Interleukin-2 variants and methods of use thereof

Abstract

The present invention relates to polypeptides that share a primary sequence with human IL-2, except for a few amino acid mutations. One IL-2 variant panel contains mutations with remarkable manufacturability that preferentially promote the proliferation, survival, activation, and / or function of immunosuppressive regulatory T cells (Treg: CD4+CD25+FoxP3+) over effector T cells and NK cells. Therapeutic uses of such IL-2-selective agents, used alone or in combination with immunomodulatory drugs or antibodies, proteins, or peptides that target diseased tissues, to treat Treg cell deficiencies, various autoimmune and inflammatory disorders, organ transplantation, and graft-versus-host disease are also encompassed. In another aspect, the present invention relates to pharmaceutical compositions comprising the disclosed polypeptides. Finally, the present invention relates to therapeutic uses of the disclosed polypeptides and pharmaceutical compositions for the selective modulation of the immune system in diseases such as autoimmune and inflammatory disorders. [Selection diagram] None

Description

[Technical field]

[0001] Related patent applications This application claims the benefit of U.S. Provisional Application No. 62 / 755,016, filed November 2, 2018, and U.S. Provisional Patent Application No. 62 / 689,055, filed June 22, 2018, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Interleukin 2 (IL-2) was the first growth factor described in association with T cells. Since its discovery, IL-2 has been shown to promote T cell proliferation and survival in vitro (Smith, K. A. (1988), Science. 240:1169-76) and to have the ability to boost immune responses in the context of T viral infection (Blattman, J. N. et al. (2003), Nat Med. 9:540-7) and vaccines (Fishman, M. et al. (2008), J Immunother. 31:72-80; Kudo-Saito, C. et al. (2007), Cancer Immunol Immunother. 56:1897-910; Lin, C. T. et al. (2007), Immunol Lett. 114:86-93).

[0003] IL-2 is used in cancer therapy. Recombinant human IL-2 is an effective immunotherapy for metastatic melanoma and renal cancer, with durable responses observed in approximately 10% of patients. However, the optimal dose of IL-2 is limited by its short half-life and high toxicity. Furthermore, IL-2 binds with greater affinity to its heterotrimeric receptor IL-2Rαβγ, leading to preferential expansion of immunosuppressive regulatory T cells (Tregs), which constitutively express high levels of IL-2Rα. Expansion of Tregs is an undesirable effect of IL-2 for cancer immunotherapy. However, the ability of IL-2 to stimulate Treg cells even at low doses could be potentially exploited in the treatment of autoimmune and various inflammatory disorders.

[0004] Tregs are central to immune system homeostasis and play a major role in maintaining peripheral immune tolerance by suppressing (autoreactive) effector T cells. Deficiencies in Treg cell numbers or Treg function have been demonstrated in several autoimmune and inflammatory diseases. As a result, there is great interest in developing therapies to enhance Treg cell numbers and / or function. One therapeutic approach under investigation for autoimmune diseases is the use of low doses of IL-2 to target Treg cells, which respond better to lower concentrations of IL-2 than many other immune cell types due to their constitutively high levels of IL-2Rα (Klatzmann D, 2015, Nat Rev Immunol. 15:283-94). Clinical trials of low-dose IL-2 treatment in various GvHD (Koreth, J. et al., 2011, N Engl J Med., 365:2055-66) and HCV-associated autoimmune vasculitis (Saadoum, D. et al., 2011, N Engl J Med., 365:2067-77) patients have shown increased Treg levels and signs of clinical benefit. However, even at these low doses, serious safety and tolerability issues have arisen. Thus, there is a need for effective autoimmune / inflammatory disease treatments that specifically target Treg cells rather than IL-2 and can enhance Treg cell numbers and function.

[0005] More recently, it has been found that IL-2 can be modified to selectively stimulate either cytotoxic effector T cells or Treg cells. Various approaches have produced IL-2 variants with improved and selective immune stimulatory capacity (see, for example, U.S. Pat. Nos. 7,186,804, 7,105,653, 6,955,807, 5,229,109, and U.S. Patent Publication No. 20050142106). In the present invention, IL-2 variants were designed to enhance the ability of this molecule to signal primarily through high affinity receptors (α, β, and γ chains) but not through intermediate affinity receptors (β and γ chains). The basic idea was to promote signaling in Treg cells but not in effector T cells and NK cells, which were believed to be responsible for the observed toxic effects. Importantly, there is no prior art disclosure of IL-2 variants that have greater therapeutic efficacy in vivo than native IL-2 based on their ability to stimulate natural regulatory T cells. However, since the early work with IL-2 variants, research in this field has provided more complete evidence that Treg cells constitutively highly express IL-2Rα (CD25) along with IL-2Rβ and γc, and IL-2 variants, as IL-2Rαβγ selective agonists, should be selective for Treg cells.

[0006] In summary, IL-2 is a highly pleiotropic cytokine highly relevant to the biological activities of various cell populations. This property makes IL-2 a key intersection in the control of immune responses and an attractive target for therapeutic approaches and complex immune modulation. Furthermore, receptor subunit-biased IL-2 variants allow the achievement of selective immune modulation via IL-2 to promote the proliferation and activity of regulatory T cells (Tregs) while minimizing helper and cytotoxic effector T (Teff) cells, and to reduce the levels of proinflammatory signaling molecules. Summary of the Invention

[0007] In one embodiment, the present invention relates to the generation of mutant IL-2 variants. These variants are characterized by enhanced selectivity in stimulating Treg (T CD4+CD25+FoxP3+) cells over normal CD4+ T cells, CD8+ T lymphocytes with cytotoxic effects, and NK cells. In particular, these variants provide a practical solution for improving IL-2 therapy in autoimmune and inflammatory disorders. The present invention relates to polypeptides that share a primary sequence with human IL-2, except for one to several amino acid mutations. These variants have amino acid substitutions at positions corresponding to the interaction of IL-2 with IL-2Rβ and / or γc, such that these variants have reduced affinity for the IL-2Rβγ receptor complex and reduced or abolished ability to activate IL-2Rβγ expressing cells, but retain the ability to bind to IL-2Rα and to bind to and activate the IL-2Rαβγ receptor complex. The present invention also encompasses therapeutic uses of these mutant variants for the treatment of autoimmune and various inflammatory disorders.

[0008] In one embodiment, the present invention relates to the generation of mutant IL-2 variants. These variants have a combination of attributes with Treg selective activity, reduced aggregation, increased expression, improved manufacturability, and improved compatibility with development. These variants also provide improved biophysical properties of the protein and reduced immunogenic risk associated with the IL-2 molecule. These variants provide a better solution for improving IL-2 therapy and reduced toxicity in autoimmune and inflammatory disorders. The present invention relates to polypeptides that share a primary sequence with human IL-2, except for one to a few amino acid mutations.

[0009] In one embodiment, the present invention provides a novel method for the preparation of a novel 'antimicrobial' agonist that mimics components of bacterial toxins. 19The present invention relates to the generation of mutant IL-2 variants characterized by the removal of the "LDL" motif (Baluna R, Rizo et al., Proc Natl Acad Sci, 1999; 96: 3957-62). This "toxic motif" is partially responsible for the direct vascular toxicity of IL-2. Mutations introduced to remove the critical residues D20 or two adjacent residues of the toxin-like domain are expected to remove the toxic motif, prevent endothelial cell damage, and greatly reduce VLS. Importantly, since this motif is located at the interface with IL-2Rβ, amino acid substitutions for this motif will reduce its affinity for IL-2Rβ, and the resulting molecule would be expected to have two beneficial properties, including selectivity in Treg cell activation and reduced endothelial damage. The present invention relates to polypeptides that share a primary sequence with human IL-2, except for one to several amino acid mutations. The present invention also encompasses the therapeutic use of these mutant variants to treat Treg cell-deficient autoimmune disorders and various inflammatory disorders.

[0010] The present invention allows for substantial improvements of current IL-2-based immunomodulatory strategies in the treatment of autoimmune and various inflammatory disorders. Specifically, replacement of native IL-2 with the mutant variants described herein results in selective stimulation of Treg cells with a bias towards CD25. In various embodiments, the IL-2 variant (or mutant) comprises an IL-2 variant (or mutant) sequence derived from the sequence of the mature human IL-2 polypeptide set forth in SEQ ID NO: 3. In various embodiments, the IL-2 variant functions as an IL-2 agonist. In various embodiments, the IL-2 variant functions as an IL-2 antagonist. In various embodiments, the IL-2 variant comprises a sequence set forth in SEQ ID NOs: 4-43, 108-146, and 193-197.

[0011] The present invention allows for substantial improvements of current IL-2-based immunomodulatory strategies in the treatment of Treg cell-deficient autoimmune disorders and various inflammatory disorders. Specifically, replacement of native IL-2 with the mutant variants described herein is expected to result in selective stimulation of Treg cells biased towards CD25, remove toxic motifs, prevent endothelial cell damage, and significantly reduce VLS. In various embodiments, the IL-2 variants (or mutants) comprise a sequence of an IL-2 variant (or mutant) derived from the sequence of the mature human IL-2 polypeptide set forth in SEQ ID NO: 3. In various embodiments, the IL-2 variants function as IL-2 agonists. In various embodiments, the IL-2 variants function as IL-2 antagonists. In various embodiments, the IL-2 variants comprise SEQ ID NOs: 5-14, 26-43, 108-111, 125-146, and 193-197.

[0012] In another aspect, the IL-2 variant of the present invention is attached to at least one heterologous protein. In various embodiments, the IL-2 variant is fused to at least one polypeptide that confers an extended half-life to the fusion molecule. Such polypeptides include other polypeptides that bind to IgG Fc or neonatal Fcγ / receptor, human serum albumin, or polypeptides that bind to proteins with long serum half-lives, including IgG, immunoglobulins other than IgG, proteins, and non-protein drugs that have an extended in vivo half-life due to the presence of an IgG constant domain, or a portion thereof that binds to FcRn, with one or more amino acid modifications that increase the affinity of the constant domain or fragment for FcRn. Such proteins and molecules with extended half-lives have the advantage that less and / or less frequent administration is required for therapeutic, prophylactic, or diagnostic use of such molecules (see, for example, U.S. Pat. No. 7,658,921).

[0013] In various embodiments, the IL-2 variant can be linked to the N-terminus or C-terminus of the heterologous protein.

[0014] In various embodiments, the IL-2 variant is fused to an IgG Fc molecule. In various embodiments, the Fc domain is a human IgG Fc domain. In various embodiments, the Fc domain is derived from a human IgG1 heavy chain constant domain sequence set forth in SEQ ID NO: 44. In various embodiments, the Fc domain is an Fc domain having an amino acid sequence set forth in SEQ ID NO: 45. In various embodiments, the Fc domain is an Fc domain having an amino acid sequence set forth in SEQ ID NO: 46. In various embodiments, the Fc domain is an Fc domain having an amino acid sequence set forth in SEQ ID NO: 47. In various embodiments, the Fc domain is derived from a human IgG2 heavy chain constant domain sequence. In various embodiments, the Fc domain is derived from a human IgG4 heavy chain constant domain sequence.

[0015] The term "Fc" refers to a molecule or sequence that includes the sequence of a non-antigen-binding fragment of a full-length antibody, which may be in monomeric or multimeric form. The original immunoglobulin source of the native Fc is preferably of human origin and may be any immunoglobulin disclosed in the art. The native Fc is composed of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent (i.e., disulfide) and non-covalent bonds. The number of intermolecular disulfide bonds between the monomeric subunits of the native Fc molecule ranges from 1 to 4, depending on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is the disulfide-linked dimer resulting from papain digestion of IgG (Ellison et al., (1982), Nucleic Acids Res., 10:4071-9). The term "native Fc" as used herein refers collectively to monomeric, dimeric, and multimeric forms of the Fc domain that contains binding sites for Protein A, Protein G, various Fc receptors, and complement proteins.

[0016] In various embodiments, the term "Fc variant" refers to a molecule or sequence that has been modified from a native Fc but still contains a binding site for the salvage receptor, FcRn. Exemplary Fc variants and interactions with the salvage receptor are described in International Publication Nos. WO 97 / 34631 (published September 25, 1997) and WO 96 / 32458, which are incorporated herein by reference. Additionally, native Fc contains sites that may be removed because they confer structural features or biological activity that are not required for the fusion molecules of the present invention. That is, in various embodiments, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity after expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than the salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0017] The term "Fc domain" encompasses native Fc and Fc variant molecules and sequences as defined above. As with Fc variants and native Fc, the term "Fc domain" encompasses molecules in monomeric or multimeric form, either digested from full-length antibodies or produced by recombinant gene expression or other means. In various embodiments, "Fc domain" refers to a dimer consisting of two Fc domain monomers (SEQ ID NO: 44), usually including all or part of the hinge region. In various embodiments, the Fc domain may be mutated to lack effector function. In various embodiments, each of the Fc domain monomers of the Fc domain comprises an amino acid substitution in the CH2 antibody constant domain to reduce the interaction or binding between the Fc domain and the Fcγ receptor. In various embodiments, each subunit of the Fc domain comprises two amino acid substitutions (L234A, L235A) that reduce binding to activating Fc receptors and / or effector function. In various embodiments, each subunit of the Fc domain contains three amino acid substitutions (L234A, L235A, and G237A) that reduce binding to activating Fc receptors and / or effector function (SEQ ID NO:45).

[0018] In various embodiments, the Fc domain may be mutated to further increase in vivo half-life. In various embodiments, each subunit of the Fc domain contains three amino acid substitutions (M252Y, S254T, and T256E) that enhance binding to human FcRn as disclosed in U.S. Pat. No. 7,658,921. In various embodiments, each subunit of the Fc domain contains one amino acid substitution (N434A) that enhances binding to human FcRn as disclosed in U.S. Pat. No. 7,371,826. In various embodiments, each subunit of the Fc domain contains one amino acid substitution (M428L and N434S) that enhances binding to human FcRn as disclosed in U.S. Pat. No. 8,546,543. In various embodiments, the half-life extending mutations can be combined with amino acid substitutions that reduce binding to activating Fc receptors and / or effector functions.

[0019] In various embodiments, the two Fc domain monomers of the Fc domain each contain an amino acid substitution that promotes heterodimerization of the two monomers. In various other embodiments, heterodimerization of the Fc domain monomers can be promoted by introducing different but compatible substitutions (such as a "knob-into-hole" residue pair) into the two Fc domain monomers. This "knob-into-hole" technique is also disclosed in U.S. Pat. No. 8,216,805. In yet another embodiment, one Fc domain monomer contains the knob-type mutation T366W and the other Fc domain monomer contains the hole-type mutations T366S, L358A, and Y407V. In various embodiments, two Cy residues that form a stabilizing disulfide bridge (S354C on the "knob" side and Y349C on the "hole" side) have been introduced. The use of heterodimeric Fc allows for monovalent IL-2 variant constructs to be obtained.

[0020] In various embodiments, the IL-2 variant Fc fusion protein will be monomeric, i.e., contains only one IL-2 mutein molecule. In such embodiments, the fusion protein is co-expressed with a heterodimeric Knob-Fc linked to an IL-2 variant and a corresponding heterodimeric Hole Fc. When a heterodimer of the two Fc-containing polypeptides is formed, the resulting protein contains only one IL-2 variant.

[0021] In various embodiments, IL-2 variants are used to generate Fc-IL-2 fusion proteins set forth in SEQ ID NOs: 69-107, 147-189, and 198-208.

[0022] In various embodiments, the IL-2 variants of the present invention can be attached to an antibody that provides an extended half-life to the fusion molecule, such as an anti-keyhole limpet hemocyanin (KLH) antibody. Such an antibody recognizes the foreign antigen and confers a longer half-life, but has no biological function or harm in humans. The IgG class could be IgG, IgA, IgE or subclasses (e.g., IgG1, IgG2, IgG3, IgA1, IgA2).

[0023] In various embodiments, the heterologous protein is attached to the IL-2 variant by a linker peptide and / or hinge linker peptide. The linker or hinge linker can be an artificial sequence of 5, 10, 15, 20, 30, 40, or more amino acids (or any number in between) that has relatively little secondary structure.

[0024] In various embodiments, the heterologous protein is attached to the IL-2 variant by a rigid linker peptide of 10, 15, 20, 30, 40 or more amino acids (or any number in between) that exhibits an alpha-helical conformation and can act as a rigid spacer between the protein domains.

[0025] In another aspect, the IL-2 variant can be linked to various non-proteinaceous polymers, including but not limited to various polyols such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, by methods described in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192, or 4,179,337. In various embodiments, amino acid substitutions can be made at various positions within the IL-2 variant to facilitate the addition of polymers such as PEG. In various embodiments, such PEGylated proteins can have extended half-lives and / or reduced immunogenicity over non-PEGylated proteins.

[0026] "Polyethylene glycol" or "PEG" refers to a polyalkylene glycol compound or derivative thereof, with or without a conjugation agent or derivatization with a conjugated or activated moiety (e.g., an aldehyde moiety, a hydroxysuccinimidyl moiety, a hydrazide moiety, a thiol moiety, a triflate moiety, a tresylate moiety, an azirdine moiety, an oxirane moiety, an orthopyridyl disulfide moiety, a vinyl sulfone moiety, an iodoacetamide moiety, or a maleimide moiety). In various embodiments, PEG encompasses substantially linear PEG, linear PEG, branched PEG, or dendritic PEG. PEG is a well-known water-soluble polymer that is commercially available or can be made by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, vol. 3, pp. 138-161).

[0027] In various embodiments, the Il-2 variants can be non-covalently or covalently linked at the N- or C-terminus to other polypeptides that bind to IgG Fc or neonatal Fcγ / receptors, to polypeptides that bind to human serum albumin, or to proteins with long serum half-lives, or to various nonproteinaceous polymers.

[0028] In another aspect, the disclosure provides a pharmaceutical composition comprising an IL-2 variant in admixture with a pharma- ceutically acceptable carrier.

[0029] In another embodiment, the IL-2 variant may be formulated for sustained release, such as by covalent or non-covalent attachment to hydrogels, nanoparticles, and the like.

[0030] In another aspect, the present disclosure provides a method for treating an autoimmune disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of the present invention. In one embodiment, the subject is a human subject. The autoimmune disease of the present invention is a disease or disorder arising from, or directed against an individual's own tissue, or a co-segregate or manifestation thereof, or a condition resulting therefrom. In various embodiments, the autoimmune disease includes graft versus host disease (GvHD), immune-related adverse events (irAEs), arthritis (including rheumatoid arthritis, reactive arthritis), systemic lupus erythematosus (SLE), atopic dermatitis, psoriasis, and inflammatory bowel disease (IBD), encephalomyelitis, uveitis, myasthenia gravis, multiple sclerosis, insulin-dependent diabetes mellitus (type 1 diabetes), various dermatitis, Addison's disease, celiac disease, chronic fatigue syndrome, autoimmune hepatitis, autoimmune alopecia, ankylosing spondylitis, ulcerative colitis, Crohn's disease, fibromyalgia, pemphigus vulgaris, Sjogren's syndrome, Kawasaki disease, hyperthyroidism / Graves' disease, hypothyroidism / Hashimoto's disease, endometriosis, scleroderma, pernicious anemia, Goodpasture's syndrome, Guillain-Barré syndrome, Wegener's disease, and the like. These include, but are not limited to, autoimmune diseases, glomerulonephritis, aplastic anemia (including patients with frequently transfused aplastic anemia), paroxysmal nocturnal hemoglobinuria, myelodysplastic syndromes, idiopathic thrombocytopenic purpura (IPT), autoimmune hemolytic anemia (AIHA), Evans syndrome, Factor VIII inhibitor syndrome, systemic vasculitis, dermatomyositis, polymyositis and rheumatic fever, autoimmune lymphoproliferative syndrome (ALPS), autoimmune bullous pemphigoid, Parkinson's disease, sarcoidosis, vitiligo, primary biliary cirrhosis, and autoimmune myocarditis.

[0031] In another aspect, the disclosure provides a method for treating an autoimmune disease in a subject, comprising administering a therapeutically effective amount of a pharmaceutical composition of the invention in combination with a second therapeutic agent capable of treating the autoimmune disease.

[0032] In another aspect, the present disclosure provides a method for treating inflammatory disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of the present invention.In one embodiment, the subject is a human subject.In various embodiments, the inflammatory disease to be treated includes, but is not limited to, Crohn's disease, colitis, dermatitis, psoriasis, diverticulitis, hepatitis, irritable bowel syndrome (IBS), lupus erythematous, nephritis, Parkinson's disease, ulcerative colitis, collagen colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet's syndrome and unspecified colitis, multiple sclerosis (MS), Alzheimer's disease, arthritis, rheumatoid arthritis, asthma, and various cardiovascular diseases such as atherosclerosis and vasculitis. In various embodiments, the inflammatory disease is selected from the group consisting of rheumatoid arthritis, diabetes, gout, cryopyrin-associated periodic syndrome, and chronic obstructive pulmonary disorder.

[0033] In another aspect, the disclosure provides a method for treating an inflammatory disease in a subject, comprising administering a therapeutically effective amount of a pharmaceutical composition of the invention in combination with a second therapeutic agent capable of treating the inflammatory disease.

[0034] In another aspect, the present disclosure provides a method for organ transplantation or related graft-versus-host disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of the present invention.In one embodiment, the subject is a human subject.In various embodiments, the transplantation is selected from heart organ transplantation, kidney organ transplantation, liver organ transplantation, lung organ transplantation, pancreas organ transplantation, intestine organ transplantation, and thymus organ transplantation, or is selected from bone tissue transplantation, tendon tissue transplantation, cornea tissue transplantation, skin tissue transplantation, heart valve tissue transplantation, nerve tissue transplantation, and vein tissue transplantation.

[0035] In another aspect, the disclosure provides the use of an IL-2 variant for the preparation of a medicament for treating an autoimmune disease.

[0036] In another aspect, the disclosure provides the use of an IL-2 variant for the preparation of a medicament for the treatment of organ transplantation and GvHD.

[0037] In another aspect, the disclosure provides the use of an IL-2 variant for the preparation of a medicament for treating an inflammatory disorder.

[0038] In another aspect, the disclosure provides an isolated nucleic acid molecule comprising a polynucleotide encoding an IL-2 variant of the disclosure. In another aspect, the disclosure provides a vector comprising a nucleic acid as described herein. In various embodiments, the vector is an expression vector. In another aspect, the disclosure provides an isolated cell comprising a nucleic acid of the disclosure. In various embodiments, the cell is a host cell comprising an expression vector of the disclosure. In another aspect, a method of producing an IL-2 variant by culturing the host cell under conditions promoting expression of the protein or polypeptide of the disclosure is provided. [Brief description of the drawings]

[0039] [Figure 1] Figure 1 shows size-exclusion chromatograms of exemplary IL-2 Fc fusion proteins, A) P-0250, B) P-0318, C) P-0317, D) P-0447, and E) P-0511, following Protein A purification, and Figures 1D and 1E show SDS-PAGE of each sample in the absence (lane 2) and presence (lane 3) of a reducing agent. [Diagram 2] FIG. 2 shows the differential effects of IL-2 variant Fc fusion proteins with an amino acid substitution at aspartic acid at position 20 (D20X) on induction of STAT5 phosphorylation in CD4+ Treg cells (A) and CD4+ Tconv cells (B) in a human PBMC assay, relative to the wild-type fusion protein (P-0250). [Diagram 3]FIG. 3 shows the differential effect of the IL-2 variant Fc fusion protein P-0375 (N88Q) on induction of STAT5 phosphorylation in CD4+ Treg cells (A) and CD4+ Tconv cells (B) relative to wild-type protein (P-0250) and reference protein controls in a human PBMC assay. [Figure 4] Figure 4 shows the differential effect of IL-2 variants with amino acid substitutions at position 19 on STAT5 phosphorylation by Fc fusion proteins compared to wild type (P-0250). The ability to induce STAT5 phosphorylation in CD4+ Treg cells (A and C) and CD4+ Tconv cells (B and D) was determined in a human PBMC assay by FACS analysis. [Diagram 5] Figure 5 shows the differential effect of IL-2 variants with combined mutations (P-419), individual amino acid substitutions at positions 19 (P-0372) or 126 (P-0303) on STAT5 phosphorylation by Fc fusion proteins compared to wild-type (P-0250) or reference-1 protein. The ability to induce STAT5 phosphorylation in CD4+ Treg cells (A, C, and E) and CD4+ Tconv cells (B, D, and F) was determined by FACS analysis. [Figure 6] Figure 6 shows the differential effects of IL-2 variants with different combinations of double amino acid substitutions on STAT5 phosphorylation by Fc fusion proteins (P-0419, P-0464, P-0471, P-0474, P-0417, and P-0322) compared to wild type (P-0250). The biological activity of P-0417 and P-0322 was also compared to their counterparts with single amino acid substitutions, P-0373 and P-0363. The ability to induce STAT5 phosphorylation in CD4+Treg cells (A and C) and CD4+Tconv cells (B and D) was determined in human PBMC assays by FACS analysis. [Figure 7]Figure 7 shows the differential effects of Fc fusion proteins of IL-2 variants with individual amino acid substitutions at positions 19 (P-0424) or 126 (P-0303) or combination mutations (P-0447) on STAT5 phosphorylation compared to wild type (P-0250) and the differential effects of Fc fusion proteins of IL-2 variants with different combinations of amino acid substitutions (P-0419, P-0447, P-0448, and P-0449) on STAT5 phosphorylation compared to wild type (P-0250) and a reference Fc fusion protein. The ability to induce STAT5 phosphorylation in CD4+Treg cells (A and C) and CD4+Tconv cells (B and D) was determined in human PBMC assays by FACS analysis. [Figure 8] Figure 8 shows the pSTAT5 stimulatory activity of IL-2 fusion proteins P-0250, P-0424, and P-447 relative to their respective S125I substitution counterparts P-0531, P-0491, and P-0511. The ability to induce STAT5 phosphorylation in CD4+ Treg cells (A, C, and E) and CD4+ Tconv cells (B, D, and F) was determined in human PBMC assays by FACS analysis. [Figure 9] Figure 9 shows the differential effect of IL-2 variant Fc fusions (P-0511 and P-0512) on STAT5 phosphorylation in three CD4+ T cell subsets: A) CD4+FoxP3+CD25+ Treg cells, B) CD4+FoxP3-CD25+ activated Tconv cells, and C) CD4+FoxP3-CD25- naive Tconv cells. STAT5 phosphorylation induction was determined in human PBMC assay by FACS analysis. [Figure 10]Figure 10 shows the differential effect of P-0511 and P-0512 on stimulating proliferation of A) CD8+ T cells and B) NK cells compared to wild type (P-0250) and reference molecule. Cell proliferation was determined in a human PBMC assay by FACS analysis of a CFSE dilution series and expressed as the percentage of dividing cells. [Figure 11] Figures 11A-11D show the differential effect of IL-2 variant Fc fusion P-0511 on STAT5 phosphorylation induction in different cell types compared to its wild type counterpart P-0531. The ability to induce STAT5 phosphorylation in A) CD4+ Treg cells, B) CD4+ Tconv cells, C) CD8+ T cells, and D) NK cells was determined in human PBMC assays by FACS analysis. Figure 11E shows the strength of binding of P-511 to the IL-2Rβ and γc complexes compared to P-0531 and reference-1 in an ELISA assay. [Figure 12] Figure 12 shows the proliferation potential and proliferation of Treg cells in IL-2 variant Fc fusion protein-treated and reference treated mice after a single subcutaneous injection. Blood was collected at the indicated time points for proliferation assay and lymphocyte phenotyping. (A) Percentage of Treg cells positive for proliferation marker Ki67; (B) Percentage of Treg cells in total CD4+ T cell population; (C) Percentage of Treg cells in total blood lymphocytes. Data are presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's post-hoc test. ****p<0.0001; ***p<0.001 compared to PBS group at each time point. [Figure 13]Figure 13 shows the proliferation potential of effector T cells and NK cells in IL-2 mutant Fc fusion protein-treated and reference-treated mice after a single subcutaneous injection. Blood was collected at the indicated time points and lymphocyte proliferation was measured. (A) Percentage of Ki67 positive CD4+ conventional T (Tconv) cells; (B) Percentage of Ki67 positive CD8+ T cells; (C) Percentage of Ki67 positive NK cells. Data are presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's post-hoc test. ****p<0.0001; ***p<0.001 compared to PBS group at each time point. [Figure 14] Figure 14 shows the proliferation of effector T cells and NK cells in IL-2 mutant Fc fusion protein and reference treated mice after a single subcutaneous injection. (A-B) Percentage of CD4+ conventional T (Tconv) cells among total CD4+ T cells (A) and total blood lymphocytes (B). (C) Percentage of CD8+ T cells among total blood lymphocytes; (D) Percentage of NK cells among total blood lymphocytes. Data are presented as mean ± SEM. [Figure 15] Figure 15 shows the ratio of Treg cells to Tconv cells based on A) percentage of Ki67 positive expression and B) cell count in IL-2 mutant Fc fusion protein treated mice and control treated mice. Data were obtained by FACS and are expressed as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's post-hoc test. ****p<0.0001; *p<0.05 compared to PBS group at each time point. [Figure 16] Figure 16 shows the expression of CD25 and Foxp3 on Treg cells in IL-2 mutant Fc fusion protein and reference treated mice after a single subcutaneous injection. A) Foxp3 and B) CD25 expression levels were analyzed by FACS analysis and are presented as mean fluorescence intensity (MFI). Data are presented as mean ± SEM. ****p<0.0001 compared to PBS group at each time point. [Figure 17]FIG. 17 shows a dose-dependent increase in the proliferation and proliferation of Treg cells in mice after a single injection of IL-2 variant Fc fusion protein P-0511. Blood was collected at the indicated time points for lymphocyte phenotyping and Ki67 proliferation marker measurement. (A) Percentage of proliferation marker Ki67 positive Treg cells; (B) Percentage of Treg cells among total CD4+ T cells; (C) Number of Treg cells per microliter of whole blood; (D) Fold change from baseline in Treg cell counts in each group. Data are presented as mean±SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's post-hoc test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05 compared to PBS group at each time point. [Figure 18] Figure 18 shows the dose-dependent effect of IL-2 variant Fc fusion protein P-0511 on the percentage of Treg cells (A), CD4+Tconv cells (B), CD8 T cells (C), and NK cells (D) among total lymphocytes in mice after a single injection. Blood was collected at the indicated time points for lymphocyte phenotyping. Data were obtained by FACS analysis and are presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's post-hoc test. ****p<0.0001, **p<0.01, *p<0.05 compared to PBS group at each time point. [Figure 19] Figure 19 shows a dose-dependent increase in A) the ratio of Treg to Tconv cell numbers, B) CD25 expression on Treg cells, and C) Foxp3 expression on Treg cells in mice after a single injection of P-0511. Data were obtained by FACS analysis and are presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's post-hoc test. ****p<0.0001, ***p<0.001, **p<0.01 compared to the PBS group at each time point. [Figure 20]Figure 20 shows that Treg cell proliferation and proliferation was maintained in mice receiving repeated injections of IL-2 variant Fc fusion proteins (P-0511 and P-0512), but not wild-type Fc fusion protein (P-0531) and control. Compounds were administered subcutaneously once every three days (Q3D), and blood was collected 3 days after the first and third injection for lymphocyte phenotyping and proliferation marker Ki67 measurement. (A) Percentage of Ki67 positive Treg cells; (B) Percentage of Treg cells in total CD4+ T cell population; (C) Percentage of Treg cells in total blood lymphocytes. Data were obtained by FACS analysis and are presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's post-hoc test. ****p<0.0001, *p<0.05 compared to each PBS group. [Figure 21] Figure 21 shows that the increase in Treg cell numbers was maintained in mice receiving repeated injections of IL-2 variant Fc fusion proteins (P-0511 and P-0512), but not wild-type Fc fusion protein (P-0531) or control. Compounds were administered subcutaneously once every three days (Q3D) and blood was collected 3 days after the first and third injection for lymphocyte phenotyping and proliferation marker Ki67 measurement. (A) Number of Treg cells per microliter of whole blood; (B) Fold change in Treg numbers compared to PBS control group. Data were obtained by FACS analysis and are presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's post-hoc test. ****p<0.0001, ***p<0.001, **p<0.01 compared to each PBS group. [Figure 22]Figure 22 shows that the elevated ratio of Treg to Tconv was maintained in mice receiving repeated injections of IL-2 variant Fc fusion proteins (P-0511 and P-0512), but not wild type Fc fusion protein (P-0531) or control. Compounds were administered subcutaneously once every three days (Q3D) and blood was collected 3 days after the first and third injection for Treg and Tconv cell phenotyping. The ratios were calculated based on %Treg and %Tconv in total CD4 cells. Data were obtained by FACS analysis and are presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's post-hoc test. ****p<0.0001 compared to the respective PBS groups. [Figure 23] FIG. 23 shows the suppression of antigen-driven inflammation by P-0511 in a mouse model of delayed-type hypersensitivity (DTH) induced by keyhole limpet hemocyanin (KLH) antigen. Mice were immunized with KLH on day 0 and re-immunized in the right ear on day 5. Mice were treated with P-0511 every 3 or 5 days starting on day -2. Kinetics of DTH response using change in ear thickness relative to baseline values ​​(Δear thickness) at various time points after KLH exposure for A) every 3 days and B) every 5 days dosing schedules are shown. Data are presented as mean±SEM. Statistical analysis was by one-way ANOVA followed by Tukey's post-hoc test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05 compared to each PBS group at each time point. [Figure 24]Figure 24 shows the suppression of antigen-driven inflammation by P-0511 compared to the reference molecule (reference-1) in a mouse model of DTH induced by KLH antigen. Mice were immunized with KLH on day 0 and re-immunized in the right ear on day 5. Mice were treated with the compounds every 5 days starting on day -2. The kinetics of the DTH response was shown using the change in ear thickness (Δ ear thickness) relative to baseline values ​​at various time points after KLH exposure. Data are presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's post-hoc test. ****p<0.0001, **p<0.01, *p<0.05 compared to the respective PBS group at each time point. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] The present invention relates to polypeptides that share a primary sequence with human IL-2, except for one to several amino acid mutations. One panel of IL-2 variants contains mutations that preferentially promote the proliferation, survival, activation and / or function of immunosuppressive regulatory T cells (T CD4+CD25+FoxP3+) over effector T cells and NK cells. The present invention also encompasses therapeutic uses of such IL-2 selective agonists, alone or in conjunction with diseased tissue targeting proteins or peptides, to treat autoimmune disorders and various inflammatory disorders. Another panel of IL-2 variants contains mutations that significantly reduce the ability of these polypeptides to stimulate Treg or Tconv cells (although they may retain receptor binding activity), making them effective as IL-2 antagonists. In another aspect, the present invention relates to pharmaceutical compositions comprising the polypeptides of the present disclosure. Finally, the present invention relates to therapeutic uses of the polypeptides and pharmaceutical compositions of the present disclosure due to their selective regulatory effect on the immune system against diseases such as autoimmune inflammatory disorders.

[0041] definition As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues. In various embodiments, a "peptide", "polypeptide" and "protein" are amino acid chains in which the alpha carbons are linked through peptide bonds. The terminal amino acid at one end of the chain (the amino terminus) i.e. has a free amino group, while the terminal amino acid at the other end of the chain (the carboxy terminus) has a free carboxyl group. As used herein, the term "amino terminus" (abbreviated N-terminus) refers to the free alpha-amino group on the amino acid at the amino terminus of a peptide, or to the alpha-amino group of an amino acid anywhere else within the peptide (when participating in a peptide bond). Similarly, the term "carboxy terminus" refers to the free carboxyl group at the carboxy terminus of a peptide, or to the carboxyl group of an amino acid anywhere else within the peptide. Peptides also encompass virtually any polyamino acid, including but not limited to peptidomimetics, such as those in which the amino acids are linked by ethers rather than amide bonds.

[0042] Polypeptides of the present disclosure include polypeptides that have been modified in any way and for any reason, for example, to (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for the purposes of protein complex formation, (4) altered binding affinity, and (5) impart or modify other physicochemical or functional properties.

[0043] An amino acid "substitution," as used herein, refers to the replacement in a polypeptide of an amino acid at a particular position in a parent polypeptide sequence with a different amino acid. Amino acid substitutions can be made using genetic or chemical techniques well known in the art. For example, a single amino acid substitution (e.g., a conservative amino acid substitution) or multiple amino acid substitutions can be made in a native sequence (e.g., in a portion of a polypeptide outside of the domains forming intermolecular contacts). A "conservative amino acid substitution" refers to the replacement of an amino acid with a functionally similar amino acid in a polypeptide. Each of the following six groups contains amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), and Threonine (T) 2) Aspartic acid (D) and glutamic acid (E) 3) Asparagine (N) and Glutamine (Q) 4) Arginine (R) and Lysine (K) 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V) 6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W)

[0044] "Non-conservative amino acid substitutions" refer to the substitution of a member of one of these classes for a member of another class. In making such changes, various embodiments may take into account the hydropathic index of amino acids. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. The respective hydropathic indices are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0045] The importance of the amino acid hydropathic index in conferring interactive biological function to a protein is understood in the art (see, for example, Kyte et al., 1982, J. Mol. Biol., 157:105-131). It is known that certain amino acids can be substituted with other amino acids having similar hydropathic indices or hydropathic scores while retaining similar biological activity. When making changes based on hydropathic indices, various embodiments include substitutions of amino acids whose hydropathic indices are within ±2 of each other. Various embodiments include those within ±1, and various embodiments include those within ±0.5.

[0046] It is also understood in the art that, particularly where the biologically functional proteins or peptides thus produced are intended for use in the immunological embodiments disclosed herein, substitutions of similar amino acids can be made efficiently on the basis of hydrophilicity. In various embodiments, the maximum local average hydrophilicity of a protein, as controlled by the hydrophilicity of adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., correlates with the biological properties of the protein.

[0047] The following hydrophilicity values ​​have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0.+-.1); glutamic acid (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5), and tryptophan (-3.4). In making changes based on similar hydrophilicity values, various embodiments include substitutions of amino acids with hydrophilicity values ​​within ±2, in various embodiments within ±1, and in various embodiments within ±0.5.

[0048] Exemplary amino acid substitutions are set forth in Table 1.

[0049] TIFF0007637415000001.tif187170

[0050] Those skilled in the art can use well-known techniques to determine suitable variants of the polypeptides described herein. In various embodiments, those skilled in the art can identify suitable regions of the molecule that can be altered without destroying activity by targeting regions that are not believed to be important for activity. In other embodiments, those skilled in the art can identify residues and portions of the molecule that are conserved between similar polypeptides. In further embodiments, even regions that may be important for biological activity or structure can be subject to conservative amino acid substitutions without destroying biological activity or adversely affecting polypeptide structure.

[0051] Additionally, one of skill in the art can investigate structure-function studies that identify residues in similar polypeptides that are important for activity or structure. Taking such comparisons into account, one of skill in the art can predict the importance of amino acid residues in a polypeptide that correspond to amino acid residues that are important for the activity or structure of the similar polypeptide. One of skill in the art can select chemically similar amino acid replacements for such predicted important amino acid residues.

[0052] Also, the skilled artisan can analyze the three-dimensional structure and amino acid sequence in relation to the structure of similar polypeptides. In light of such information, the skilled artisan can predict the sequence of amino acid residues of a polypeptide with respect to its three-dimensional structure. In various embodiments, the skilled artisan can select to avoid radical changes to amino acid residues predicted to be present on the surface of the polypeptide, since such residues may be involved in important interactions with other molecules. Furthermore, the skilled artisan can generate test variants containing single amino acid substitutions at each of the desired amino acid residues. The variants can then be screened using activity assays known to those skilled in the art. Such variants may be used to gather information about suitable variants. For example, if it is found that a change to a particular amino acid residue results in the destruction of activity, an undesirable reduction in activity, or inappropriate activity, variants with such changes can be avoided. In other words, based on the information gathered from such routine experiments, the skilled artisan can easily identify amino acids for which further substitutions should be avoided, alone or in combination with other mutations.

[0053] The terms "polypeptide fragment" and "truncated polypeptide" as used herein refer to a polypeptide having a deletion at the amino terminus and / or carboxy terminus compared to the corresponding full-length protein. In various embodiments, a fragment can be, for example, 5 or more, 10 or more, 25 or more, 50 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1000 or more amino acids in length. In various embodiments, a fragment can be, for example, 1000 or less, 900 or less, 800 or less, 700 or less, 600 or less, 500 or less, 450 or less, 400 or less, 350 or less, 300 or less, 250 or less, 200 or less, 150 or less, 100 or less, 50 or less, 25 or less, 10 or less, or 5 or less amino acids in length. The fragment may further comprise one or more additional amino acids at one or both termini, for example, an amino acid sequence derived from a different naturally occurring protein (e.g., an Fc or leucine zipper domain) or an artificial amino acid sequence (e.g., an artificial linker sequence).

[0054] The terms "polypeptide variant," "hybrid polypeptide," and "polypeptide variant," as used herein, refer to a polypeptide comprising an amino acid sequence in which one or more amino acid residues have been inserted, deleted, and / or substituted into the amino acid sequence compared to another polypeptide sequence. In various embodiments, the number of inserted, deleted, or substituted amino acid residues can be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids in length. Hybrids of the present disclosure encompass fusion proteins.

[0055] A "derivative" of a polypeptide is a polypeptide that has been chemically modified, such as, for example, conjugation to another chemical moiety, such as polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.

[0056] As used herein, the term "% sequence identity" is used synonymously with the term "% identity" and refers to the value of amino acid sequence identity between two or more peptide sequences, or the value of nucleotide sequence identity between two or more nucleotide sequences, when aligned using a sequence alignment program. For example, as used herein, 80% identity means the same as 80% sequence identity measured by a defined algorithm, meaning that a given sequence has at least 80% identity to another sequence of another length. In various embodiments, the % identity is selected from, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence identity to a given sequence. In various embodiments, the percent identity is within the range of, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

[0057] As used herein, the term "% sequence identity" is used synonymously with the term "% homology" and refers to the value of amino acid sequence identity between two or more peptide sequences or the value of nucleotide sequence identity between two or more nucleotide sequences when aligned using a sequence alignment program. For example, as used herein, 80% identity means the same as 80% sequence identity measured by a defined algorithm, i.e., a homolog of a given sequence has more than 80% sequence identity over a length of the given sequence. In various embodiments, the % identity is selected from, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence identity to the given sequence. In various embodiments, the percent homology is within the range of, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

[0058] Exemplary computer programs that can be used to determine identity between two sequences include, but are not limited to, the suite of BLAST programs publicly available on the Internet at the NCBI website, such as BLASTN, BLASTX, and TBLASTX, BLASTP, and TBLASTN. See also Altschul et al., J. Mol. Biol., 215:403-10, 1990 (with particular reference to the published default settings, i.e., parameters w=4, t=17), and Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997. When evaluating a given amino acid sequence against amino acid sequences in GenBank Protein Sequences or other public databases, sequence searches are typically performed using the BLASTP program. The BLASTX program is preferred for searching nucleic acid sequences translated in all reading frames against amino acid sequences in GenBank Protein Sequences or other public databases. Both BLASTP and BLASTX are run using default parameters of open gap penalty=11.0, gap extension penalty=1.0 and utilize the BLOSUM-62 matrix.

[0059] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA, vol. 90: pp. 5873-5787, 1993). One of the similarity measures provided by the BLAST algorithm is the smallest sum probability (P(N)), which indicates the probability that a match will occur by chance between two nucleotide sequences or two amino acid sequences. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability when comparing a test nucleic acid with the reference nucleic acid is, for example, less than about 0.1, less than about 0.01, or less than about 0.001.

[0060] The term "modification", as used herein, refers to any manipulation of the peptide backbone (eg, amino acid sequence) or post-translational modification of a polypeptide (eg, glycosylation).

[0061] The term "knob-into-hole modification" as used herein refers to a modification in the interface between two immunoglobulin heavy chains at the CH3 domain. In one embodiment, the "knob-into-hole modification" comprises the amino acid substitutions T366W and optionally S354C in one antibody heavy chain and the amino acid substitutions T366S, L368A, Y407V and optionally Y349C in the other antibody heavy chain. Knob-into-hole technology is described, for example, in U.S. Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng, 9, 617-621 (1996); and Carter, J Immunol Meth, 248, 7-15 (2001).

[0062] The term "fusion protein," as used herein, refers to a fusion polypeptide molecule that contains two or more genes that originally encoded separate proteins, and whose components are linked to each other by peptide bonds, either directly or through a peptide linker. The term "fused," as used herein, refers to the components being linked to each other by peptide bonds, either directly or through one or more peptide linkers.

[0063] A "linker" refers to a molecule that links two other molecules, either covalently or through ionic, van der Waals, or hydrogen bonds, such as a nucleic acid molecule that hybridizes to the 5' end of one complementary sequence and to the 3' end of another complementary sequence, thereby linking two non-complementary sequences. A "cleavable linker" refers to a linker that can be degraded or otherwise cleaved to separate the two components linked by the cleavable linker. Cleavable linkers are generally cleaved by enzymes, typically peptidases, proteases, nucleases, lipases, and the like. Cleavable linkers can also be cleaved by environmental factors, such as changes in temperature, pH, and salt concentration.

[0064] The term "peptide linker" as used herein refers to a peptide containing one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides include (G4S)n peptide linkers, (SG4)n peptide linkers, or G4(SG4)n peptide linkers. "n" is usually a number from 1 to 10, typically a number from 2 to 4.

[0065] "Pharmaceutical composition" refers to a composition suitable for pharmaceutical use in animals. A pharmaceutical composition contains a pharmacologically effective amount of an active agent and a pharma- ceutical acceptable carrier. "Pharmacologically effective amount" refers to an amount of an agent effective to obtain an intended pharmacological result. "Pharmaceutical acceptable carrier" refers to any standard pharmaceutical carrier, solvent, buffer, and excipient, such as phosphate buffered saline, 5% dextrose in water, emulsions such as oil-in-water emulsions or water-in-oil emulsions, and various types of wetting agents and / or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 21st Edition, 2005, Mack Publishing Co, Easton. "Pharmaceutical acceptable salt" refers to a salt that can be incorporated into a compound for pharmaceutical use, such as metal salts (sodium salts, potassium salts, magnesium salts, calcium salts, etc.), ammonia salts, and organic amine salts.

[0066] As used herein, "treatment" (and grammatical variations thereof, such as "treat" and "treating") refers to a clinical intervention to alter the natural course of a disease in the individual being treated, and can be performed prophylactically or during the course of clinical pathology. Desirable therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or reduction of disease symptoms, and remission or improvement of prognosis. As used herein, "alleviating" a disease, disorder, or condition means reducing the severity and / or frequency of occurrence of symptoms of the disease, disorder, or condition. Furthermore, references to "treatment" herein encompass references to curative, symptomatic, and prophylactic treatment.

[0067] The term "effective amount" or "therapeutically effective amount" as used herein refers to an amount of a compound or composition sufficient to treat a particular disorder, condition, or disease, such as improving, alleviating, reducing, and / or delaying one or more of its symptoms. With respect to cancer, or other undesirable cell proliferation, an effective amount includes an amount sufficient to: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, slow, slow to some extent, preferably stop, cancer cell invasion into peripheral organs; (iv) inhibit (i.e., slow to some extent, preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) inhibit or delay tumor onset and / or recurrence; and / or (vii) relieve to some extent one or more of the symptoms associated with cancer. An effective amount can be administered in one or more administrations.

[0068] The phrases "administer" or "cause to be administered" refer to the act performed by a medical professional (e.g., physician) or a person managing the medical care of a patient, of managing and / or authorizing the administration of the agent / compound in question to a patient. Administering may include diagnosing and / or determining an appropriate treatment regimen, and / or prescribing a particular agent / compound to a patient. Such prescribing may include, for example, drafting a prescription form, annotating a medical record, and the like. Where administering is described herein, "causing to be administered" is also contemplated.

[0069] The terms "patient," "individual," and "subject" may be used interchangeably and refer to a mammal, preferably a human or non-human primate, but may also refer to domesticated mammals (e.g., dogs or cats), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and agricultural mammals (e.g., horses, cows, pigs, sheep). In various embodiments, the patient may be a human (e.g., adult male, adult female, adolescent male, adolescent female, boy, girl) receiving treatment from a physician or other medical personnel in a hospital, psychiatric care facility, as an outpatient, or in other clinical settings. In various embodiments, the patient may be an immunocompromised patient or a patient with a weakened immune system, including, but not limited to, primary immunodeficiency patients, AIDS patients; cancer patients and transplant patients taking certain immunosuppressants; and patients with genetic diseases that affect the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency). In various embodiments, the patient has an immunogenic cancer, including, but not limited to, bladder cancer, lung cancer, melanoma, and other cancers with reported high mutation rates (Lawrence et al., Nature, 499(7457):214-218, 2013).

[0070] The term "Fc domain" or "Fc region" as used herein is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term encompasses native sequence Fc regions and variant Fc regions. The Fc region of an IgG is composed of an IgG CH2 domain and an IgG CH3 domain. The CH3 region, as used herein, may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain in which a "protrusion" ("knob") is introduced in one chain and a corresponding "recess" ("hole") is introduced in the other chain; see U.S. Pat. No. 5,821,333, expressly incorporated herein by reference). Such variant CH3 domains may be used to facilitate heterodimerization of two non-identical immunoglobulin heavy chains as described herein. Unless otherwise specified herein, the numbering of amino acid residues in an Fc region or constant region is according to the EU numbering system.

[0071] The term "effector function" as used herein refers to the biological activity attributable to the Fc region of immunoglobulin, and varies depending on the isotype of immunoglobulin. Examples of effector functions of immunoglobulin include complement-dependent cytotoxicity (CDC) by binding with C1q, binding with Fc receptor, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, antigen uptake by antigen-presenting cells via immune complexes, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0072] The term "regulatory T cells" or "Treg cells," as used herein, refers to a special type of CD4+ T cell that can suppress the responses of other T cells (effector T cells). Treg cells are characterized by expression of CD4, the a subunit of the IL-2 receptor (CD25), and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol, 22:531-62 (2004)), and play a critical role in the induction and maintenance of peripheral self-tolerance to antigens, including those expressed by tumors.

[0073] The term "normal CD4+ T cells" as used herein means CD4+ T cells other than regulatory T cells.

[0074] The term "selective activation of Treg cells" as used herein refers to activation of Treg cells essentially without activation of other T cell subsets (e.g., CD4+ helper T cells, CD8+ cytotoxic T cells) or natural killer (NK) cells. Methods for identifying and distinguishing these cell types are described in the Examples. Activation may include induction of IL-2 receptor signaling (e.g., as measured by detection of phosphorylated STAT5), induction of proliferation (e.g., as measured by detection of Ki-67), and / or upregulation of expression of activation markers (e.g., CD25) and increased cell numbers.

[0075] As used herein, "specific binding" means that the binding is selective for the antigen and can be distinguished from undesired or non-specific interactions. The ability of an immunoglobulin to bind to a specific antigen can be measured through enzyme-linked immunosorbent assay (ELISA) or other methods known to those skilled in the art, such as surface plasmon resonance (SPR) methods.

[0076] The term "affinity" or "binding affinity" as used herein refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). In general, the affinity of a molecule X to its partner Y can be expressed as a dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (koff and kon, respectively). A particular method for measuring affinity is surface plasmon resonance (SPR).

[0077] The term "decreased binding," as used herein, refers to a decrease in affinity for the respective interaction, such as measured by SPR. Conversely, "increased binding" refers to an increase in binding affinity for the respective interaction.

[0078] The term "polymer," as used herein, generally includes, but is not limited to, homopolymers; copolymers, such as block copolymers, graft copolymers, random copolymers, and alternating copolymers; and terpolymers; and mixtures and modifications thereof. Furthermore, unless otherwise specifically limited, the term "polymer" is intended to include all possible geometric configurations of the material. These configurations include, but are not limited to, isotactic, syndiotactic, and random symmetries.

[0079] "Polynucleotide" refers to a polymer composed of nucleotide units. Polynucleotides include not only natural nucleic acids such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), but also nucleic acid analogs. Nucleic acid analogs include those that contain unnatural bases, which are nucleotides that participate in bonds with other nucleotides other than natural phosphodiester bonds, and those that contain bases added through bonds other than phosphodiester bonds. That is, nucleic acid analogs include, but are not limited to, for example, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonic acids, chiral-methylphosphonic acids, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized using automated DNA synthesizers and the like. The term "nucleic acid" generally refers to large polynucleotides. The term "oligonucleotide" generally refers to short polynucleotides, usually no more than about 50 nucleotides. Where a nucleotide sequence is represented as a DNA sequence (i.e., A, T, G, C), it is understood that this also encompasses RNA sequences in which "U" is replaced with "T" (i.e., A, U, G, C).

[0080] Conventional notation is used herein to describe polynucleotide sequences. The left-hand end of a single-stranded polynucleotide sequence is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The addition of nucleotides from the 5' to the 3' direction to the nascent RNA transcript is referred to as the transcription direction. The DNA strand having the same sequence as the mRNA is referred to as the "coding strand;" the sequence on the DNA strand having the same sequence as the mRNA transcribed from the DNA and located 5' to the 5' end of the RNA transcript is referred to as the "upstream sequence;" the sequence on the DNA strand having the same sequence as the RNA and located 3' to the 3' end of the coding RNA transcript is referred to as the "downstream sequence."

[0081] "Complementary" refers to the topological compatibility, i.e., the matching of the interacting surfaces of two polynucleotides. That is, the two molecules can be described as complementary, and furthermore, the contact surface features are complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, or if the first polynucleotide is capable of hybridizing to the second polynucleotide under stringent hybridization conditions.

[0082] "Specific hybridization to" or "specific hybridization" or "selective hybridization to" refers to a nucleic acid molecule that preferentially binds, duplexes, or hybridizes under stringent conditions to a particular nucleotide sequence when that sequence is present in a mixture (e.g., whole cell) DNA or RNA. The term "stringent conditions" refers to conditions under which a probe will hybridize preferentially to its target material, but will hybridize to a lesser extent or not at all to other sequences. In the context of nucleic acid hybridization experiments such as Southern and Northern hybridization, "stringent hybridization" and "stringent hybridization wash conditions" are sequence-dependent and will be different under different environmental parameters. Detailed introductions to nucleic acid hybridization can be found in Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, Inc., NY; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 3rd Edition, NY; and Ausubel et al. (eds.), Current Edition, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.

[0083] Typically, highly stringent hybridization conditions and highly stringent washing conditions are selected to be about 5°C lower than the melting temperature (Tm) of a specific sequence at a defined ionic strength and pH. Tm is the temperature (under a defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be the same as the Tm of a specific probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids having more than about 100 complementary residues on a filter in a Southern or Northern blot is 50% formalin + 1 mg heparin, 42°C, overnight hybridization run. An example of highly stringent washing conditions is 0.15M NaCl, 72°C, about 15 minutes. An example of stringent washing conditions is 0.2xSSC wash, 65°C, 15 minutes. See Sambrook et al. for a description of SSC buffer. A low stringency wash can be used prior to a high stringency wash to remove background probe signal. An example of a moderately stringent wash, for example for a duplex of more than about 100 nucleotides, is 1×SSC at 45°C for 15 minutes. An example of a low stringency wash, for example for a duplex of more than about 100 nucleotides, is 4-6×SSC at 40°C for 15 minutes. Typically, a signal-to-noise ratio of 2x (or more) than that observed for an unrelated probe in a particular hybridization assay indicates detection of specific hybridization.

[0084] "Primer" refers to a polynucleotide capable of specifically hybridizing to a designated polynucleotide template and providing a site for the initiation of synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is conducive, i.e., in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization, such as DNA polymerase. Primers are typically single-stranded, but may be double-stranded. Primers are typically deoxyribonucleic acids, although a wide variety of synthetic and natural primers are useful in many applications. Primers are designed to be complementary to a template and hybridize to the template to serve as a site for initiation of synthesis, but need not exactly reflect the sequence of the template. In such cases, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled, such as with chromogenic, radioactive, or fluorescent moieties, and used as detectable moieties.

[0085] "Probe", when used in reference to a polynucleotide, refers to a polynucleotide that can specifically hybridize to a designated sequence of another polynucleotide. A probe specifically hybridizes to a complementary polynucleotide of a target, but does not necessarily reflect the exact complementary sequence of the template. In such a case, specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. Probes can be labeled, such as with chromogenic, radioactive, or fluorescent moieties, and can be used as detectable moieties. A probe can also be a primer, where it provides a point of initiation for the synthesis of a complementary polynucleotide.

[0086] A "vector" is a polynucleotide that can be used to introduce another nucleic acid linked to it into a cell. One type of vector is a "plasmid," which is a linear or circular double-stranded DNA molecule into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication-defective retroviruses, replication-defective adenoviruses, and replication-defective adeno-associated viruses), whose viral genome can include additional DNA segments. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors containing a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after introduction into the host cell and are replicated along with the host genome. An "expression vector" is a type of vector that can direct the expression of a selected polynucleotide.

[0087] A "control sequence" is a nucleic acid that affects the expression (e.g., amount, timing, or location) of a nucleic acid to which it is operably linked. A control sequence can, for example, exert its effect directly on the regulated nucleic acid or through the action of one or more other molecules (e.g., a polypeptide that binds to the control sequence and / or the nucleic acid). Examples of control sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Further examples of control sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology, vol. 185, Academic Press, San Diego, Calif., and Baron et al., 1995, Nucleic Acids Res., vol. 23, pp. 3605-06. A nucleotide sequence is "operably linked" to a control sequence if the control sequence affects the expression (e.g., amount, timing, or location) of the nucleotide sequence.

[0088] A "host cell" is a cell that can be used to express the polynucleotides of the present disclosure. A host cell can be prokaryotic, e.g., E. coli, or a host cell can be eukaryotic, e.g., a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., a tobacco plant cell or a tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell), or a hybridoma. Typically, a host cell is a cultured cell that can be transformed or transfected with a polypeptide-encoding nucleic acid, allowing expression of the polypeptide-encoding nucleic acid in the host cell. The term "recombinant host cell" is sometimes used to refer to a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell can also be a cell that contains the nucleic acid but does not express it at a desired level, unless a control sequence has been introduced into the host cell so that it is operably linked to the nucleic acid. It is understood that the term host cell refers not only to the particular subject cell, but also to the progeny and potential progeny of such a cell. Because certain modifications may occur in subsequent generations due to mutations, environmental influences, and the like, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term as used herein.

[0089] The term "isolated molecule" (where the molecule is a polypeptide or polynucleotide, etc.) refers to a molecule that is (1) free from associated naturally occurring components that accompany it in the natural state, (2) substantially free from other molecules from the same species, (3) expressed by cells from a different species, or (4) not naturally occurring, based on its origin or source of derivation. That is, a molecule that is chemically synthesized or expressed in a cellular system different from the cell in which it occurs in nature will be "isolated" from its naturally occurring associated components. A molecule may be rendered substantially free of naturally occurring associated components by isolation using purification methods well known in the art. The purity or homogeneity of a molecule can be assessed by several means well known in the art. For example, the purity of a polypeptide sample can be assessed by visualization of the polypeptide by polyacrylamide gel electrophoresis and gel staining using methods well known in the art. For certain purposes, higher resolution may be obtained by using HPLC or other purification means well known in the art.

[0090] A protein or polypeptide is "substantially pure," "substantially homogeneous," or "substantially purified" when at least about 60%-75% of a sample represents a single polypeptide species. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein will usually comprise about 50% (W / W), about 60% (W / W), about 70% (W / W), about 80% (W / W), or about 90% (W / W) of a protein sample, more frequently about 95%, and preferably greater than 99% pure. Protein purity or homogeneity can be demonstrated by several means well known in the art, such as subjecting a protein sample to polyacrylamide gel electrophoresis followed by visualization of a single polypeptide band by staining the gel with staining methods well known in the art. For certain purposes, higher resolution may be obtained by using HPLC or other purification means well known in the art.

[0091] The term "label" or "labeling" as used herein refers to the incorporation of another molecule into an antibody. In one embodiment, the label is a detectable marker, such as the incorporation of a radioactively labeled amino acid or the addition of a biotinyl moiety to a polypeptide that is detectable by a marked avidin (e.g., streptavidin containing a fluorescent marker or an enzymatic activity detectable by optical or calorimetric methods). In another embodiment, the label or marker can be a therapeutic label or marker, such as a drug conjugate or a toxin. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131I), fluorescent labels (e.g., FITC fluorophores, rhodamine fluorophores, lanthanide fluorophores), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope labels), magnetic agents, e.g., gadolinium chelates, toxins, e.g., pertussis toxin, taxol, Cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. In various embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0092] The term "heterologous" as used herein refers to a configuration or state that is not native or does not exist in nature, e.g., a configuration or state that can be achieved by replacing an existing native configuration or state with a configuration or state from another source. Similarly, expression of a protein in an organism other than the organism in which the protein is naturally expressed results in a heterologous expression system and a heterologous protein.

[0093] Aspects and embodiments of the present disclosure described herein are understood to include "consisting of" and / or "consisting essentially of" aspects and embodiments.

[0094] Any statement herein referring to "about" a value or parameter encompasses (and accounts for) the variation that is directed to the value or parameter itself. For example, a statement referring to "about X" encompasses the statement "X."

[0095] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include the plural unless the context clearly indicates otherwise. The aspects and modifications of the disclosure described herein are understood to include "consisting of" and / or "consisting essentially of" aspects and modifications.

[0096] IL-2 Interleukin-2 (IL-2) is a classical Th1 cytokine, produced by T cells after activation via the T cell antigen receptor and the costimulatory molecule CD28. Regulation of IL-2 occurs through activation of signaling pathways and transcription factors that act on the IL-2 promoter to induce gene transcription de novo, but also involves regulation of IL-2 mRNA stability. IL-2 binds to a highly regulated multichain receptor containing α, β, and γ chains that mediate signaling through the Jak-STAT pathway. IL-2 delivers activation, proliferation, and differentiation signals to T, B, and NK cells. IL-2 is also important in mediating activation-induced T cell cell death, a function that provides an essential mechanism for terminating immune responses. A commercially available nonglycosylated human recombinant IL-2 product, aldesleukin (available under the trade name PROLEUKIN® des-alanyl-1, serine-125 human interleukin-2 from Prometheus Laboratories Inc., San Diego, CA), has been approved for administration to patients with metastatic renal cell carcinoma and metastatic melanoma. IL-2 has also been suggested for administration in patients with hepatitis C virus (HCV) infection, human immunodeficiency virus (HIV) infection, acute myeloid leukemia, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, juvenile rheumatoid arthritis, atopic dermatitis, breast cancer, and bladder cancer. Unfortunately, the short half-life and high toxicity limit the optimal dose of IL-2.

[0097] As used herein, the terms "native IL-2" and "native interleukin-2" refer to any naturally occurring mammalian interleukin-2 amino acid sequence, including immature or precursor forms, and mature forms, in the context of a protein or polypeptide. Non-limiting examples of GenBank accession numbers for the amino acid sequences of various native mammalian interleukin-2 species include NP_032392.1 (Mus musculus, immature form), NP_001040595.1 (Macaca mulatta, immature form), NP_000577.2 (human, precursor form), CAA01199.1 (human, immature form), AAD48509.1 (human, immature form), and AAB20900.1 (human). In various embodiments of the present invention, native IL-2 is the immature or precursor form of naturally occurring mammalian IL-2. In other embodiments, the native IL-2 is the mature form of naturally occurring mammalian IL-2. In various embodiments, the native IL-2 is the precursor form of naturally occurring human IL-2. In various embodiments, the native IL-2 is the mature form of naturally occurring human IL-2. In various embodiments, the IL-2 is derived from the amino acid sequence of the human IL-2 precursor sequence set forth in SEQ ID NO:1 below: MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 1).

[0098] In various embodiments, the IL-2 comprises the amino acid sequence of the human IL-2 mature wild-type sequence set forth in SEQ ID NO:3 below, which contains a cysteine ​​to serine substitution at position 125, but does not alter binding to the IL-2 receptor compared to native IL-2: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 3).

[0099] IL-2 variants The present invention relates to polypeptides that share a primary sequence with human IL-2, except for one to several amino acid mutations. One panel of IL-2 variants contains mutations that preferentially promote proliferation, survival, activation and / or function of immunosuppressive regulatory T cells (T CD4+CD25+FoxP3+) over effector T cells and NK cells. Also included are therapeutic uses of such IL-2 selective agonists, alone or in combination with diseased tissue targeting proteins or peptides, for treating Treg cell deficient autoimmune disorders and various inflammatory disorders. In another aspect, the present invention relates to pharmaceutical compositions comprising the polypeptides of the present disclosure. Finally, the present invention relates to therapeutic uses of the polypeptides and pharmaceutical compositions of the present disclosure, due to their selective regulatory effect on the immune system against autoimmune and inflammatory disorders or diseases such as cancer and various infectious diseases.

[0100] The present invention relates to polypeptides having an apparent molecular weight of at least 15 kD, between 100 and 500 amino acids in length, preferably 140 residues in size. These polypeptides retain a high sequence identity of more than 90% with native IL-2. At these positions, these polypeptides are mutated to introduce amino acid residues different from those at the same positions in native IL-2.

[0101] The polypeptides of the invention may be referred to as immunomodulatory polypeptides, IL-2 analogs, or IL-2 variants, among other names. These polypeptides are designed based on the three-dimensional structure of the IL-2 receptor complex (available in the PDB public database) and contain mutations primarily at positions in IL-2 that correspond to amino acids that interact with the receptor subunits β, γ, or βγ.

[0102] In various embodiments, the IL-2 variant (or mutant) comprises a sequence derived from the sequence of the mature human IL-2 polypeptide set forth in SEQ ID NO:3. In various embodiments, the IL-2 variant comprises an amino acid sequence that differs from the native (or wild-type) IL-2 protein. In various embodiments, the IL-2 variant binds to an IL-2Rα polypeptide and functions as an IL-2 agonist or antagonist. In various embodiments, an IL-2 variant that has agonist activity has superagonist activity. In various embodiments, an IL-2 variant can function as an IL-2 agonist or antagonist independent of binding to IL-2Rα. An IL-2 agonist is exemplified by equivalent or increased biological activity compared to wild-type IL-2. An IL-2 antagonist is exemplified by decreased biological activity compared to wild-type IL-2 or the ability to inhibit an IL-2 mediated response. In various embodiments, the sequence of the IL-2 variant has at least one amino acid change, e.g., a substitution or deletion, when compared to the native IL-2 sequence, which confers IL-2 agonist or IL-2 antagonist activity. In various embodiments, the IL-2 variant as an Fc fusion protein has the amino acid sequence set forth in SEQ ID NOs: 4-43, 108-146, and 193-197, and has reduced binding to IL-2Rβ and / or γc and enhanced selectivity in the activation and proliferation of regulatory T cells (Treg).

[0103] Exemplary IL-2 variants are shown in Tables 2A-2F.

[0104] TIFF0007637415000002.tif164170

[0105] TIFF0007637415000003.tif84170

[0106] TIFF0007637415000004.tif149170

[0107] TIFF0007637415000005.tif66170

[0108] TIFF0007637415000006.tif144170TIFF0007637415000007.tif156170

[0109] TIFF0007637415000008.tif147170

[0110] The present invention also encompasses additional modifications to the above IL-2 variant classes, particularly those listed in Tables 2A-2F. As will be appreciated by those skilled in the art, additional combinatorial mutations combining the preferred mutations listed in Tables 2A-2F may result in IL-2 agonists with higher Treg cell selectivity. Any additional combinatorial mutations, whether they increase its affinity for a particular component of the IL-2 receptor or improve its in vivo pharmacodynamic properties (prolonging its half-life or reducing its internalization by T cells), are within the spirit and scope of the present invention. These additional mutations may be obtained by rational design using bioinformatics tools or by using combinatorial molecular libraries of various nature (phage libraries, libraries of gene expression in yeast or bacteria). In another embodiment, the present invention relates to a fusion protein comprising any of the above immunomodulatory polypeptides conjugated to a carrier protein. The carrier protein may be albumin or the Fc region of a human immunoglobulin.

[0111] Fc domain Immunoglobulins of the IgG class are the most abundant proteins in human blood. The circulating half-life of IgG immunoglobulins can reach as long as 21 days. Fusion proteins have been reported that combine the Fc region of IgG with domains of other proteins, such as various cytokines and receptors (see, e.g., Capon et al., Nature, vol. 337: pp. 525-531, 1989; Chamow et al., Trends Biotechnol., vol. 14: pp. 52-60, 1996; U.S. Pat. Nos. 5,116,964 and 5,541,087). The prototype of a fusion protein is a homodimeric protein linked through cysteine ​​residues in the hinge region of the Fc of IgG, resulting in a molecule similar to an IgG molecule without the variable region and CH1 domain of the heavy chain and the light chain. The dimeric nature of fusion proteins containing the Fc domain can be advantageous in achieving higher order interactions (i.e., bivalent or bispecific binding) with other molecules. Due to their structural homology, Fc fusion proteins exhibit in vivo pharmacokinetic profiles comparable to those of human IgG of the similar isotype.

[0112] The term "Fc" refers to a molecule or sequence that includes the sequence of a non-antigen-binding fragment of a full-length antibody, which may be in monomeric or multimeric form. The original immunoglobulin source of the native Fc is preferably of human origin and may be any immunoglobulin, with IgG1 and IgG2 being preferred. The native Fc is composed of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent (i.e., disulfide) and non-covalent bonds. The number of intermolecular disulfide bonds between the monomeric subunits of the native Fc molecule ranges from 1 to 4, depending on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is the disulfide-linked dimer resulting from papain digestion of IgG (Ellison et al., (1982), Nucleic Acids Res., 10:4071-9). The term "native Fc" as used herein refers collectively to monomeric, dimeric, and multimeric forms of the Fc domain that contains binding sites for Protein A, Protein G, various Fc receptors, and complement proteins.

[0113] In various embodiments, the term "Fc variant" refers to a molecule or sequence that has been modified from a native Fc but still contains a binding site for the salvage receptor, FcRn. Exemplary Fc variants and interactions with the salvage receptor are described in International Publication Nos. WO 97 / 34631 (published September 25, 1997) and WO 96 / 32478, which are incorporated herein by reference. Additionally, native Fc contains sites that may be removed because they confer structural features or biological activity not required for the fusion molecules of the present invention. That is, in various embodiments, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity after expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than the salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0114] The term "Fc domain" encompasses native Fc and Fc variant molecules and sequences as defined above. As with Fc variants and native Fc, the term "Fc domain" encompasses molecules in monomeric or multimeric form, either digested from full-length antibodies or produced by recombinant gene expression or other means. In various embodiments, "Fc domain" refers to a dimer consisting of two Fc domain monomers (SEQ ID NO: 44), usually including all or part of the hinge region. In various embodiments, the Fc domain may be mutated to lack effector function. In various embodiments, each of the Fc domain monomers of the Fc domain contains amino acid substitutions in the CH2 antibody constant domain to reduce the interaction or binding between the Fc domain and the Fcγ receptor. In various embodiments, each subunit of the Fc domain contains two amino acid substitutions (L234A and L235A) that reduce binding to activating Fc receptors and / or effector function. In various embodiments, each subunit of the Fc domain contains three amino acid substitutions (L234A, L235A, and G237A) that reduce binding to activating Fc receptors and / or effector function (SEQ ID NO:45).

[0115] In various embodiments, the two Fc domain monomers of the Fc domain each contain an amino acid substitution that promotes heterodimerization of the two monomers. In various other embodiments, heterodimerization of the Fc domain monomers can be promoted by introducing different but compatible substitutions (such as a "knob-into-hole" residue pair) into the two Fc domain monomers. This "knob-into-hole" technique is also disclosed in U.S. Pat. No. 8,216,805. In yet another embodiment, one Fc domain monomer contains the knob-type mutation T366W and the other Fc domain monomer contains the hole-type mutations T366S, L358A, and Y407V. In various embodiments, two Cy residues that form a stabilizing disulfide bridge (S354C on the "knob" side and Y349C on the "hole" side) have been introduced. The use of heterodimeric Fc allows for monovalent IL-2 variants to be obtained.

[0116] In various embodiments, the Fc domain may be mutated to further increase in vivo half-life. In various embodiments, each subunit of the Fc domain contains three amino acid substitutions (M252Y, S254T, and T256E) that enhance binding to human FcRn as disclosed in U.S. Pat. No. 7,658,921. In various embodiments, each subunit of the Fc domain contains one amino acid substitution (N434A) that enhances binding to human FcRn as disclosed in U.S. Pat. No. 7,371,826. In various embodiments, each subunit of the Fc domain contains one amino acid substitution (M428L and N434S) that enhances binding to human FcRn as disclosed in U.S. Pat. No. 8,546,543. In various embodiments, the half-life extending mutations can be combined with amino acid substitutions that reduce binding to activating Fc receptors and / or effector functions.

[0117] In various embodiments, the Fc domain sequence used to generate the IL-2 variant Fc fusion is a human IgG1-Fc domain sequence with reduced / eliminated effector function as set forth in SEQ ID NO: 45 below: DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 45)

[0118] SEQ ID NO:45 contains amino acid substitutions (underlined) that disrupt FcγR binding and C1q binding.

[0119] In various embodiments, the Fc domain sequence used to generate the IL-2 variant Fc fusion is the IgG1-Fc domain sequence with extended half-life and reduced / eliminated effector function as set forth in SEQ ID NO: 46 below: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 46)

[0120] SEQ ID NO:46 contains amino acid substitutions (underlined) that abolish FcγR binding and C1q binding, and substitutions (bold) that increase the serum half-life of the fusion protein.

[0121] In various embodiments, the Fc domain sequence used to generate the IL-2 variant Fc fusion is an IgG1-Fc domain with reduced / eliminated effector function and extended half-life, having the amino acid sequence set forth in SEQ ID NO: 47 below: DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPG (SEQ ID NO: 47)

[0122] SEQ ID NO:47 contains amino acid substitutions (underlined) that abolish FcγR binding and C1q binding, and substitutions (bold) that increase the serum half-life of the fusion protein.

[0123] In various embodiments, the heterodimeric Fc domain sequence used to generate the IL-2 variant Fc fusion is the extended in vivo half-life Knob-Fc domain set forth in SEQ ID NO: 212 below: DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPG (SEQ ID NO: 212)

[0124] SEQ ID NO:212 contains amino acid substitutions that disrupt FcγR binding, C1q binding (underlined) and extend half-life (bold).

[0125] In various embodiments, the heterodimeric Fc domain sequence used to generate the IL-2 variant Fc fusion is the extended in vivo half-life Hole-Fc domain set forth in SEQ ID NO: 213 below: DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPG (SEQ ID NO: 213) SEQ ID NO:213 contains amino acid substitutions that disrupt FcγR binding, C1q binding (underlined) and extend half-life (bold).

[0126] Linker In various embodiments, the heterologous protein is attached to the IL-2 variant by a linker peptide and / or hinge linker peptide. The linker or hinge linker can be an artificial sequence of 5, 10, 15, 20, 30, 40, or more amino acids (or any number in between) that has relatively little secondary structure or exhibits an alpha-helical conformation.

[0127] Peptide linkers provide covalent bonds and additional structural and / or spatial flexibility between protein domains. As is known in the art, peptide linkers include flexible amino acid residues such as glycine and serine. In various embodiments, peptide linkers can include 1-100 amino acids. In various embodiments, the spacer can include the motif GGGSGGGS (SEQ ID NO: 55). In other embodiments, the linker can include the motif (GGGGS) nThe linker may include a motif (n is an integer between 1 and 10). In other embodiments, the linker may include amino acids other than glycine and serine. In another embodiment, the linker may include other protein motifs, including but not limited to, alpha helical conformation sequences such as AEAAAKEAAAKEAAAKA (SEQ ID NO: 53). In various embodiments, the length and composition of the linker may be tailored to optimize activity or development suitability, including but not limited to expression levels and aggregation tendency. In another embodiment, the peptide linker may be a simple chemical bond, such as an amide bond (e.g., by chemical attachment of PEG).

[0128] Exemplary peptide linkers are shown in Table 3.

[0129] TIFF0007637415000009.tif123170

[0130] Polynucleotides In another aspect, the present disclosure provides an isolated nucleic acid molecule comprising a polynucleotide encoding the IL-2, IL-2 variant, IL-2 fusion protein, or IL-2 variant fusion protein of the present disclosure. The subject nucleic acid may be single-stranded or double-stranded. Such nucleic acid may be a DNA molecule or an RNA molecule. DNA may include, for example, cDNA, genomic DNA, synthetic DNA, PCR amplified DNA, and combinations thereof. Genomic DNA encoding IL-2 polypeptides can be obtained from genomic libraries corresponding to many species. Synthetic DNA can be obtained by chemical synthesis of overlapping oligonucleotide fragments followed by assembly of the fragments to reconstitute some or all of the coding region and flanking sequences. RNA can be obtained from a prokaryotic expression vector that directs high-level mRNA synthesis, such as a vector using a T7 promoter, and an RNA polymerase. cDNA can be obtained from a library prepared from mRNA isolated from various tissues that express IL-2. The DNA molecules of the present disclosure include not only full-length genes, but also polynucleotides and fragments thereof. The full-length gene may also include sequences encoding an N-terminal signal sequence. Such nucleic acids may be used, eg, in methods for making novel IL-2 variants.

[0131] In various embodiments, the isolated nucleic acid molecule comprises a polynucleotide as described herein and further comprises a polynucleotide encoding at least one heterologous protein as described herein. In various embodiments, the nucleic acid molecule further comprises a polynucleotide encoding a linker or hinge linker as described herein.

[0132] In various embodiments, the recombinant nucleic acid of the present disclosure may be operably linked to one or more control nucleotide sequences in an expression construct. Control sequences are art-recognized and selected to direct the expression of the IL-2 variant. That is, the term control sequence includes promoters, enhancers, and other expression control elements. Exemplary control sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990). Typically, the one or more control nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription start and end sequences, translation start and end sequences, and enhancer or activator sequences. Constitutive or inducible promoters known in the art are contemplated by the present disclosure. The promoter may be a naturally occurring promoter or a hybrid promoter that combines two or more promoter elements. The expression construct may be present intracellularly or episomally, such as a plasmid, or the expression construct may be inserted into a chromosome. In various embodiments, the expression vector contains a selectable marker gene to allow for the selection of transformed host cells. Selectable marker genes are well known in the art and vary depending on the host cell used.

[0133] In another embodiment of the present disclosure, the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence operably linked to at least one regulatory sequence encoding an IL-2 variant. The term "expression vector" refers to a plasmid, phage, virus, or vector for expressing a polypeptide from a polynucleotide sequence. Vectors suitable for expression in a host cell are readily available, and insertion of a nucleic acid molecule into a vector is performed using standard recombinant DNA techniques. Such vectors can include a wide variety of expression regulatory sequences that can be used in these vectors to express a DNA sequence encoding an IL-2 variant, which regulates the expression of the DNA sequence when operably linked to the vector. Such useful expression control sequences include, for example, the SV40 early and late promoters, the tet promoter, the adenovirus or cytomegalovirus immediate early promoters, the RSV promoter, the lac, trp, TAC or TRC systems, the T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of lambda phage, the control region for the fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoter for acid phosphatase (e.g., PhoS), the promoter for yeast alpha mating factor, the polyhedrin promoter for baculovirus systems, and other sequences known to regulate the expression of genes in prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It will be understood that the design of the expression vector may vary depending on factors such as the choice of the host cell to be transformed and / or the type of protein desired to be expressed. Additionally, consideration should be given to the copy number of the vector, the ability to regulate that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers.Exemplary expression vectors suitable for expressing vIL-2 include pDSRa (described in WO 90 / 14363, incorporated herein by reference), and derivatives thereof, containing the vIL-2 polynucleotide and any additional suitable vectors known in the art or described below.

[0134] The cloned gene or a portion thereof can be ligated into a vector suitable for expression in either prokaryotic or eukaryotic cells (yeast, avian, insect, or mammalian cells), or both, to produce the recombinant nucleic acid of the present disclosure. Expression vehicles for producing recombinant IL-2 polypeptides include plasmids and other vectors. For example, suitable vectors include the following plasmids for expression in prokaryotic cells, such as E. coli: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids.

[0135] Some mammalian expression vectors contain both prokaryotic sequences that facilitate the propagation of the vector in bacteria and one or more eukaryotic transcription units that are expressed in eukaryotic cells. pcDNAI / amp-derived vectors, pcDNAI / neo-derived vectors, pRc / CMV-derived vectors, pSV2gpt-derived vectors, pSV2neo-derived vectors, pSV2-dhfr-derived vectors, pTk2-derived vectors, pRSVneo-derived vectors, pMSG-derived vectors, pSVT7-derived vectors, pko-neo-derived vectors, and pHyg-derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors have been modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, for transient expression of proteins in eukaryotic cells, derivatives of viruses such as bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived, and p205) can be used. Examples of other viral (including retroviral) expression systems can be found in the description of gene therapy delivery systems below. The various methods used in the preparation of plasmids and transformation of host organisms are well known in the art. For other expression systems suitable for both prokaryotic and eukaryotic cells, as well as general recombinant methods, see Molecular Cloning A Laboratory Manual, 2nd Edition, Sambrook, Fritsch, and Maniatis (eds.) (Cold Spring Harbor Laboratory Press, 1989), Chapters 16 and 17. In some cases, it may be desirable to express recombinant polypeptides using a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as the B-gal containing pBlueBacIII).

[0136] In various embodiments, vectors will be designed for production of a subject IL-2 variant in CHO cells, such as, for example, the Pcmv-Script vector (Stratagene, La Jolla, Calif.), the pcDNA4 vector (Invitrogen, Carlsbad, Calif.), and the pCI-neo vector (Promega, Madison, Wis.) As described below, the subject genetic constructs can be used to direct expression of a subject IL-2 variant in cells grown in culture, for example, to produce and purify a protein, including a fusion protein or a variant protein.

[0137] The present disclosure also relates to a host cell transfected with a recombinant gene comprising a nucleotide sequence encoding the amino acid sequence of one or more of the subject IL-2 variants. The host cell may be any prokaryotic or eukaryotic cell. For example, the IL-2 variants of the present disclosure may be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cells are known to those skilled in the art.

[0138] Accordingly, the present disclosure further relates to methods of producing the subject IL-2 variants. For example, IL-2 variants can be expressed by culturing host cells transfected with an expression vector encoding the IL-2 variant under appropriate conditions. After secretion, the IL-2 variant can be isolated from a mixture of cells and medium containing the IL-2 variant. Alternatively, the IL-2 variant may be retained in the cytoplasm or membrane fraction, and the cells harvested, lysed, and the protein isolated. The cell culture medium includes host cells, culture medium, and other by-products. Suitable media for cell culture are well known in the art.

[0139] The polypeptides and proteins of the present disclosure can be purified according to protein purification methods well known to those skilled in the art. These methods include, at some level, crude fractionation of proteinaceous and non-proteinaceous fractions. After separation of the peptide polypeptide from other proteins, the peptide or polypeptide of interest can be further purified by chromatographic and electrophoretic methods to achieve partial or complete purification (i.e., purification to homogeneity). The term "isolated polypeptide" or "purified polypeptide" as used herein is intended to refer to a composition that can be isolated from other components, in which the polypeptide is purified to any degree relative to its naturally available state. A purified polypeptide therefore also refers to a polypeptide that has been separated from the environment in which it may naturally occur. In general, "purified" refers to a polypeptide composition that has been fractionated to remove various other components and that substantially retains its expressed biological activity. When the term "substantially purified" is used, this designation refers to a peptide or polypeptide composition in which the polypeptide or peptide forms a majority of the composition, e.g., constitutes about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 85% or more, or about 90% or more of the protein in the composition.

[0140] Various methods suitable for use in purification are well known to those skilled in the art. These methods include, for example, precipitation using ammonium sulfate, PEG, antibodies (immunoprecipitation), or precipitation by heat denaturation followed by centrifugation; chromatography such as affinity chromatography (protein A column), ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, hydroxyapatite chromatography, hydrophobic interaction chromatography; isoelectric focusing; gel electrophoresis; and combinations of these methods. It is well known in the art that the order in which the various purification steps are performed may be changed, or certain steps may be omitted, and still be suitable for preparing a substantially purified polypeptide.

[0141] Pharmaceutical Compositions In another aspect, the disclosure provides a pharmaceutical composition comprising an IL-2 variant or an IL-2 variant fusion protein in admixture with a pharma- ceutically acceptable carrier. Such pharma-ceutically acceptable carriers are well known and understood by those skilled in the art and have been described extensively (see, for example, Remington's Pharmaceutical Sciences, 18th ed., AR Gennaro (ed.), Mack Publishing Co., 1990). Pharmaceutically acceptable carriers may be included for the purpose of altering, maintaining or preserving the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution rate, release rate, adsorption, or permeability of the composition. Such pharmaceutical compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the polypeptide.Suitable pharma- ceutically acceptable carriers include amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, other organic acids, etc.); bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants; flavoring agents and diluting agents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEGs, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal, etc.); stabilizers (sucrose or sorbitol); tonicity enhancing agents (alkali metal halides (preferably sodium or potassium chloride, mannitol, sorbitol, etc.); delivery vehicles vehicle); diluent; excipient; and / or pharmaceutical adjuvant.

[0142] The primary solvent or carrier in a pharmaceutical composition may be aqueous or non-aqueous in nature. For example, a suitable solvent or carrier may be water for injection, physiological saline, or artificial cerebrospinal fluid, to which other substances normally included in compositions for parenteral administration may be added. Further exemplary solvents include neutral buffered saline or saline mixed with serum albumin. Other exemplary pharmaceutical compositions include Tris buffer at about pH 7.0-8.5, or acetate buffer at about pH 4.0-5.5, which may further include sorbitol or a suitable substitute thereof. In one embodiment of the present disclosure, the composition may be prepared for storage in the form of a lyophilized cake or aqueous solution by mixing the selected composition having the desired purity with any formulation agent (Remington's Pharmaceutical Sciences, supra). Additionally, the therapeutic composition may be formulated as a lyophilizate using suitable pharmaceutical excipients such as sucrose. The optimal pharmaceutical composition is determined by one of skill in the art based on the intended route of administration, mode of delivery, and desired dose, etc.

[0143] When parenteral administration is intended, the therapeutic pharmaceutical composition may be in the form of a pyrogen-free parenterally acceptable aqueous solution containing the desired IL-2 polypeptide or IL-2 polypeptide fusion protein in a pharma- ceutical acceptable solvent. A particularly suitable parenteral injection solvent is sterile distilled water, in which the polypeptide is formulated as a properly preserved, sterile, isotonic solution. In various embodiments, pharmaceutical preparations suitable for injection administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Additionally, suspensions of the active compound may be prepared as appropriate oily injection suspensions. Optionally, the suspension may contain suitable stabilizers, i.e., agents that increase the solubility of the compounds, thereby allowing for the preparation of highly concentrated solutions.

[0144] In various embodiments, the therapeutic pharmaceutical composition can be formulated for targeted delivery using colloidal dispersion systems. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Examples of lipids useful in liposome production include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Exemplary phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine. Liposome targeting can also be based on, for example, organ-specific, cell-specific, and organelle-specific, and is known in the art.

[0145] In various embodiments, oral administration of the pharmaceutical composition is contemplated. Pharmaceutical compositions administered in this manner can be formulated with or without carriers typically used in the preparation of solid dosage forms such as tablets and capsules. In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.), one or more therapeutic compounds of the present disclosure may be mixed with one or more pharma- ceutically acceptable carriers, such as, for example, sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin. (6) absorption enhancers such as quaternary ammonium compounds; (7) wetting agents such as, for example, acetyl alcohol and glycerol monostearate; (8) adsorbents such as kaolin clay and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical compositions may also contain buffering agents. Similar types of solid compositions may also be employed as fillers in soft and hard gelatin capsules, using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols. Liquid dosage forms for oral administration include pharma-ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.In addition to the active ingredient, the liquid dosage form may contain inert excipients commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (specifically cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol sorbitan fatty acid esters, and mixtures thereof. In addition to the inert excipients, oral compositions may contain auxiliary agents such as wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, coloring agents, fragrances, and preservatives.

[0146] In various embodiments, topical administration of the pharmaceutical composition to the skin or mucosa is contemplated. The topical formulation may further include one or more of a variety of agents known to be effective as skin or stratum corneum penetration enhancers. Examples of these include 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methyl alcohol, isopropyl alcohol, dimethylsulfoxide, and azone. Additional agents may further be included to make the formulation cosmetically acceptable. Examples of these include fats, waxes, oils, dyes, fragrances, preservatives, stabilizers, and surfactants. Keratolytic agents may be included, such as those known in the art. Examples include salicylic acid and sulfur. Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharma- ceutically acceptable carrier, and, if necessary, with any preservatives, buffers, or propellants. Ointments, pastes, creams, and gels may contain, in addition to the subject compounds of the present disclosure (e.g., IL-2 variants), pharmaceutical additives such as animal fats, vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0147] Additional pharmaceutical compositions contemplated for use herein include formulations that contain the polypeptide in sustained or controlled delivery formulations.Methods for formulating various other sustained or controlled delivery means, such as liposome carriers, biodegradable microparticles or porous beads, and depots, are also known to those skilled in the art.

[0148] The effective amount of the pharmaceutical composition used therapeutically will vary depending on the content and purpose of the treatment. That is, as will be understood by those skilled in the art, the dosage level suitable for treatment will vary, in part, depending on the molecule being delivered, the indication for which the polypeptide is intended to be used, the route of administration, and the size (weight, body surface, or organ size) and condition (age and overall health) of the patient. Thus, the clinician can adjust the dosage and modify the route of administration to obtain the optimal therapeutic effect. Typical dosages can range from about 0.001 mg / kg to about 100 mg / kg or more, depending on the factors mentioned above. Preferably, the polypeptide composition may be injected or administered intravenously. A sustained release pharmaceutical composition may be administered every 3 to 4 days, every week, or every other week, depending on the half-life and clearance rate of the particular formulation. The frequency of administration will depend on the pharmacokinetic parameters of the polypeptide in the formulation used. Typically, the composition is administered until a dose is reached that achieves the desired effect. The composition may therefore be administered as a single dose, or as multiple doses over time (same or different concentrations per dose), or as a continuous infusion. Further refinement of the appropriate dosage is performed periodically. Appropriate dose-response data may be used to confirm the appropriate dosage.

[0149] The route of administration of the pharmaceutical composition is according to known methods, for example, oral, intravenous, intraperitoneal, intracerebral (intracerebroparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, intralesional injection, intramedullary, intrathecal, intraventricular, percutaneous, subcutaneous, or intraperitoneal, or intratumoral; as well as intranasal, enteral, topical, sublingual, transurethral, ​​vaginal, or rectal means, sustained release systems, or implanted devices. If desired, the composition may be administered in a bolus or continuously by infusion, or by implanted devices. Alternatively or additionally, the composition may be administered locally by implantation of a membrane, sponge, or another suitable material to which the molecule of interest is adsorbed or encapsulated. Where an implantation device is used, the device may be implanted into any suitable tissue or organ and delivery of the molecule of interest may be via diffusion, timed-release bolus, or continuous administration.

[0150] therapeutic use The present disclosure provides a method of treating an autoimmune disease in a subject comprising administering to the subject a therapeutically effective amount (as a monotherapy regimen or in a combination therapy regimen) of an IL-2 variant or IL-2 variant fusion protein of the present disclosure in a pharma- ceutically acceptable carrier. An autoimmune disease according to the present invention is a disease or disorder arising from, directed against an individual's own tissues, or a co-segregate or manifestation thereof, or a condition resulting therefrom. In various embodiments, the autoimmune disease includes arthritis (including rheumatoid arthritis, reactive arthritis), systemic lupus erythematosus (SLE), psoriasis and inflammatory bowel disease (IBD), encephalomyelitis, uveitis, myasthenia gravis, multiple sclerosis, insulin-dependent diabetes mellitus, Addison's disease, celiac disease, chronic fatigue syndrome, autoimmune hepatitis, autoimmune alopecia, ankylosing spondylitis, ulcerative colitis, Crohn's disease, fibromyalgia, pemphigus vulgaris, Sjogren's syndrome, Kawasaki disease, hyperthyroidism / Graves' disease, hypothyroidism / Hashimoto's disease, endometriosis, scleroderma, pernicious anemia, Goodpasture's syndrome, Guillain-Barré syndrome, Wegener's disease, These include, but are not limited to, autoimmune diseases, glomerulonephritis, aplastic anemia (including patients with frequently transfused aplastic anemia), paroxysmal nocturnal hemoglobinuria, myelodysplastic syndrome, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, Evans syndrome, Factor VIII inhibitor syndrome, systemic vasculitis, dermatomyositis, polymyositis and rheumatic fever, autoimmune lymphoproliferative syndrome (ALPS), autoimmune bullous pemphigoid, Parkinson's disease, sarcoidosis, vitiligo, primary biliary cirrhosis, and autoimmune myocarditis.

[0151] The present disclosure provides a method of treating an inflammatory disease in a subject, comprising administering to the subject a therapeutically effective amount (as a monotherapy regimen or in a combination therapy regimen) of an IL-2 variant or an IL-2 variant fusion protein of the present disclosure in a pharma- ceutically acceptable carrier. An "inflammatory disease" includes any disease involving acute inflammation or various inflammations. Acute inflammation is the body's initial response to a harmful stimulus and is caused by increased migration of plasma and white blood cells (e.g., granulocytes, etc.) from the blood to the injured tissue. Several biochemical events, including the local vasculature, immune system, and various cells within the injured tissue, propagate and mature the inflammatory response. Prolonged inflammation, referred to as various inflammations, is characterized by gradual changes in the types of cells present at the site of inflammation and by the simultaneous destruction and recovery of tissue from the inflammatory process. In another aspect, the present disclosure provides a method of treating an inflammatory disease in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a pharmaceutical composition of the present invention. In one embodiment, the subject is a human subject. In various embodiments, the inflammatory diseases to be treated include, but are not limited to, Crohn's disease, colitis, dermatitis, psoriasis, diverticulitis, hepatitis, irritable bowel syndrome (IBS), lupus erythematous, nephritis, Parkinson's disease, ulcerative colitis, collagen colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behcet's syndrome and unspecified colitis, multiple sclerosis (MS), Alzheimer's disease, arthritis, rheumatoid arthritis, asthma, and various cardiovascular diseases such as atherosclerosis and vasculitis. In various embodiments, the inflammatory disease is selected from the group consisting of rheumatoid arthritis, diabetes, gout, cryopyrin-associated periodic syndrome, and chronic obstructive pulmonary disorder.

[0152] In another aspect, the disclosure provides a method for organ transplantation or related graft-versus-host disease in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of the present invention. In one embodiment, the subject is a human subject. In various embodiments, the transplant is selected from heart organ transplantation, kidney organ transplantation, liver organ transplantation, lung organ transplantation, pancreas organ transplantation, intestine organ transplantation, and thymus organ transplantation, or selected from bone tissue transplantation, tendon tissue transplantation, corneal tissue transplantation, skin tissue transplantation, heart valve tissue transplantation, nerve tissue transplantation, and vein tissue transplantation. As used herein, the term "graft-versus-host disease" or "GvHD" refers to a condition, including acute and chronic, caused by the effect of transplanted (graft) cells on host cells and tissues resulting from GVH. In other words, donor immune cells injected into the graft or donor immune cells generated from stem cells may recognize patient (host) cells as foreign and mount an immune response against them. Acute graft-versus-host disease (GvHD) is specifically a disorder caused by donor immune cells in patients who have received allogeneic bone marrow or blood cell transplants. The most commonly affected tissues are the skin, intestine, and liver. In severe cases, GvHD can cause blistering of the skin, excessive diarrhea, and wasting. Prednisone and / or other immunosuppressive drugs are used to treat acute graft-versus-host disease.

[0153] First, E.C. 50 The therapeutically effective amount can be evaluated from cell culture assays by measurement. The EC measured in cell culture medium in animal models can then be used to evaluate the therapeutically effective amount. 50 The dose can be formulated to achieve a circulating plasma concentration range that includes. Such information can be used to more accurately determine a useful dose in humans. Plasma levels can be measured by HPLC or the like. The exact composition, route of administration, and dose can be selected by the individual physician in view of the subject's condition.

[0154] The dosage regimen can be adjusted to obtain the optimum desired response (e.g., therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses (multiple doses or repeated doses or maintenance doses) can be administered over a long period of time, and the dose can be increased or decreased proportionally to the needs of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. Dosage unit form, as used herein, refers to physically discrete units suitable as a uniform dose for the mammalian subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present disclosure are primarily determined by the unique characteristics of the antibody and the particular therapeutic or prophylactic effect to be achieved.

[0155] That is, as will be understood by those skilled in the art, based on the disclosure provided herein, dosage and administration schedules are adjusted according to methods well known in the therapeutic field.That is, the maximum tolerated dose can be easily established, and the effective amount that gives a detectable therapeutic effect to a subject can be determined, as well as the time requirement for administering each agent to give a detectable therapeutic effect to a subject can be determined.Thus, although certain dosage and administration schedules are exemplified herein, these examples do not limit the dosage and administration schedules that can be given to a subject when implementing the present disclosure.

[0156] It should be noted that dosage values ​​vary depending on the type and severity of the condition to be improved and may include single or repeated doses. It should further be understood that for any particular subject, the specific dosing regimen should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the composition, and that the dosage ranges set forth herein are for illustrative purposes only and are not intended to limit the scope or practice of the claimed compositions. Furthermore, dosing regimens using the compositions of the present disclosure may be based on a variety of factors, including the type of disease, the age, weight, sex, medical condition of the subject, the severity of the condition, the route of administration, and the particular antibody used. That is, dosing regimens may vary, but can be routinely determined using standard methods. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and / or laboratory values. That is, the present disclosure encompasses intra-subject dose escalation as required by those of skill in the art. It is understood that the determination of appropriate doses and dosing regimens is well known in the relevant art and can be accomplished by one of skill in the art given the teachings disclosed herein.

[0157] Exemplary, non-limiting daily dose ranges for therapeutically or prophylactically effective amounts of an IL-2 variant or IL-2 variant fusion protein of the present disclosure can range from about 0.1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. When administered repeatedly over several days or more, depending on the condition, treatment will usually be sustained until a desired suppression of disease symptoms occurs.In various embodiments, the dosage is approximately 0.0001-100 mg / kg body weight, 0.0001-90 mg / kg body weight, 0.0001-80 mg / kg body weight, 0.0001-70 mg / kg body weight, 0.0001-60 mg / kg body weight, 0.0001-50 mg / kg body weight, 0.0001-40 mg / kg body weight, 0.0001-30 mg / kg body weight, 0.0001-20 mg / kg body weight, 0.0001-10 mg / kg body weight, 0.0001-5 mg / kg body weight, body weight, 0.0001-4mg / kg body weight, 0.0001-3mg / kg body weight, 0.0001-2mg / kg body weight, 0.0001-1mg / kg body weight, 0.001-50mg / kg body weight, 0.001-40mg / kg body weight, 0.001-30mg / kg body weight, 0.001-20mg / kg body weight, 0.001-10mg / kg body weight, 0.001-5mg / kg body weight, 0.001-4mg / kg body weight, 0.001-3mg / kg body weight, 0.001-2mg / kg body weight, 0.001-1mg / kg body weight, 0.010-50mg / kg body weight, 0.010-40mg / kg body weight, 0.010-30mg / kg body weight, 0.010-20mg / kg body weight, 0.010-10mg / kg body weight, 0.010-5mg / kg body weight, 0.010-4mg / kg body weight, 0.010-3mg / kg body weight, 0.010-2mg / kg body weight, 0.010-1mg / kg body weight, 0.1-50mg / kg body weight, 0.1-40mg / kg body weight, 0.1-30mg / kg body weight, 0.1-20mg / kg body weight , 0.1-10 mg / kg body weight, 0.1-5 mg / kg body weight, 0.1-4 mg / kg body weight, 0.1-3 mg / kg body weight, 0.1-2 mg / kg body weight, 0.1-1 mg / kg body weight, 1-50 mg / kg body weight, 1-40 mg / kg body weight, 1-30 mg / kg body weight, 1-20 mg / kg body weight, 1-10 mg / kg body weight, 1-5 mg / kg body weight, 1-4 mg / kg body weight, 1-3 mg / kg body weight, 1-2 mg / kg body weight, or 1-1 mg / kg body weight. That is, one or more doses of about 0.5 mg / kg, about 2.0 mg / kg, about 5.0 mg / kg, or about 10 mg / kg (or any combination thereof) may be administered to the patient. It should be noted that the dosage value may vary depending on the type and severity of the condition to be improved.It is further to be understood that for any particular subject, specific dosage regimens will be adjusted over time according to the individual need and professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

[0158] The toxicity and therapeutic index of the pharmaceutical compositions of the present disclosure are 50 (a dose lethal to 50% of the population) and ED 50 This can be determined by standard pharmaceutical techniques in cell cultures or experimental animals to determine the dose that is therapeutically effective in 50% of a population. The dose ratio between the toxic dose and the therapeutically effective dose is the therapeutic index, and the LD 50 / ED 50 Compositions that exhibit large therapeutic indices are generally preferred.

[0159] The frequency of administration of the IL-2 variant or IL-2 variant fusion protein pharmaceutical composition will depend on the nature of the treatment method and the particular disease being treated. The subject can be treated at regular intervals, such as twice weekly, once weekly, or once monthly, until the desired therapeutic result is achieved. Exemplary administration frequencies include, but are not limited to, once every week; once every two weeks; once every three weeks; once every two weeks, then once a month; once every three weeks, then once a month; once a month; once every two months; once every three months; once every four months; once every five months; or once every six months, or once a year.

[0160] Combination therapy As used herein, the terms "co-administration," "co-administered," and "in combination with," with reference to an IL-2 variant or IL-2 variant fusion protein of the present disclosure and one or more other therapeutic agents, are intended to mean and include: simultaneous administration of such a combination of an IL-2 variant or IL-2 variant fusion protein of the present disclosure and a therapeutic agent to a subject in need of treatment, where such components are combined into a single dosage form and each of said components is released to said subject at substantially the same time; substantially simultaneous administration of such a combination of an IL-2 variant or IL-2 variant fusion protein of the present disclosure and a therapeutic agent to a subject in need of treatment, where such components are formulated separately from one another into separate dosage forms and each of said components is delivered to said subject at substantially the same time when ingested by said subject at substantially the same time. sequential administration of such a combination of an IL-2 variant or an IL-2 variant fusion protein of the present disclosure and a therapeutic agent to a subject in need of treatment, where such components are formulated separately from one another into separate dosage forms which, when ingested by the subject at successive times with a significant time interval between each administration, are released to the subject at substantially different times; and sequential administration of such a combination of an IL-2 variant or an IL-2 variant fusion protein of the present disclosure and a therapeutic agent to a subject in need of treatment, where such components are combined into a single dosage form and, when released in a sustained manner, are released to the subject at the same and / or different times in a concurrent, sequential, and / or overlapping manner, and each portion may be administered by the same route or by a different route.

[0161] In another aspect, the disclosure provides a method for treating an autoimmune disease in a subject, comprising administering a therapeutically effective amount of a pharmaceutical composition of the present invention in combination with a second therapeutic agent capable of treating the autoimmune disease. In various embodiments, the second therapeutic agent is selected from the group consisting of: immunosuppressants, such as corticosteroids, cyclosporine, cyclophosphamide, prednisone, azathioprine, methotrexate, rapamycin, tacrolimus, biologic agents, such as blockers or antagonists of TNF-α, IL-10 agonists or long-term half-life extended IL-10 (PEGylated, antibody- or Fc-fused IL-10); immunosuppressants, such as antibodies that inhibit other lymphocyte surface markers (e.g., for example, antibodies against CD40, alpha-4 integrin, or antibodies against cytokines), other fusion proteins (e.g., CTLA-4-Ig (ORENCIA®), TNFR-Ig (ENBREL®)), TNF-alpha blockers, e.g., ENBREL®, REMICADE®, CIMZIA®, and HUMIRA®, cyclophosphamide (CTX) (i.e., ENDOXAN®, CYTOXAN®, NEOSAR®, PROCYTOX®, REVIMMUNE®), methotrexate (MTX) (i.e., RHEUMATREX®, TREXALL®), rituximab, belimumab (i.e., BENLYSTA, TA)®), anti-IL-6 antibodies (e.g., sarilumab), or anti-IL-6 receptor antibodies (e.g., tocilizumab), or other immunosuppressants (e.g., cyclosporine A, FK506-like compounds, rapamycin compounds, or steroids), anti-proliferatives, cytotoxic drugs, or other compounds that may aid in immunosuppression, or any other biological agent that targets any inflammatory cytokine, nonsteroidal anti-inflammatory agents / Cox-2 inhibitors,Hydroxychloroquine, sulphasalazopryine, gold salts, etanercept, infliximab, mycophenolate mofetil, basiliximab, atacicept, rituximab, cytoxan, interferon beta-1a, interferon beta-1b, glatiramer acetate, mitoxantrone hydrochloride, anakinra, and / or other biologics, and / or intravenous immunoglobulin (IVIG). Non-limiting examples of such known therapeutic agents include interferons, such as IFN-β-1a (REBIF®, AVONEX®, and CINNOVEX®), and IFN-β-1b (BETASERON®, EXTAVIA®, BETAFERON®, ZIFERON®); glatiramer acetate (COPAXONE®), a polypeptide; natalizumab (TYSABRI®); and mitoxantrone (NOVANTRONE®), a cytotoxic drug.

[0162] In another aspect, the disclosure provides a method for treating an inflammatory disease in a subject, comprising administering a therapeutically effective amount of a pharmaceutical composition of the present invention in combination with a second therapeutic agent, said second therapeutic agent being capable of inhibiting or suppressing the differentiation of Th1 cells, Th17 cells, Th22 cells, and / or other cells that secrete or cause other cells to secrete inflammatory molecules, including but not limited to, IL-1β, TNF-α, TGF-β, IFN-γ, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs; inhibiting or suppressing the activity of Th1 cells, Th17 cells, Th22 cells, and / or other cells that secrete or cause other cells to secrete inflammatory molecules, including but not limited to, IL-1β, TNF-α, TGF-β, IFN-γ, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs. the therapeutic agent capable of inhibiting or suppressing the proliferation of Th1 cells, Th17 cells, Th22 cells, and / or other cells that secrete or cause other cells to secrete inflammatory molecules, including but not limited to, IL-1β, TNF-α, TGF-β, IFN-γ, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs.In various embodiments, the second therapeutic agent is a nonsteroidal anti-inflammatory agent, for example, an oxicam, such as piroxicam, isoxicam, tenoxicam, sudoxicam, etc.; a salicylates, such as aspirin, disalcid, benorate, trilisate, safapryn, solprin, diflunisal, and fendosal; an acetic acid derivative, such as diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac, furofenac, tiopinac, zidometacin, acematacin, fentiazac, zomepirac, clmdanac, oxepinac, felbmac, and ketorolac; a mefenacin, such as ketoconazole, ... Nonsteroidal anti-inflammatory agents include, but are not limited to, fenamates such as namic acid, meclofenamic acid, flufenamic acid, niflumic acid, and tolfenamic acid; propionic acid derivatives such as ibuprofen, naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen, indopropfen, pirprofen, carprofen, oxaprozin, pranoprofen, miloprofen, tioxaprofen, suprofen, aluminoprofen, and tiaprofenic acid; pyrazoles such as phenylbutazone, oxyphenbutazone, feprazone, azapropazone, and trimethazone. Mixtures of these nonsteroidal anti-inflammatory agents may also be used.In various embodiments, the second therapeutic agent is a steroidal anti-inflammatory agent, for example, a corticosteroid, such as hydrocortisone, hydroxyl-triamcinolone, alpha-methyldexamethasone, dexamethasone phosphate, beclomethasone dipropionate, clobetasol valerate, desonide, desoxymethasone, desoxycorticosterone acetate, dexamethasone, dichlorisone, diflorasone acetate, diflucortolone valerate, fluadrenolone, fluclorolone acetonide, fludrocortisone, flumethasone pivalate, fluocinolone acetonide, fluocinonide, fluocortin butyl ester ... butylester), fluocortolone, fluprednidene acetate, flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolone, fludrocortisone, diflurozone diacetate diacetate), fluradrenolon acetonide, medrysone, amcinafel, amcinafide, betamethasone and the balance of its esters, chloroprednisone, chloroprednisone acetate, clocortelone, clescinolone, dichlorisone, diflurprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone, paramethasone, prednisolone prednisone, beclomethasone dipropionate, triamcinolone, and mixtures thereof.

[0163] In various embodiments, the combination therapy comprises administering the IL-2 variant and the second pharmaceutical composition simultaneously, either in the same pharmaceutical composition or in separate pharmaceutical compositions. In various embodiments, the IL-2 variant and the second pharmaceutical composition are administered sequentially, i.e., the IL-2 variant composition is administered before or after the administration of the second pharmaceutical composition. In various embodiments, the administration of the IL-2 variant composition and the second pharmaceutical composition is concurrent, i.e., the administration of the IL-2 variant composition overlaps with the administration of the second pharmaceutical composition. In various embodiments, the administration of the IL-2 variant composition and the second pharmaceutical composition is not concurrent. For example, in various embodiments, the administration of the IL-2 variant composition is terminated before the second pharmaceutical composition is administered. In various embodiments, the administration of the second pharmaceutical composition is terminated before the IL-2 variant composition is administered. EXAMPLES

[0164] The following examples are provided in order to more fully illustrate the present disclosure and are not to be construed as limiting the scope of the disclosure.

[0165] Example 1 Designing IL-2 variants to selectively target Treg cells In one embodiment, the present invention is directed to one or more mutations to weaken the affinity of IL-2 to IL-2Rβ and / or γc receptor subunits. In the context of weakened IL-2Rβγ interaction, the enhanced IL-2 sensitivity of Treg conferred by IL-2Rα expression may provide this cell subset with a distinct growth advantage. As a result, these mutants could act as Treg promoters in autoimmune and inflammatory diseases.

[0166] Each variant was computationally designed based on the structure of human IL-2 reported in the Protein Data Bank (PDB code 2B5I). A panel of variants was generated that contained one to three mutations (introducing conservative and non-conservative amino acid substitutions) in residues that are present at or near the interface that makes direct contact with the IL-2Rβ or γc receptor subunits. For example, D20 is involved in an extensive hydrogen bond network to the receptor subunit side chains at the IL-2Rβ interface. Similarly, N88 is a potential hotspot for IL-2 / IL-2Rβ interactions and is involved in a critical hydrogen bond with the receptor chain. Q126 is essential for γc interactions, and Q22 is also present at the γc interface. We hypothesized that mutations at or near the residues at these sites may result in defects in the ability of IL-2 to interact with the intermediate affinity receptor for IL-2, IL-2Rβγ.

[0167] Interestingly, the proposed " 19 The "LDL" motif (Baluna R, Rizo et al., Proc Natl Acad Sci, 1999;96:3957-62) overlaps with the IL-2Rβ interface. This "toxicity motif" is partially responsible for the direct vascular toxicity of IL-2. As a result, mutations introduced to replace residue D20 of the critical toxicity motif, or the adjacent residues L19 and L21 with non-aliphatic residues, were expected to eliminate the toxicity motif, inhibit endothelial cell injury, and also significantly reduce VLS.

[0168] In the present invention, the following 1 to 3 amino acid substitutions (D20T, D20E, D20N, D20Q, D20S, D20Y, D20I, L19Y, L19N, L19R, L19Q, L19H, L19D, L19P, L19S, L21S, L21N, L21R, N88R, N88G, N88I, N88Q, N88E, N88T, N88M, Q126E, Q126L, Q126N, Q126R ... 6D, Q126M, Q126K, Q126H, Q126Y, Q125E, S125K, S125H, S125W, S125I, Q22N, Q22H, Q22K, Q22Y, Q22I, D2 0I / N88G, D20I / N88R, D20T / N88R, D20I / N88I, D20T / Q126E, D20T / N88R / Q126E, D20T / Q126L, D20T / N88R / Q126L, L19N / Q126E, L19R / Q126E, L19Y / Q126E, L19H / Q126E, L19Q / Q126E, L19S / Q126E, L19Y / Q126K, L19Y / Q126H, L19Y / Q126Y, L19Y / S125E, L19Y / S125K, L19Y / S125H, L19Y / S125W, L19Y / S125I, L19Y / Q22 N, L19Y / Q22H, L19Y / Q22K, L19Y / Q22Y, L19Y / Q22I, L19H / Q126K, L19H / S125I, L19D / S125I, D20E / S25I , D20T / S125I, L19Y / S125I / Q126E, L19H / S125I / Q126E, L19H / S125I / Q126K, L19Q / S125I / Q126E, L19Q / A panel of IL-2 variants (SEQ ID NOs: 4-43, 108-146, and 193-197) with the amino acid substitutions Q126K, L19Q / S125I / Q126K, D20T / S125I / Q126K, L19N / S125I / Q126K, and L19R / S125I / Q126K) were expressed C-terminally fused to the Fc homodimer via a "GGGSGGGS" linker (SEQ ID NO: 55). Additionally, IL-2 variants with the amino acid substitutions D20I, D20I / N88G, D20E, or L19N were expressed N-terminally fused to the Fc homodimer via a rigid "AEAAAKEAAAKEAAAKA" linker (SEQ ID NO: 53).Collectively, the sequences of these IL-2 variant Fc fusion constructs are listed with SEQ ID NOs: 73 to 107, 147 to 189, and 198 to 211. Constructs containing the same Fc fusions (both C- and N-terminal) to wild-type IL-2 were also generated (SEQ ID NOs: 71 and 72).

[0169] All of the above IL-2 variant Fc fusion molecules are designed to confer a growth advantage to cells that highly express IL-2Rα, and to prioritize Treg cells over the proliferation of other lymphocytes, including CD4+ conventional T cells, CD8+ T cells, and NK cells. In addition, mutations at positions 19, 20, or 21 are expected to remove toxic motifs involved in vascular toxicity, so that the resulting molecules may have two beneficial properties, including enhanced selectivity of Treg activation and reduced endothelial cell damage. However, optimal mutations or combinations of mutations are crucial to maximize the window of selective targeting of Treg subsets by modulating the level of impairment to effector T and NK cell activation while maintaining a sufficiently high potency to activate Treg cells.

[0170] Furthermore, single point mutations were identified that resulted in improved protein stability, enhanced expression levels, and reduced aggregation tendency. The combination of aggregation-reducing mutations with Treg-targeting mutations made the protein more suitable for development and conferred a proliferation advantage on Treg cells.

[0171] Example 2 Construction and production of IL-2 Fc fusion constructs All genes were codon-optimized for expression in mammalian cells, synthesized, and subcloned into a recipient mammalian expression vector (GenScript). Protein expression is driven by a CMV promoter, and a synthetic SV40 polyA signal sequence is present at the 3' end of the CDS. A leader sequence is provided at the N-terminus of the construct to ensure proper signaling and processing for secretion.

[0172] The constructs were produced by co-transfecting HEK293-F cells growing in suspension with the mammalian expression vectors using polyethyleneimine (PEI, 25,000 molecular weight, linear, Polysciences). When more than one expression vector was present, the vectors were transfected at a 1:1 ratio. For transfection, HEK293 cells were cultured in serum-free FreeStyle™ 293 Expression Medium (ThermoFisher). For production in 1000 ml shake flasks (working volume 330 mL), 0.8 × 10 HEK293 cells were cultured at 100 °C for 24 h prior to transfection. 6 Cells were seeded at a density of 10 ...

[0173] For affinity chromatography, each supernatant was loaded onto a HiTrap MabSelectSure column (CV=5 mL, GE Healthcare) equilibrated with 25 ml of phosphate buffered saline (pH 7.2) (Thermo Fisher Scientific). Unbound proteins were removed by washing with 5 column volumes of PBS (pH 7.2), and the target proteins were eluted with 25 mM sodium citrate, 25 mM sodium chloride (pH 3.2). The protein solution was neutralized by the addition of 3% 1 M Tris (pH 10.2). If necessary, the Protein A material was also polished using ion exchange chromatography or mixed mode chromatography including, but not limited to, CaptoMMC (GE Healthcare), ceramic hydroxyapatite, or ceramic fluoroapatite (Bio-Rad). The target proteins were concentrated using an Amicon® Ultra-15 concentrator 10 KDa NMWC (Merck Millipore).

[0174] Purification and molecular weight of the purified constructs were analyzed by SDS-PAGE in the presence and absence of reducing agents and stained with Coomassie (Imperial™ Stain). NuPAGE® Pre-Cast Gel System (4-12% or 8-16% Bis-Tris, Thermo Fisher Scientific) was used according to the manufacturer's instructions. Protein concentration of purified protein samples was determined by measuring UV absorbance at 280 nm (Nanodrop spectrophotometer, Thermo Fisher Scientific) and dividing by the molar extinction coefficient calculated based on the amino acid sequence. Agilent 1200 high performance liquid chromatography (HPLC) system analyzed the amount of aggregation of the constructs. Samples were injected onto an AdvanceBio size exclusion column (300 Å, 4.6 × 150 mm, 2.7 μm, LC column, Agilent) at 25 °C with 150 mM sodium phosphate (pH 7.0) as the mobile phase.

[0175] It is noteworthy that the expression profiles and aggregation propensities of IL-2 variant Fc fusions differ markedly between constructs with different mutation sites or between variants sharing the same mutation site but with different residue substitutions.

[0176] Example 3 A single amino acid substitution in IL-2 results in a universal improvement in the suitability of fusion compounds for development The design approach to find combinations of mutations that result in variant proteins with desired biological properties faced great challenges when applied to IL-2. It is known in the art that naturally occurring IL-2 proteins tend to be less stable and more prone to aggregation. This was demonstrated in experiments where wild-type IL-2 Fc fusion protein (P-0250) was expressed at low levels (~3 mg / L transiently in HEK-293F cells) and showed a high tendency to aggregate, as shown by the SEC chromatogram illustrated in Figure 1A. Painstaking efforts to design amino acid substitutions in IL-2 aimed at the desired biological activity generally resulted in mutant proteins with even less stability. A significant portion of the IL-2 variants in this study were expressed at very low levels, and some variants had a significantly higher tendency to aggregate, as illustrated by the SEC chromatogram of P-0318 (SEQ ID NO: 95) shown in Figure 1B. This is problematic in the manufacture and storage of therapeutics.

[0177] We also observed that the expression profiles and aggregation propensity of IL-2 variant fusions differed significantly between constructs with different mutation sites or between variants sharing the same mutation site but with different residue substitutions. This finding is exemplified by P-0317 (SEQ ID NO: 94) and P-0318 (SEQ ID NO: 95). Both variant fusions share the same mutation sites at residues 20 and 88, differing by only one amino acid. P-0317 contains D20I and N88R amino acid substitutions, while P-0318 contains D20I and N88I mutations. Both variant fusions were expressed at similarly low levels. As can be seen in Figure 1B, P-0318 is highly aggregation-prone, with 65% high molecular weight species, which results in the expected peak being a smaller species in the chromatogram (indicated by the arrow). In contrast, P-0317 is relatively pure, with 7.5% aggregates (Figure 1C). It would be inferred that the N88R mutation may reduce the aggregation tendency of the resulting fusion protein. However, P-0254 (SEQ ID NO: 71) and P-0324 (SEQ ID NO: 96), fusion proteins derived from IL-2 with the N88R single mutation or IL-2 with the D20T / N88R double mutation, respectively, were prone to aggregation, with 30-40% aggregates. Therefore, the contribution of individual amino acid substitutions to protein stability appears to be context-dependent.

[0178] The fact that amino acid substitutions for IL-2 often reduce protein stability is further complicated by the unpredictable contribution of different residue substitutions to protein stability, making it highly desirable to find residue substitutions that can confer universal enhancements to protein suitability for development, including improved stability, increased expression levels, and reduced aggregation tendency.

[0179] The amino acid substitution at position 125 was originally intended to modulate the selectivity of IL-2, since this residue is in close proximity to Q126, which is essential for γc interaction. Native IL-2 contains an unpaired cysteine ​​at position 125, which was replaced by serine in Proleukin, but in the present invention, S125 is considered as the wild-type IL-2 residue. IL-2 containing an alanine substitution at position 125 is also widely used. Since serine or alanine substitutions for cysteine ​​at position 125 retained full biological activity, bulky charged or hydrophobic residues, including Glu, Lys, Tyr, His, and Ile, were introduced at position 125 to replace Ser in P-0372 (SEQ ID NO: 79), with the aim of interfering with the interaction of Q126 with γc to modify biological activity. All the resulting fusion molecules, except for P-0471 (SEQ ID NO: 178), could not be characterized because their expression levels were too low. P-0471, in contrast, was expressed at significantly higher levels (titers of 19.3 mg / L versus 4.0 mg / L) and had greatly reduced aggregation propensity (1% aggregation versus 21.7%) when compared to its S125 counterpart (P-0372).The impressive improvements in development suitability, particularly with respect to product purity, prompted us to evaluate whether such enhancements by isoleucine substitution at position 125 could be reproduced under different mutational contexts.

[0180] Therefore, the S125I substitution was introduced into several IL-2 variant Fc fusion molecules. Constructs with Ile-125 substitutions in IL-2 were expressed in the same vector and under the same culture conditions as their Ser-125 counterparts and purified using MabSelectSure. The expression levels (mg / L) and purity (aggregation rate) assessed by SEC chromatography of exemplary molecules are summarized in Table 4. Two molecules in the same row of Table 4 share the same other amino acid substitutions and differ only in whether residue 125 is a serine or an isoleucine. As an example, the SEC chromatograms and SDS-PAGE images of P-0447 (SEQ ID NO: 168) and its Ile-125 counterpart P-0511 (SEQ ID NO: 198) are further illustrated in Figure 1D and Figure 1E. As is evident from Table 4, the isoleucine substitution at position 125 resulted in 4- to 11-fold enhanced expression levels and uniformly lower aggregation propensity.

[0181] TIFF0007637415000010.tif83170

[0182] It is clear from the present invention that the substitution of isoleucine at position 125 results in a universal improvement in the suitability of IL-2 fusion constructs for development, while retaining full biological activity. Ile substitutions at position 125 of wild-type IL-2 and IL-2 variants of different mutation configurations in Fc fusion formats all resulted in 4-11 fold enhanced expression levels and uniformly low aggregation tendency. Expression levels (mg / L) and purity (aggregation rate) of Protein A purified products assessed by SEC chromatography for exemplary molecules are summarized in Table 4. This finding is particularly valuable because engineering of IL-2 for favorable biological properties has been hindered by the fact that modifications of the marginally stable wild-type IL-2 often result in less stable mutant proteins. The problems associated with engineering IL-2 can be alleviated by a single amino acid substitution at position 125 with isoleucine. In summary, the substitution of isoleucine at position 125 of IL-2, the IL-2 variant, significantly improves the development suitability profile of the protein, as demonstrated by increased protein expression and a significantly reduced aggregation tendency of the IL-2 construct.

[0183] Example 4 Identification of IL-2 variants with a single amino acid substitution that show differential selectivity for Treg lymphocytes Single amino acid substitutions were introduced into IL-2 at positions corresponding to amino acids interacting with the β, γ, or βγ receptor subunits. These substitutions were intended to reduce the ability of IL-2 to signal through the intermediate affinity IL-2Rβγ complex and confer specificity to signaling from the high affinity IL-2Rαβγ. IL-2 variants containing single amino acid substitutions were examined for their differential ability to stimulate STAT5 phosphorylation in CD4+ Treg and Tconv cells. STAT5 is known to be involved in downstream signaling cascades following IL-2 binding to the transmembrane IL-2 receptor. STAT5 phosphorylation in defined lymphocyte subpopulations was measured using fresh human peripheral blood mononuclear cells (PBMCs), and Treg populations were identified using the forkhead transcription factor FOXP3 in FACS analysis.

[0184] Briefly, human PBMCs were isolated by Ficoll-Hypaque centrifugation from buffy coats of healthy donors. PBMCs were starved for 1 h in serum-free MACS buffer at 4 °C. Then, 2 × 10 5 PBMCs were treated with serial dilutions of test compounds for 30 min at 37°C. Cells were fixed and permeabilized using Foxp3 / Transcription Factor Staining Buffer Set (EBIO) by incubation with 1x Foxp3 Fixation / Permeabilization Working Solution for 30 min and washing with 1x Permeabilization Buffer. Cells were then fixed with Cytofix buffer, permeabilized with Perm Buffer III (BD Biosciences), and washed. After blocking Fc receptors by adding human TruStain FcX (1:50 dilution), cells were stained with a mixture of anti-CD25-PE, anti-FOXP3-APC, anti-pSTAT5-FITC, and anti-CD4-PerCP-Cy5.5 antibodies at the manufacturer's recommended concentrations for 45 min at room temperature. Cells were collected by centrifugation, washed, resuspended in FACS buffer, and analyzed by flow cytometry. Flow cytometry data were gated into CD4+ / Foxp3+ / CD25 high and CD4+ / Foxp3+ / CD25 low populations (Treg and CD4 normal T cell subsets, respectively), and data are presented as the percentage of pStat5 positive cells in the gated populations.

[0185] Figure 2 shows the dose-response effect of exemplary IL-2 variant Fc fusion proteins on STAT5 phosphorylation in CD4 positive Treg and Tconv cells, compared to wild-type fusion protein. Wild-type IL-2 Fc fusion protein (P-0250) induced STAT5 phosphorylation in both Treg and Teff cells, with EC50 values ​​of 0.1 pM and 25.4 pM, respectively. Wild-type IL-2 activity was approximately 250-fold greater in Treg cells than in CD4+Tconv cells, consistent with the higher expression levels of high affinity trimeric receptors in Treg cells.

[0186] Various substitutions of aspartic acid at position 20, P-0364 (D20E), P-0363 (D20T), P-0365 (D20N), P-0366 (D20Q), and P-0367 (D20S), showed the ability to induce STAT5 phosphorylation in Treg cells, but such activity was largely attenuated or abolished in CD4+Tconv cells (Figures 2A and 2B). These variants may be Treg-biased IL-2 agents to activate Treg cells for the treatment of autoimmune diseases. Furthermore, mutations at D20, a key residue in the proposed toxin-like motif, are expected to remove the toxic motif and suppress endothelial cell damage. Thus, these variants are expected to have Treg-selective activity and improved safety profile for VLS. Furthermore, P-0368 showed no biological activity (Figures 2A and 2B).

[0187] Figure 3 shows the ability of IL-2 variant P-0375 (N88Q) to induce STAT5 phosphorylation in CD4+ Treg and CD4+ Tconv cells compared to reference-1 and reference-2 compounds with V91K and N88R mutations, respectively. The activity profile of the N88Q variant was similar to that of reference-2.

[0188] Figure 4 shows the biological activity of IL-2 variants with various mutations at position 19 compared to wild type. Variants P-0372 (L19Y), P-0373 (L19N), P-0374 (L19R), P-0423 (L19Q), P-0424 (L19H), and P-0427 (L19S) showed similar activity to wild type in inducing STAT5 phosphorylation in Treg cells (Figure 4A and Figure 4C). Variants P-0372, P-0374, P-0423, and P-0427 also largely retained biological activity in CD4+Tconv cells (Figure 4B and Figure 4D), whereas such activity was reduced in CD4+Tconv for variants P-0373 and P-0424. Mutant P-0425(L19D) showed a slight reduction in the activity of inducing STAT5 phosphorylation in Treg cells, but such activity was significantly impaired in CD4+Tconv cells (Figure 4C and Figure 4D). The selective activation of Treg cells by mutants P-0373, P-0424, and P-0425 over CD4+Tconv cells shown above, especially the wide window of selective targeting of Treg subsets by P-0373 and P-0425, make the mutants Treg-biased IL-2 drug candidates to activate Treg cells for the treatment of autoimmune diseases. Importantly, L19 is part of the proposed toxin-like motif, and mutation of this site is also expected to improve the safety profile and reduce VLS.

[0189] Example 5 IL-2Rβ and γ in IL-2 for differential selectivity towards Treg lymphocytes c Combination of targeted amino acid substitutions In Example 4, it was shown that specific mutations aimed at reducing the affinity of IL-2 to the IL-2Rβ or γc receptor subunits can result in IL-2 variants with differential selectivity for Treg lymphocytes. It was concluded that modulating the affinity of IL-2 to both the IL-2Rβ and γc receptor subunits through a combination of certain amino acid substitutions targeted to the β receptor and other substitutions targeted to the γ receptor can result in a window of favorable activity and selectivity for Treg lymphocytes.

[0190] Such a rationale is shown in Figure 5. Figures 5A and 5B show the effect of the IL-2Rβ-targeting variant P-0372 (L19Y) on STAT5 phosphorylation in Treg and CD4+Tconv cells, with wild-type IL-2 fusion protein P-0250 as a control. Similarly, Figures 5C and 5D show the STAT5 phosphorylation activity in the case of P-0303 (Q126E), which has an amino acid substitution targeted to disrupt interaction with the gamma receptor. The data showed that each single amino acid substitution had minimal impact on pSTAT5 activation capacity, but also showed only a slight improvement in the window of selectivity for the Treg lymphocyte subset compared to the wild type. As shown in Figures 5E and 5F, the combination of the L19Y and Q126E mutations in P-0419 significantly widened the window of selective activation of Treg cells. P-0419 largely retained its Treg activation potential, and this activity profile of the P-0419 variant was highly similar to that of the reference-1 molecule containing the V91K mutation. This strategy is particularly attractive because 19L is also part of a proposed toxin-like motif, and mutations at this site are also expected to improve the safety profile and reduce VLS.

[0191] However, combining one amino acid substitution targeting the β receptor with another substitution targeting the γ receptor does not provide a favorable window of activity and selectivity. It requires the right amount of activity modulation for each embodiment. In Figures 6A and 6B, the four IL-2 variants share the same L19Y substitution targeting the β receptor, and the additional mutations designed to target the γc receptor are Q126E in P-0419, Q126K in P-0464, S125I in P-0471, and Q22K in P-0474. All variants retained comparable activity in inducing STAT5 phosphorylation in Treg cells (Figure 6A), but the activity was significantly variable in CD4+Tconv cells (Figure 6B), indicating different abilities in selectively modulating Treg activation via combinations of amino acid substitutions.

[0192] However, by combining the Q126E substitution with additional receptor attenuation of IL-2 variants that already showed biased specificity for Treg subsets, activity against Treg cells could be significantly attenuated or eliminated. As shown in (Figure 6C and Figure 6D), both variants P-0373(L19N) and P-0363(D20T) already showed some or significant bias in the selectivity window for Treg cells (Figure 6C and Figure 6D). Their respective counterparts P-0417 and P-0322 with the additional Q126E substitution showed a significant reduction in Treg cell activation capacity. Thus, it is important to find the right combination of residue substitutions to tune activity so as to obtain favorable activation capacity and specificity bias for Treg cells.

[0193] Additional variants with double amino acid substitutions at positions L19 and Q126, including P-0447 (L19H / Q126E), P-0448 (L19Q / Q126E), and P-0449 (L19S / Q126E), were evaluated and their activity is shown in Figures 7A-7D. Compared to IL-2 variants P-0424 (L19H) and P-0303 (Q126E), each of which contains one amino acid substitution, variant P-0447 (L19H, Q126E), which has a combination of two amino acid substitutions, showed robust biological activity in stimulating STAT5 phosphorylation in Treg cells, but such activity was almost completely lost in Tconv cells (Figures 7A and 7B). In another study evaluating P-0419, P-0447, P-0448, and P-0449 against two benchmark compounds, all four variants were significantly able to induce STAT5 phosphorylation in Treg cells, but this activity was largely lost in CD4+Tconv cells (Figures 7C and 7D).Similar to what was shown in Figures 5E and 5F, P-0419 had an activity profile comparable to benchmark-1, while P-0447, P-0448, and P-0449 were comparable to benchmark-3 in terms of activity and selectivity window for Treg cells.

[0194] All these variants are potential Treg-biased IL-2 agents to activate Treg cells for the treatment of autoimmune diseases. Moreover, these variants are expected to have improved safety profiles and reduced VLS due to the removal of potential toxic motifs.

[0195] Example 6 IL-2 variants with isoleucine substitutions at position 125 retain full biological activity In Example 3, it was shown that an isoleucine substitution at position 125 results in a universal improvement in the development suitability of IL-2 fusion constructs. In order to make the S125I substitution a viable approach to alleviate the development suitability problem of IL-2 engineering, it will be important to show that such an amino acid substitution does not impair the biological activity of the resulting fusion protein in comparison to its Ser-125 counterpart.

[0196] Therefore, the S125I substitution was introduced into wild-type IL-2 or into IL-2 variants that already had one or two mutations targeting the receptor subunits β, γ, or βγ. The resulting IL-2 variants with isoleucine at position 125 were tested for their ability to stimulate STAT5 phosphorylation in Treg and Tconv cells, with their serine 125 counterparts as controls. Table 5 shows the activity and selectivity of IL-2 variants in Treg cells. Two molecules in the same row in Table 5 share the same amino acid substitutions with respect to other amino acid substitutions, and only differ in whether serine or isoleucine is at position 125. The data showed that the S125I substitution fully retained or slightly improved the biological activity of the various tested IL-2 variants without changing the Treg specificity.

[0197] TIFF0007637415000011.tif85170

[0198] Data from three exemplary constructs, P-0250, P-0424, and P-0447, and their S125I counterparts, P-0531, P-0491, and P-0511, are shown in FIG. 8. P-0250 is a wild-type IL-2 Fc fusion molecule, P-0424 contains one amino acid substitution L19H, and P-0447 contains two amino acid substitutions L19H / Q126E. Their dose-dependent effects on STAT5 phosphorylation in Treg and CD4+Tconv cells are shown in FIG. 8. As shown in FIG. 8A-F, the S125I substitution slightly increased the activity of the three tested compounds, did not change Treg selectivity for P-0531 and P-0491, and for P-0511, the S125I substitution further widened the window of Treg selectivity.

[0199] The data thus show that the S125I substitution in IL-2 preserves the IL-2 activity profile of IL-2 fusion proteins with different mutational backgrounds. In summary, the isoleucine substitution at position 125 of IL-2 provided universal development compatibility improvements (increased production yield, reduced aggregation, reduced potential immunogenicity) for IL-2, IL-2 fusions, IL-2 variants, and IL-2 variant fusions, while fully retaining biological activity and selectivity. This particular amino acid substitution represents a viable mitigation strategy to address problems associated with IL-2 engineering.

[0200] Example 7 Effects of IL-2 variants on CD25+CD4+ T cells, CD8 cytotoxic T cells, and NK cells In addition to assessing the differential ability to stimulate STAT5 phosphorylation in CD4+ Treg cells (CD4+ / Foxp3+ / CD25-high) and Tconv cells (CD4+ / Foxp3-negative / CD25-low), the two variants, P-0511 and P-0512, were further assayed for their ability to stimulate other effector T cells and NK cells, including CD4+ Teff cells (CD4+ / Foxp3-negative / CD25-positive), CD8 cytotoxic effector T cells, and NK cells, in comparison to wild-type IL-2 (P-0250) and three IL-2 reference molecules, including V91K, N88R, and N88D, respectively.

[0201] The IL-2 variants of the present invention attenuate IL-2Rβγ interaction, and the marked proliferation advantage of these variants over CD4+ Tconv is conferred by the constitutive high expression of IL-2Rα (CD25) on Treg. CD25 expression can be induced on CD4+ effector T cells after immune stimulation. Therefore, it is desirable to ensure that the IL-2 variants retain Treg specificity over other CD25+ lymphocyte subsets. An example of a lymphocyte subset with intermediate to high CD25 expression levels is CD4+ effector T cells (Teff).

[0202] Human PBMC cells were treated with serial dilutions of test compounds, fixed, permeabilized, washed, and stained with a mixture of anti-CD25-PE, anti-FOXP3-APC, anti-pSTAT5-FITC, and anti-CD4-PerCP-Cy5.5 antibodies, followed by flow cytometry analysis with gating to define CD4+ / Foxp3+ / CD25+, CD4+ / Foxp3- / CD25+, and CD4+ / Foxp3- / CD25- as Treg, CD4 effector, and CD4 naive T cell subsets, respectively. Data are expressed as the percentage of pSTAT5 positive cells in the gated population and are shown in Figure 9. P-0512 has a comparable activity profile to criterion-1 in all three T cell subsets, and P-511 outperforms both criterion-2 and criterion-3 in terms of activity and window of selectivity for Treg cells compared to both Teff and naive CD4 T cells. Criterion-2 showed a much weaker activation capacity for each of the three subsets. Figures 9A and 9B clearly show preferential activation of Treg over Teff by IL-2 variants with attenuated IL-2Rβγ interaction, especially P-0511, despite the moderate to high expression of CD25 on Teff.

[0203] Furthermore, P-0511 and P-0512 were tested for their ability to stimulate proliferation of NK cells and CD8+ T cells in comparison to wild-type and reference molecules. The intracellular fluorescent label, carboxyfluorescein diacetate succinimidyl ester (CFSE) method was used. Briefly, human PBMCs (1 × 10 5100 cells / well) were labeled with CFSE, seeded in 96-well plates, and incubated with increasing concentrations of various IL-2 compounds. After 5 or 7 days of incubation, cells were harvested and stained with anti-CD56-APC antibody for NK cells and anti-CD8-APC antibody for CD8+ T cells, and subjected to flow cytometry analysis. Data are presented as the percentage of dividing cells and are shown in Figure 10A for CD8+ T cell proliferation and Figure 10B for NK cell proliferation.

[0204] As expected, all IL-2 variants showed attenuated ability to stimulate both CD8+ T and NK cells compared to the wild-type IL-2 fusion molecule, P-0250. In support of the STAT5 phosphorylation assay (Figure 9), P-0512 had a comparable activity profile to Criteria-1, P-0511 was comparable to Criteria-3 in terms of activity against both lymphocyte subsets, while Criteria-2 showed much weaker activity.

[0205] The STAT5 phosphorylation activity of P-0511 on responder cells other than CD4+T cell subsets, including CD8+T cells and NK cells, was compared with that of P-0531, the S125I counterpart of wild-type P-0250. P-0511, like P-0531, showed significant activity in stimulating STAT5 phosphorylation in Treg cells (Figure 11A), but such activity was almost completely abolished in CD4+Tconv cells (Figure 11B), CD8+T cells (Figure 11C), and NK cells (Figure 11D). The IL-2 receptor expressed on CD4+Tconv cells, CD8+T cells, and NK cells is mainly a dimeric IL-2R, containing IL-2Rβ and γc. To confirm whether the significant attenuation of pSTAT5 signaling by P-0511 on CD8+T cells and NK cells was due to impaired interaction with IL-2Rβ and γc, an ELISA assay was developed.

[0206] Briefly, non-covalent complexes of IL-2Rβ-ECD (NP_000869) and γc-ECD (NP_000197) via heterodimeric Fc chains were coated at 2 μg / well on each well of a Nunc Maxisorp 96-well microplate. After overnight incubation at 4°C and blocking with SuperBlock (Thermo Fisher Scientific), 3-fold serial dilutions of IL-2 Fc fusion proteins starting from 100 nM or 270 nM were added to each well at 100 μl / well. After 1 h incubation at room temperature, 1 μg / ml of biotin mouse anti-human IL-2 antibody (BD Biosciences) was added to each well, followed by incubation with 1 μg / ml of HRP-avidin (Thermo Fisher Scientific) for 1 h. After each step, wells were thoroughly aspirated and washed three times with PBS / 0.05% Tween-20. Finally, 100 μl of TMB substrate was added to each well, plates were developed for 10 min in the dark at room temperature, and 100 μl / well of stop solution (2N sulfuric acid, Ricca Chemical) was added. Absorbance at 450 nm was measured and curve fitting was performed using Prism software (GraphPad) and is illustrated in Figure 11E.

[0207] As shown in Figure 11E, P-0531, a wild-type IL-2 fusion protein with improved development compatibility, binds the IL-2 dimeric receptor complex with sub-nM affinity (EC 50 = 0.06 nM); the reference-1 molecule showed a decrease in binding (EC 50=1.6 nM), consistent with the molecule's attenuated ability to stimulate STAT5 phosphorylation in CD8+ T cells and NK cells (Figures 10A-B). In contrast, P-0511 showed no appreciable binding to the IL-2Rβ and γc complex, and two IL-2 mutations in P-0511 at both the β and γc receptor subunit interfaces dramatically impaired interaction with the complex. Surprisingly, despite virtually abolishing binding to the dimeric IL-2 receptor complex, P-0511 showed only a modest decrease in activity against Tregs compared to the wild-type IL-2 fusion. P-0511 exemplifies an IL-2 variant with a window of activity and selectivity favorable for Treg lymphocytes.

[0208] In summary, a series of IL-2 variants listed in Tables 2A-2F were constructed, expressed, and tested in in vitro assays. The biological activity of exemplary IL-2 variants in Treg cells compared to other lymphocyte subsets, including CD4+Tconv cells, CD4+Teff cells, CD8+T cells, and NK cells, is shown in Figures 2-11. Many variants retained high activity in Treg cells, but reduced or lost activity in Tconv cells and other lymphocyte subsets. Some variants had activity profiles similar to those of criterion-1, while others resembled activity characteristics of criteria-2 or criteria-3. In addition, most of these IL-2 variants had proposed toxin-like motifs removed with the aim of reducing VLS. Importantly, the incorporation of the S125I amino acid substitution resulted in IL-2 variant fusions with better development suitability profiles while retaining biological activity and selectivity in Treg cells. These variants are potent Treg-biased IL-2 agents with improved safety profiles for the treatment of autoimmune diseases.

[0209] Example 8 IL-2 variant Fc fusion proteins preferentially stimulate the proliferation and expansion of Treg cells in mice IL-2 variant Fc fusion proteins were administered to mice and their ability to preferentially stimulate the proliferation and proliferation of regulatory T cells (CD4+CD25+FoxP3+ T cells) over effector T cells and NK cells in vivo was determined.

[0210] Female C57 / BL6 mice (7 weeks old) were obtained from Charles River Laboratories and acclimated in-house for at least 7 days prior to testing. On day 0, mice were subcutaneously dosed with vehicle (PBS), 0.3 mg / kg of each test compound, or IL-2 reference compound. Peripheral blood samples were collected and placed in heparinized tubes on days 3, 5, and 7 after treatment. Each group contained six mice, and baseline blood was collected 2 days prior to treatment (day -2). After lysis of red blood cells, total viable mononuclear blood cells were counted by trypan blue dead cell exclusion and proceeded to intracellular staining for immune cell phenotypes and Ki67 proliferation marker using flow cytometry analysis. Cells were stained separately with two antibody panels as described: 1) for CD4+ regulatory T cells (Tregs), anti-mouse Foxp3-FITC, Ki67-PE, anti-mouse CD25-APC, and anti-mouse CD4-Percpcy5.5 (1:50 dilution); 2) for CD8+ T cells and NK cells, anti-mouse CD3-FITC, Ki67-PE, anti-mouse CD335-APC, and anti-mouse CD8-Percpcy5.5 (1:50 dilution).

[0211] All tested IL-2 compounds stimulated the proliferation and proliferation of Treg cells, as shown by the increase in Ki67 positive Treg cells and the increase in the percentage of Treg cells relative to total CD4+ T cells or total lymphocytes (Figures 12A-C). After one single injection, this effect was observed 3 days after injection and persisted until the 5th or 7th day. In contrast to the ex vivo confirmation that benchmark-1 consistently showed the highest activity among IL-2 variants in inducing Treg phosphorylation, all three tested variants, P-0511, P-0512, and P-0514, showed stronger in vivo efficacy in stimulating the proliferation and proliferation of Treg cells than the benchmark molecules in mice. P-0511, P-0512, and P-0514 showed comparable activity. The relative in vivo activity ranked among the three standards was consistent with the ex vivo human PBMC cell assay, i.e., standard-1 was the most active, followed by standard-3, and standard-2 was much weaker in stimulating the proliferation and proliferation of Treg cells (Figures 12A-C).

[0212] For effector T cells and NK cells, Criterion-1 showed strong Ki67 stimulation for cytotoxic CD8 T cells and NK cells, whereas Criterion-2 and Criterion-3 showed less effect for CD8 T cells and NK cells (Figure 13A-C). Variant P-0514 showed Ki67 stimulation for CD8+ T cells similar to Criterion-1, while variants P-0511 and P-0512 showed mild Ki67 stimulation for CD8 T cells and NK cells like Criterion-2 and Criterion-3 (Figure 13A-C). The data suggest that variants P-0511 and P-0512 show better bioactivity and selectivity for Treg compared to Criterion-1 and Criterion-2. Criterion-3 was not effective in stimulating and expanding both Treg and effector cells.

[0213] The increase in the Treg population led to a decrease in the percentage of CD4+ conventional T cells in all IL-2 variant treatment groups (Figure 14A). No significant increase in CD4+ Tconv cells, CD8 T cells, or NK cells was observed in mice treated with any of the Treg-biased IL-2 variants (P-0511, P-0512, and P-0514) or in mice treated with the three criteria (Figures 14B-D).

[0214] Compared to the three benchmarks, all three variants, P-0511, P-0512, and P-0514, also showed the most beneficial Treg / Tconv ratios in terms of both Ki67 stimulation and cell proliferation based on cell numbers at all measurement time points (Figures 15A and 15B).

[0215] Foxp3 expression was increased on Treg cells by all tested IL-2 compounds 3 days after injection (Figure 16A), and all three variants showed similarly higher expression of CD25 and Foxp3 markers than the three baselines (Figures 16A and 16B), suggesting better Treg activation and functionality.

[0216] Body weight was monitored before and during treatment and no significant changes in body weight were noted (data not shown).

[0217] Collectively, these data show that variants P-0511, P-0512, and P-0514 display the ability to promote the activation, proliferation, and expansion of immunosuppressive Treg cells, while sparing CD4+ conventional cells, cytotoxic effector T cells, and NK cells. These data also demonstrated the superiority of these three variants over the reference molecules in terms of both efficacy and selectivity for Treg proliferation and expansion. These variants may serve as therapeutic agents to combat autoimmune and inflammatory diseases, as well as organ transplant rejection.

[0218] Example 9 Dose-response pharmacodynamic study with IL-2 variant Fc fusion proteins in mice after a single injection Vehicle (PBS) or P-0511 (1 mg / kg, 0.3 mg / kg, 0.1 mg / kg, or 0.03 mg / kg) was administered subcutaneously once to female Balb / C mice (n=5 mice / group), and peripheral blood was collected on day -2 as baseline and on days 3, 5, and 7 after administration. On day 7, mice were sacrificed and spleens were removed. Blood lymphocyte phenotype, proliferation capacity, and proliferation were measured by flow cytometry using fresh whole blood at each time point.

[0219] There were no significant changes in body or spleen weights in any treatment group (data not shown).

[0220] As shown in Figure 17, a dose-dependent increase in the proliferation potential of Treg cells was observed in mice treated with P-0511 at dose levels of 1 mg / kg, 0.3 mg / kg, or 0.1 mg / kg, as reflected by an increase in the percentage of Ki67-positive cells (Figure 17A). Treatment with 0.03 mg / kg was the least effective. At the three highest dose levels, stimulation of Ki67 expression in Treg cells peaked on day 3, remained at a plateau until day 5, and then decreased. As a result, P-0511 treatment dose-dependently increased the percentage of Treg to total CD4+ T cells (Figure 17B), the absolute number of Treg cells (Figure 17C), and the fold change in cell number from baseline (Figure 17D). The increase in Treg cell proliferation followed a similar kinetic pattern as the proliferation potential / activation Ki67 marker (Figure 17), i.e., peaked on day 3 and continued until day 5. Administration of 1 mg / kg stimulated Tregs of greater magnitude and duration, with the signal persisting through the fifth day.

[0221] Treatment with P-0511 also resulted in a dose-dependent and statistically significant increase in the proportion of Tregs relative to total lymphocytes (Figure 18A), while no statistically significant changes were observed in the proportion of CD4+Tconv cells (Figure 18B), CD8 Teff cells (Figure 18C), or NK cells (Figure 18D) relative to total lymphocytes. At peak, Tregs accounted for 4.5% of total lymphocytes in the 1 mg / kg single dose treatment, compared with 3.1% in the 0.3 mg / kg treatment and 1.4% in the 0.1 mg / kg treatment. In the vehicle control group, Tregs accounted for 0.5% of total lymphocytes (Figure 18A).

[0222] The Treg / Tconv ratio was calculated based on cell counts (Figure 19A). The Treg / Tconv ratio peaked at 0.27 with 1 mg / kg treatment, 0.18 with 0.3 mg / kg, and 0.06 with 0.1 mg / kg, compared with 0.027 without treatment (Figure 19A), suggesting preferential expansion of Treg cells over Tconv cells by P-0511. In addition, the expression of Treg cell functional markers, including CD25 (Figure 19B) and FoxP3 (Figure 19C), was also increased in a dose-dependent manner. The increase in mean fluorescence intensity (MFI) of CD25 and FoxP3 peaked on day 3 and attenuated to lower levels on day 5.

[0223] Collectively, these data indicate that P-0511 exerts potent and preferential Treg activation and expansion in a dose-dependent manner. Careful consideration is needed to achieve an optimized dosing strategy aimed at maximizing activity to promote the activation, proliferative capacity, and proliferation of immunosuppressive Treg cells while sparing cytotoxic effector T cells and NK cells.

[0224] Example 10 Pharmacodynamic study in mice following repeated administration of IL-2 variant Fc fusion proteins Female Balb / C mice (7 weeks old) were acclimated in house for 5–7 days prior to testing. Mice (n=5 / group) were subcutaneously administered vehicle (PBS), 0.3 mg / kg P-0511, P-0512, P-0531, or Reference-1 compound on days 0, 3, and 6. Peripheral blood was collected on day 3, 3 days after the first injection, and on day 9, 3 days after multiple (3) injections. Based on previous in vivo experiments, Treg cell activation, proliferation capacity, and proliferation were expected to peak on day 3, so 3 days post-injection were selected for data collection and analysis. Changes in blood lymphocyte activation, proliferation capacity, and proliferation were measured by flow cytometry. P-0531 is the S125I counterpart of the wild-type IL-2 fusion protein. Reference-1 contains the V91K mutation.

[0225] Three days after a single subcutaneous administration of IL-2 fusion protein, nearly 90% of Treg cells showed positive Ki67 expression in all test groups, and these Ki67 positive cells remained significantly higher after the third dose of all test compounds (Figure 20A). Interestingly, Treg cells, expressed as the percentage of Tregs to total CD4 T cells or the percentage of Tregs to total lymphocytes, were dramatically reduced to near control levels in P-0531- and criterion-1-treated mice after three consecutive every-third-day treatments compared to a single treatment, but remained at significantly higher levels in P-0511- and P-0512-treated mice (Figures 20B-C). The data suggest that wild-type IL-2 or criterion-1 may promote Treg cell exhaustion or induce Treg desensitization due to their stronger activity on Treg stimulation. Further explanations could include differences in half-life or "receptor sinks" on non-lymphocytes that could result in altered drug exposure, leading to little or no Treg selectivity of wild-type IL-2 or canonical-1.

[0226] Similar findings were obtained for Treg cell counts and fold changes over PBS control (Figures 21A-B), as well as Treg / Tconv ratios (Figure 22). P-0511 and P-0512 showed a better ability to maintain the Treg pool and maintain Treg selectivity compared to P-0531 and Reference-1.

[0227] Collectively, these data indicate that P-0511 and P-0512 are superior IL-2 molecules that show preferential and sustained in vivo Treg expansion after multiple dosing. By adjusting the dosing regimen, such as the dose and dosing frequency, of the IL-2 variant Fc fusions, further optimization of the desired activity and selectivity towards Tregs beyond proinflammatory immune activation can be achieved.

[0228] Example 11 Suppression of antigen-driven inflammation by IL-2 variant Fc fusion proteins in a mouse model of delayed-type hypersensitivity (DTH) The in vivo ability of Treg cells induced by IL-2 variants to suppress T cell antigen-driven inflammation was evaluated in a delayed type hypersensitivity (DTH) model. Female Balb / C mice (7 weeks old) were acclimated in-house for 7 days and randomly divided into groups. Subcutaneous administration of vehicle (PBS), 0.1 mg / kg or 0.3 mg / kg P-0511 was started on day -2 and administered once every 3 days (every 3 days) for three injections or once every 5 days (every 5 days) for two injections. Mice were then sensitized on day 0 by subcutaneous administration of 100 μg keyhole limpet hemocyanin (KLH) in 200 μl saline. For every 3 days administration, two further subcutaneous injections of PBS or P-0511 (0.1 mg / kg or 0.3 mg / kg) were administered on days 1 and 4; for every 5 days administration, one additional subcutaneous injection was administered. On day 3, mice were administered an injection of PBS, 0.1 mg / kg, or 0.3 mg / kg P-0511. On day 5, mice were administered KLH (5 μg / 10 μl saline) intradermally in the right ear. Right ear thickness was measured using a caliper before KLH administration on day 5, and every day from day 6 to day 8, which correspond to 24 hours, 48 ​​hours, and 72 hours after KLH administration. As a positive control, one group of mice was also administered 5 mg / kg dexamethasone intraperitoneally every day from day 5 to day 8.

[0229] FIG. 23 shows the kinetics of the DTH response in terms of the change in ear thickness relative to baseline values ​​(Δear thickness) at various time points after KLH exposure.

[0230] After subcutaneous KLH antigen sensitization, significant ear inflammation and swelling peaked 24 hours after intradermal KLH administration of the pinna, and this ear swelling was prolonged for 72 hours in the PBS group. It is clear that the immunosuppressive steroid dexamethasone strongly inhibited the KLH-induced inflammatory response with four consecutive daily doses at 5 mg / kg, achieving approximately 85% inhibition 72 hours after KLH administration. Suppression of antigen-driven inflammation by Treg cells induced by P-0511 was also evident in mice treated with 0.3 mg / kg P-0511 every 3 or 5 days at all time points after KLH administration (Figures 23A-B). At 0.1 mg / kg, a similar trend toward reduced DTH inflammatory responses was observed in both the every 3 and every 5 day doses, but this effect did not reach statistical significance at most time points. Both every 3 day and every 5 day dosing schedules were effective.

[0231] In another study, the dose-dependent response of P-0511 (0.1 mg / kg, 0.3 mg / kg, and 1 mg / kg, every 5 days) to suppression of KLH-induced DTH was determined and compared to Reference-1 (0.3 mg / kg, every 5 days). As shown in Figure 24, P-0511 demonstrated a dose-dependent inhibition of ear inflammation. Mice administered 1 mg / kg P-0511 exhibited strong resistance to KLH-induced DTH and exhibited the least ear swelling after KLH administration. Intermediate and mild inhibitory effects were observed for P-0511 at 0.3 mg / kg and P-0511 at 0.1 mg / kg, respectively. Reference-1 showed mild inhibition of ear swelling, and the effect of Reference-1 at 0.3 mg / kg was similar to that achieved with P-0511 at 0.1 mg / kg (Figure 24).

[0232] In summary, Treg cells induced by P-0511 administration were effective in suppressing T cell antigen-driven inflammation in the DTH model. In addition, Treg suppression was maintained after KLH administration without repeated administration. This example makes it clear that it is also very important to adjust the administration schedule to achieve optimal efficacy.

[0233] Example 12 Pharmacodynamic / pharmacokinetic effects of P-0511 in cynomolgus monkeys The PK / PD properties of P-0511, an IL-2 variant Fc fusion protein, in cynomolgus monkeys will be evaluated following a 3 dose dosing schedule every 14 days. Drug-naive cynomolgus monkeys will be acclimated for 3-4 weeks and randomized into 4 groups (n=3-4 / group) followed by a pre-dose baseline week. On days 1, 15, and 29, one group will receive vehicle (PBS) subcutaneously and the other groups will receive 100 μg / kg, 30 μg / kg, or 10 μg / kg P-0511 subcutaneously.

[0234] Blood samples will be collected on days -7, -3, 2, 4, 6, 8, 11, 14, 18, 21, 28, 32, 35, 43, 50, and 57. Whole blood will be used for immunophenotyping by FACS of peripheral blood Tregs, non-regulatory CD4 T cells, CD8 T cells, and NK cells, naive cells, and memory cells to determine their pharmacodynamic properties. Cell activation and proliferation will also be monitored by measuring CD69 and Ki67. Whole blood will also be used for a complete blood count (CBC) with a 5-fold differential (neutrophils, lymphocytes, monocytes, eosinophils, and basophils).

[0235] The PK properties of P-0511 will be evaluated in cynomolgus monkey plasma samples by measuring full-length intact P-0511 using mouse anti-human IL-2 monoclonal antibody (BD Pharmingen) to coat 96-well plates for P-0511 capture. P-0511 will be detected using goat anti-human Fc polyclonal-HRP (Thermo Fisher Scientific) and its plasma concentration will then be quantified. In addition to plasma samples taken on days -7, -3, 2, 4, 6, 8, 11, 14, 18, 21, 28, 32, 35, 43, 50, and 57, three additional plasma samples were taken on day 1 at 0.5, 3, and 6 hours after the first dose of P-0511.

[0236] The following clinical chemistry parameters will also be assessed using plasma samples obtained from days -8, 8, 21, 35, 43, and 57: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma glutamyl transferase, albumin, total bilirubin, creatinine, blood urea nitrogen, and C-reactive protein.

[0237] In addition, the weight and temperature of each animal will be monitored weekly or twice weekly for the entire study period.

[0238] All of the articles and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the articles and methods of the present disclosure have been described in terms of preferred embodiments, it will be apparent to one skilled in the art that modifications may be applied to the articles and methods without departing from the spirit and scope of the present disclosure. All such modifications and equivalents, whether now existing or later developed, that are apparent to one skilled in the art are deemed to be within the spirit and scope of the present disclosure as defined by the appended claims. All patents, patent applications, and publications mentioned in this specification are indicative of the level of skill in the art to which this disclosure pertains. All patents, patent applications, and publications are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. The disclosure illustratively described herein may be suitably practiced in the absence of any element not specifically disclosed herein. That is, while the present disclosure has been specifically disclosed by preferred embodiments and optional features, it is to be understood that modifications and variations of the concepts disclosed herein may be made by those skilled in the art, and that such modifications and variations are deemed to be within the scope of the present disclosure as defined by the appended claims. [Sequence table] In the nucleic acid and amino acid sequences listed in the accompanying sequence listing, nucleotide bases are shown using standard abbreviations and amino acids are shown using the single-letter code, as provided in 37 CFR §1.822. SEQ ID NO:1 is the amino acid sequence of the human IL-2 precursor. SEQ ID NO:2 is the native amino acid sequence of the mature form of human IL-2. SEQ ID NO:3 is the wild-type amino acid sequence of mature human IL-2. SEQ ID NOs: 4 to 43, 108 to 146, and 193 to 197 are the amino acid sequences of various IL-2 variants. SEQ ID NO: 44 is the amino acid sequence of human IgG1-Fc. SEQ ID NO: 45 is the amino acid sequence of human IgG1-Fc with reduced / eliminated effector function. SEQ ID NOs: 46, 47, and 212-213 are the amino acid sequences of human IgG1-Fc with reduced effector function and extended half-life. SEQ ID NOs: 48 to 67 are the amino acid sequences of various peptide linker sequences. SEQ ID NO:68 is the amino acid sequence of the human IL-2 receptor alpha Sushi domain. SEQ ID NOs: 69 to 107, 147 to 189, and 198 to 211 are the amino acid sequences of various Fc-IL-2 fusion proteins. SEQ ID NOs: 190 to 192 are the amino acid sequences of reference Fc-IL-2 variant fusion proteins. SEQ ID NOs:214-222 are the nucleotide sequences of various Fc-IL-2 fusion proteins. Sequence Listing Human IL-2 precursor sequence MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 1) Human IL-2 mature native sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 2) Human IL-2 mature wild type sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 3) IL-2 N88R variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 4) IL-2 D20T variant sequence APTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 5) IL-2 D20E variant sequence APTSSSTKKTQLQLEHLLLELQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 6) IL-2 D20N variant sequence APTSSSTKKTQLQLEHLLLNLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 7) IL-2 D20Q variant sequence APTSSSTKKTQLQLEHLLLQLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 8) IL-2 D20S variant sequence APTSSSTKKTQLQLEHLLLSLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 9) IL-2 D20Y variant sequence APTSSSTKKTQLQLEHLLLYLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 10) IL-2 D20I variant sequence APTSSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 11) IL-2 L19Y variant sequence APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 12) IL-2 L19N variant sequence APTSSSTKKTQLQLEHLLNDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 13) IL-2 L19R variant sequence APTSSSTKKTQLQLEHLLRDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 14) IL-2 N88G variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 15) IL-2 N88I variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISIINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 16) IL-2 N88Q variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISQINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 17) IL-2 N88E variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISEINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 18) IL-2 N88T variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 19) IL-2 N88M variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISMINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 20) IL-2 Q126E variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 21) IL-2 Q126L variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSLSIISTLT (SEQ ID NO: 22) IL-2 Q126N variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSNSIISTLT (SEQ ID NO: 23) IL-2 Q126D variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSDSIISTLT (SEQ ID NO: 24) IL-2 Q126M variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSMSIISTLT (SEQ ID NO: 25) IL-2 D20I / N88G variant sequence APTSSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 26) IL-2 D20I / N88R variant sequence APTSSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 27) IL-2 D20T / N88R variant sequence APTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 28) IL-2 D20I / N88I variant sequence APTSSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISIINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 29) IL-2 D20T / Q126E variant sequence APTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 30) IL-2 D20T / N88R / Q126E variant sequence APTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 31) IL-2 D20T / Q126L variant sequence APTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSLSIISTLT (SEQ ID NO: 32) IL-2 D20T / N88R / Q126L variant sequence APTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSLSIISTLT (SEQ ID NO: 33) IL-2 L19N / Q126E variant sequence APTSSSTKKTQLQLEHLLNDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 34) IL-2 L19R / Q126E variant sequence APTSSSTKKTQLQLEHLLRDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 35) IL-2 L19Y / Q126E variant sequence APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 36) IL-2 L19Q variant sequence APTSSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 37) IL-2 L19H variant sequence APTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 38) IL-2 L19D variant sequence APTSSSTKKTQLQLEHLLDDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 39) IL-2 L19P variant sequence APTSSSTKKTQLQLEHLLPDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 40) IL-2 D20T / S125I / Q126K variant sequence APTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT (SEQ ID NO: 41) IL-2 L19N / S125I / Q126K variant sequence APTSSSTKKTQLQLEHLLNDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT (SEQ ID NO: 42) IL-2 L19R / S125I / Q126K variant sequence APTSSSTKKTQLQLEHLLRDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT (SEQ ID NO: 43) Human IgG1-Fc DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 44) Human IgG1-Fc with reduced / aborted effector functions DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 45) Human IgG1-Fc with reduced / eliminated effector functions and extended half-life DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 46) Human IgG1-Fc with reduced / eliminated effector functions and extended half-life DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPG (SEQ ID NO: 47) Peptide linker sequence GGGSGGGSGGGS (SEQ ID NO:48) Peptide linker sequence GGGS (SEQ ID NO:49) Peptide linker sequence GSSGGSGGSGGSG (SEQ ID NO:50) Peptide linker sequence GSSGT (SEQ ID NO:51) Peptide linker sequence GGGGSGGGGSGGGS (SEQ ID NO:52) Peptide linker sequence AEAAAKEAAAKEAAAKA (SEQ ID NO:53) Peptide linker sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:54) Peptide linker sequence GGGSGGGS (SEQ ID NO:55) Peptide linker sequence GS (SEQ ID NO:56) Peptide linker sequence GGS (SEQ ID NO:57) Peptide linker sequence GGGGS (SEQ ID NO:58) Peptide linker sequence GGSG (SEQ ID NO:59) Peptide linker sequence SGGG (SEQ ID NO:60) Peptide linker sequence GSGS (SEQ ID NO:61) Peptide linker sequence GSGSGS (SEQ ID NO:62) Peptide linker sequence GSGSGSGS (SEQ ID NO:63) Peptide linker sequence GSGSGSGSGS (SEQ ID NO:64) Peptide linker sequence GSGSGSGSGSGS (SEQ ID NO: 65) Peptide linker sequence GGGGSGGGGS (SEQ ID NO:66) Peptide linker sequence GGGGSGGGGSGGGGS (SEQ ID NO:67) Human IL-2Rα sushi domain sequence ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVG QMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTG (SEQ ID NO: 68) P-0250 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 69) P-0305 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTAEAAAKEAAAKEAAAKACPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 70) P-0254 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 71) P-0363 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 72) P-0364 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLELQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 73) P-0365 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLNLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 74) P-0366 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLQLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 75) P-0367 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLSLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 76) P-0368 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLYLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 77) P-0252 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 78) P-0372 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 79) P-0373 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLNDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 80) P-0374 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLRDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 81) P-0253 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 82) P-0302 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISIINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 83) P-0375 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISQINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 84) P-0376 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISEINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 85) P-0377 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 86) P-0378 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISMINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 87) P-0303 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 88) P-0304 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSLSIISTLT (SEQ ID NO: 89) P-0369 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSNSIISTLT (SEQ ID NO: 90) P-0370 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSDSIISTLT (SEQ ID NO: 91) P-0371 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSMSIISTLT (SEQ ID NO: 92) P-0251 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 93) P-0317 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 94) P-0318 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISIINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 95) P-0324 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 96) P-0322 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 97) P-0323 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSLSIISTLT (SEQ ID NO: 98) P-0325 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 99) P-0326 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSLSIISTLT (SEQ ID NO: 100) P-0417 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLNDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 101) P-0418 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLRDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 102) P-0419 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 103) P-0416 APTSSSTKKTQLQLEHLLNDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTAEAAAKEAAAKEAAAKACPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 104) P-0412 APTSSSTKKTQLQLEHLLLELQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTAEAAAKEAAAKEAAAKACPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 105) P-0306 APTSSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTAEAAAKEAAAKEAAAKACPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 106) P-0319 APTSSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTAEAAAKEAAAKEAAAKACPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 107) IL-2 L19S variant sequence APTSSSTKKTQLQLEHLLSDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 108) IL-2 L21S variant sequence APTSSSTKKTQLQLEHLLLDSQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 109) IL-2 L21N variant sequence APTSSSTKKTQLQLEHLLLDNQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 110) IL-2 L21R variant sequence APTSSSTKKTQLQLEHLLLDRQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 111) IL-2 Q126K variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSKSIISTLT (SEQ ID NO: 112) IL-2 Q126H variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSHSIISTLT (SEQ ID NO: 113) IL-2 Q126Y variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSYSIISTLT (SEQ ID NO: 114) IL-2 S125E variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFEQSIISTLT (SEQ ID NO: 115) IL-2 S125K variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFKQSIISTLT (SEQ ID NO: 116) IL-2 S125H variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFHQSIISTLT (SEQ ID NO: 117) IL-2 S125W variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFWQSIISTLT (SEQ ID NO: 118) IL-2 S125I variant sequence APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 119) IL-2 Q22N variant sequence APTSSSTKKTQLQLEHLLLDLNMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 120) IL-2 Q22H variant sequence APTSSSTKKTQLQLEHLLLDLHMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 121) IL-2 Q22K variant sequence APTSSSTKKTQLQLEHLLLDLKMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 122) IL-2 Q22Y variant sequence APTSSSTKKTQLQLEHLLLDLYMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 123) IL-2 Q22I variant sequence APTSSSTKKTQLQLEHLLLDLIMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 124) IL-2 L19H / Q126E variant sequence APTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 125) IL-2 L19Q / Q126E variant sequence APTSSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 126) IL-2 L19S / Q126E variant sequence APTSSSTKKTQLQLEHLLSDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 127) IL-2 L19Y / Q126K variant sequence APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSKSIISTLT (SEQ ID NO: 128) IL-2 L19Y / Q126H variant sequence APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSHSIISTLT (SEQ ID NO: 129) IL-2 L19Y / Q126Y variant sequence APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSYSIISTLT (SEQ ID NO: 130) IL-2 L19Y / S125E variant sequence APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFEQSIISTLT (SEQ ID NO: 131) IL-2 L19Y / S125K variant sequence APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFKQSIISTLT (SEQ ID NO: 132) IL-2 L19Y / S125H variant sequence APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFHQSIISTLT (SEQ ID NO: 133) IL-2 L19Y / S125W variant sequence APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFWQSIISTLT (SEQ ID NO: 134) IL-2 L19Y / S125I variant sequence APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 135) IL-2 L19Y / Q22N variant sequence APTSSSTKKTQLQLEHLLYDLNMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 136) IL-2 L19Y / Q22H variant sequence APTSSSTKKTQLQLEHLLYDLHMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 137) IL-2 L19Y / Q22K variant sequence APTSSSTKKTQLQLEHLLYDLKMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 138) IL-2 L19Y / Q22Y variant sequence APTSSSTKKTQLQLEHLLYDLYMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 139) IL-2 L19Y / Q22I variant sequence APTSSSTKKTQLQLEHLLYDLIMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 140) IL-2 L19H / Q126K variant sequence APTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSKSIISTLT (SEQ ID NO: 141) IL-2 L19H / S125I variant sequence APTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 142) IL-2 L19D / S125I variant sequence APTSSSTKKTQLQLEHLLDDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 143) IL-2 D20E / S125I variant sequence APTSSSTKKTQLQLEHLLLELQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 144) IL-2 D20T / S125I variant sequence APTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 145) IL-2 L19Y / S125I / Q126E variant sequence APTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIESIISTLT (SEQ ID NO: 146) P-0423 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 147) P-0424 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 148) P-0425 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLDDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 149) P-0426 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLPDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 150) P-0427 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLSDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 151) P-0428 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDSQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 152) P-0429 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDNQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 153) P-0430 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDRQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 154) P-0497 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSKSIISTLT (SEQ ID NO: 155) P-0498 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSHSIISTLT (SEQ ID NO: 156) P-0499 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSYSIISTLT (SEQ ID NO: 157) P-0500 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFEQSIISTLT (SEQ ID NO: 158) P-0501 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFKQSIISTLT (SEQ ID NO: 159) P-0502 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFHQSIISTLT (SEQ ID NO: 160) P-0503 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFWQSIISTLT (SEQ ID NO: 161) P-0531 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 162) P-0505 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLNMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 163) P-0506 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLHMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 164) P-0507 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLKMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 165) P-0508 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLYMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 166) P-0509 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLIMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 167) P-0447 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 168) P-0448 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 169) P-0449 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLSDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 170) P-0464 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSKSIISTLT (SEQ ID NO: 171) P-0465 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSHSIISTLT (SEQ ID NO: 172) P-0466 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSYSIISTLT (SEQ ID NO: 173) P-0467 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFEQSIISTLT (SEQ ID NO: 174) P-0468 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFKQSIISTLT (SEQ ID NO: 175) P-0469 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFHQSIISTLT (SEQ ID NO: 176) P-0470 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFWQSIISTLT (SEQ ID NO: 177) P-0471 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 178) P-0472 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLYDLNMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 179) P-0473 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLYDLHMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 180) P-0474 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLYDLKMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 181) P-0475 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLYDLYMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 182) P-0476 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLYDLIMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 183) P-0480 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSKSIISTLT (SEQ ID NO: 184) P-0491 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 185) P-0492 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLDDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 186) P-0493 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLELQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 187) P-0494 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(SEQ ID NO: 188) P-0495 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIESIISTLT(SEQ ID NO: 189) P-0496(Standard-2) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGSCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 190) Standard-1 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINKIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 191) standard-3 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 192) IL-2 L19H / S125I / Q126E variant sequence APTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIESIISTLT (SEQ ID NO: 193) IL-2 L19H / S125I / Q126K variant sequence APTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT (SEQ ID NO: 194) IL-2 L19Q / Q126K variant sequence APTSSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSKSIISTLT (SEQ ID NO: 195) IL-2 L19Q / S125I / Q126E variant sequence APTSSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIESIISTLT (SEQ ID NO: 196) IL-2 L19Q / S125I / Q126K variant sequence APTSSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT (SEQ ID NO: 197) P-0511 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIESIISTLT (SEQ ID NO: 198) P-0512 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT (SEQ ID NO: 199) P-0513 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSKSIISTLT (SEQ ID NO: 200) P-0514 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIESIISTLT (SEQ ID NO: 201) P-0515 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT (SEQ ID NO: 202) P-0582 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT (SEQ ID NO: 203) P-0583 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLNDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT (SEQ ID NO: 204) P-0584 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLRDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT (SEQ ID NO: 205) P-0585 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIESIISTLT (SEQ ID NO: 206) P-0586 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT (SEQ ID NO: 207) P-0616 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIESIISTLT (SEQ ID NO: 208) P-0672 Knob-Fc chain DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIESIISTLT (SEQ ID NO: 209) P-0673 Knob-Fc chain APTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIESIISTLTGGGGSGGGGSGGGGSGGGGSCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPG (SEQ ID NO: 210) P-0674 APTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIESIISTLTGGGGSGGGGSGGGGSGGGSCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPG (SEQ ID NO: 211) Knob-Fc domain with extended in vivo half-life DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPG (SEQ ID NO: 212) Hole-Fc domain with extended in vivo half-life DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPG (SEQ ID NO: 213) P-0511 atggagtttgggctgagctggctttttcttgtggctattttaaaaggtgtccagtgt P-0512 atggagtttgggctgagctggctttttcttgtggctattttaaaaggtgtccagtgt P-0514 atggagtttgggctgagctggctttttcttgtggctattttaaaaggtgtccagtgt P-0582 atggagtttgggctgagctggctttttcttgtggctattttaaaaggtgtccagtgt P-0583 atggagtttgggctgagctggctttttcttgtggctattttaaaaggtgtccagtgt P-0584 atggagtttgggctgagctggctttttcttgtggctattttaaaaggtgtccagtgt P-0585 atggagtttgggctgagctggctttttcttgtggctattttaaaaggtgtccagtgt P-0586 atggagtttgggctgagctggctttttcttgtggctattttaaaaggtgtccagtgt P-0616 atggagtttgggctgagctggctttttcttgtggctattttaaaaggtgtccagtgt

Claims

1. 1. An isolated interleukin-2 (IL-2) variant polypeptide comprising: said IL-2 variant polypeptide has a reduced ability to activate the IL-2Rβγ receptor complex compared to the polypeptide set forth in SEQ ID NO:3, but retains the ability to activate the IL-2Rαβγ receptor complex; The IL-2 variant polypeptide comprises only three amino acid substitutions at amino acid residues at positions L19, S125, and Q126 of SEQ ID NO:3, the amino acid substitutions being selected from the group consisting of an L19H substitution at position 19, an S125I substitution at position 125, and a Q126E and Q126K substitution at position 126. An IL-2 variant polypeptide, characterized in that:

2. 2. The IL-2 variant polypeptide of claim 1, comprising an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 193-194.

3. 3. The IL-2 variant polypeptide of claim 2, comprising the amino acid sequence set forth in SEQ ID NO:

193.

4. 3. The IL-2 variant polypeptide of claim 2, comprising the amino acid sequence set forth in SEQ ID NO:

194.

5. 5. The IL-2 variant polypeptide according to claim 3 or 4, characterized in that stimulation with said IL-2 variant polypeptide results in a regulatory T cell (Treg) / conventional T cell (Tconv) ratio of at least 2.

5.

6. 5. The IL-2 variant polypeptide according to claim 3 or 4, wherein stimulation with said IL-2 variant polypeptide results in greater stimulation of Treg than stimulation of Tconv.

7. 1) an IL-2 variant polypeptide according to any one of claims 1 to 6; and 2) a heterologous protein, wherein the isolated fusion protein is in either a dimeric or monomeric form, the heterologous protein is an Fc domain having the amino acid sequence set forth in SEQ ID NO:45, and the isolated fusion protein comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs:198 to 199.

8. 1. An isolated fusion protein comprising an IL-2 variant polypeptide selected from the group consisting of an IL-2 variant polypeptide comprising the amino acid sequence set forth in SEQ ID NO:193 and an IL-2 variant polypeptide comprising the amino acid sequence set forth in SEQ ID NO:194 fused to an Fc domain comprising the amino acid sequence set forth in SEQ ID NO:45, 13. An isolated fusion protein, wherein the IL-2 variant polypeptide is fused to the Fc domain by a peptide linker having the amino acid sequence set forth in SEQ ID NO:

55.

9. 8. The isolated fusion protein of claim 7, comprising the amino acid sequence set forth in SEQ ID NO:

198.

10. 8. The isolated fusion protein of claim 7, comprising the amino acid sequence set forth in SEQ ID NO:

199.

11. A pharmaceutical composition comprising an IL-2 variant polypeptide or an isolated fusion protein according to any one of claims 1 to 10 in admixture with a pharma- ceutically acceptable carrier.

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