide.

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