Interleukin-2 variants and methods of use thereof

Abstract

To provide a pharmaceutical composition comprising a fusion protein for use in the treatment of autoimmune diseases, graft rejection of organ transplant or related graft versus host disease (GvHD), inflammatory disease, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, atopic dermatitis, diabetes and immune related adverse effect (irAE).SOLUTION: The disclosure provides an isolated fusion protein comprising: 1) IL-2 variant polypeptide; and 2) Fc domain, where the fusion protein is a dimeric form, and each of the IL-2 variants of the fusion protein is fused to the Fc domain via a peptide linker. Also provided is a pharmaceutical composition comprising the isolated fusion protein in a mixture with a pharmaceutically acceptable carrier.SELECTED DRAWING: 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 boost immune responses in the context of T cell-mediated viral infection (Blattman, J.N. et al. (2003), Nat Med. 9:540-547) 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 a highly effective immunotherapy for metastatic melanoma and renal carcinoma, producing durable responses in approximately 10% of patients. However, the optimal dose of IL-2 is limited by its short half-life and significant 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. Treg expansion is an undesirable effect of IL-2 for cancer immunotherapy. However, the ability of IL-2 to stimulate Treg cells even at low doses has potential applications in the treatment of autoimmune and various inflammatory disorders.

[0004] Tregs are central to immune system homeostasis and play a key 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 has been considerable 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-dose IL-2 to target Treg cells, because Treg cells respond better to lower concentrations of IL-2 than many other immune cell types due to constitutively elevated levels of IL-2Rα (Klatzmann D, 2015, Nat Rev Immunol., 15:283-94). Clinical trials of low-dose IL-2 treatment in various patients with 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) have shown increased Treg levels and signs of clinical benefit. However, even at these low doses, serious safety and tolerability issues arose. Therefore, 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 shown 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 immunostimulatory properties (see, e.g., U.S. Patent Nos. 7,186,804, 7,105,653, 6,955,807, 5,229,109, and U.S. Patent Application Publication No. 20050142106). In the present invention, IL-2 variants were designed to enhance the molecule's ability 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 enhance signaling in Treg cells but not in effector T cells and NK cells, which were thought to be responsible for the observed toxic effects. Importantly, the prior art does not disclose 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 initial studies of IL-2 variants, research in this field has provided more complete evidence that Treg cells constitutively express high levels of 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 involved in the biological activities of various cell populations. This property makes IL-2 a critical intersection in the control of immune responses and an attractive target for therapeutic approaches and complex immunomodulation. Furthermore, receptor subunit-biased IL-2 variants have enabled the achievement of selective IL-2-mediated immunomodulation 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 for stimulating Treg (T CD4+CD25+FoxP3+) cells over normal CD4+ T cells, cytotoxic CD8+ T lymphocytes, and NK cells. Specifically, 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 or several amino acid mutations. These variants contain amino acid substitutions at positions corresponding to the interaction of IL-2 with IL-2Rβ and / or γc. As a result, these variants have reduced affinity for the IL-2Rβγ receptor complex and reduced or eliminated ability to activate IL-2Rβγ-expressing cells, while retaining 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 treating autoimmune and various inflammatory disorders.

[0008] In one embodiment, the present invention relates to the generation of mutant IL-2 variants. These variants possess a combination of attributes, including Treg-selective activity, reduced aggregation, increased expression, improved manufacturability, and improved development suitability. These variants also offer improved biophysical properties of the protein and reduced immunogenicity risk associated with the IL-2 molecule. These variants offer 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 or several amino acid mutations.

[0009] In one embodiment, the present invention provides a proposed " 19The present invention relates to the creation of mutated 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 residue D20 of the toxin-like domain or two adjacent residues are expected to remove the toxic motif, prevent endothelial cell damage, and significantly reduce VLS. Importantly, because this motif is located at the interface with IL-2Rβ, amino acid substitutions in this motif will reduce its affinity for IL-2Rβ, and the resulting molecule is expected to have two beneficial properties, including selectivity for 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 therapeutic uses of these mutated variants for treating Treg cell-deficient autoimmune disorders and various inflammatory disorders.

[0010] The present invention enables substantial improvements over current IL-2-based immunomodulatory strategies for the treatment of autoimmune and various inflammatory disorders. Specifically, replacement of native IL-2 with the mutant variants described herein results in selective CD25-biased stimulation of Treg cells. 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 the sequence set forth in SEQ ID NOs: 4-43, 108-146, and 193-197.

[0011] The present invention enables substantial improvements over current IL-2-based immunomodulatory strategies for 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 with a bias toward CD25, remove toxic motifs, prevent endothelial cell damage, and significantly reduce VLS. 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 SEQ ID NOs: 5-14, 26-43, 108-111, 125-146, and 193-197.

[0012] In another aspect, the IL-2 variants of the present invention are attached to at least one heterologous protein. In various embodiments, the IL-2 variants are 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 / receptors, 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 reduced amounts and / or less frequent administration are required for therapeutic, prophylactic, or diagnostic uses of such molecules (see, e.g., U.S. Patent 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 the 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 the amino acid sequence set forth in SEQ ID NO: 45. In various embodiments, the Fc domain is an Fc domain having the amino acid sequence set forth in SEQ ID NO: 46. In various embodiments, the Fc domain is an Fc domain having the 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 containing the sequence of a non-antigen-binding fragment of a full-length antibody, which may be in a monomeric or multimeric form. The original immunoglobulin source of a native Fc is preferably human and may be any immunoglobulin disclosed in the art. Native Fc is composed of monomeric polypeptides that can be linked by covalent (i.e., disulfide) and non-covalent bonds into dimeric or multimeric forms. The number of intermolecular disulfide bonds between the monomeric subunits of a native Fc molecule ranges from one to four, 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 the monomeric, dimeric, and multimeric forms of the Fc domain, which 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 their 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, the native Fc contains sites that may be removed because they confer structural features or biological activity not required for the fusion molecules of the 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 salvage receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0017] The term "Fc domain" encompasses native Fc and Fc variant molecules and sequences, as defined above. Similar to Fc variants and native Fc, the term "Fc domain" encompasses molecules in monomeric or multimeric form, either digested from a full-length antibody 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), typically 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 Fc domain monomer of the Fc domain contains an amino acid substitution in the CH2 antibody constant domain to reduce interaction or binding between the Fc domain and Fcγ receptors. In various embodiments, each subunit of the Fc domain contains 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 extend 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. Patent 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. Patent 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. Patent No. 8,546,543. In various embodiments, half-life extending mutations can be combined with amino acid substitutions that reduce binding to activating Fc receptors and / or effector function.

[0019] In various embodiments, 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 Fc domain monomers can be promoted by introducing different but compatible substitutions (e.g., a "knob-into-hole" pair of residues) 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 stabilizing disulfide bridges (S354C on the "knob" side and Y349C on the "hole" side) have been introduced. The use of heterodimeric Fc allows for the generation of monovalent IL-2 variant constructs.

[0020] In various embodiments, the IL-2 variant Fc fusion protein will be monomeric, i.e., contain 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 the two Fc-containing polypeptides form a heterodimer, the resulting protein contains only one IL-2 variant.

[0021] In various embodiments, IL-2 variants are used to generate the 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 confers 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 a subclass (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 therebetween) 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 nonproteinaceous 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 compared to 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 includes 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 prepared 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 proteins with long serum half-lives, or to various non-proteinaceous polymers.

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

[0029] In another embodiment, the IL-2 variant can 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, the method comprising administering to a 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. An autoimmune disease according to the present invention is a disease or disorder arising from, directed against, or co-segregates or manifestations of an individual's own tissues, or a condition resulting therefrom. In various embodiments, autoimmune diseases include graft-versus-host disease (GvHD), immune-related adverse events (irAEs), arthritis (including rheumatoid arthritis and 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 syndrome, 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 present 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.

[0032] In another aspect, the present disclosure provides a method for treating an inflammatory disease in a subject, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a subject in need thereof.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 erythematosus, nephritis, Parkinson's disease, ulcerative colitis, collagen-related 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 present 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 capable of treating the inflammatory disease.

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

[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 present 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 present disclosure provides an isolated nucleic acid molecule comprising a polynucleotide encoding an IL-2 variant of the present disclosure. In another aspect, the present disclosure provides a vector comprising a nucleic acid described herein. In various embodiments, the vector is an expression vector. In another aspect, the present disclosure provides an isolated cell comprising a nucleic acid of the present disclosure. In various embodiments, the cell is a host cell comprising an expression vector of the present disclosure. In another aspect, a method for producing an IL-2 variant by culturing the host cell under conditions promoting expression of the protein or polypeptide is provided. [Brief explanation 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, after 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. [Figure 2] Figure 2 shows the differential effects of IL-2 variant Fc fusion proteins with an aspartic acid substitution at position 20 (D20X) on the induction of STAT5 phosphorylation in CD4+ Treg cells (A) and CD4+ Tconv cells (B) in a human PBMC assay, compared with the wild-type fusion protein (P-0250) as a control. [Figure 3]Figure 3 shows the differential effects 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) in a human PBMC assay, compared with the wild-type protein (P-0250) and a reference protein. [Figure 4] Figure 4 shows the differential effects of IL-2 variants with amino acid substitutions at position 19 on STAT5 phosphorylation by Fc fusion proteins compared to wild-type (P-0250) control. 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. [Figure 5] Figure 5 shows the differential effects 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 with wild-type (P-0250) or reference-1 proteins. 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 variant Fc fusion proteins with different combinations of double amino acid substitutions (P-0419, P-0464, P-0471, P-0474, P-0417, and P-0322) on STAT5 phosphorylation, compared with wild-type (P-0250). The biological activities of P-0417 and P-0322 were also compared with their single-amino acid substitution counterparts, P-0373 and P-0363. Their 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 a combination mutation (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 a human PBMC assay by FACS analysis. [Figure 8] Figure 8 shows the pSTAT5-stimulating 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 a human PBMC assay by FACS analysis. [Figure 9] Figure 9 shows the differential effects of IL-2 variant Fc fusions (P-0511 and P-0512) on STAT5 phosphorylation compared to wild-type (P-0250) and three reference molecules 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 was determined in a human PBMC assay by FACS analysis. [Figure 10]Figure 10 shows the differential effects 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 molecules. 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 effects of the 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 binding intensity of P-0511 to the IL-2Rβ and γc complex in an ELISA assay compared to P-0531 and reference-1. [Figure 12] Figure 12 shows the proliferation and expansion of Treg cells in mice treated with IL-2 variant Fc fusion proteins and control mice after a single subcutaneous injection. Blood was collected at the indicated time points and subjected to proliferation assays and lymphocyte phenotyping. (A) Percentage of Treg cells positive for the proliferation marker Ki67; (B) Percentage of Treg cells among the total CD4+ T cell population; (C) Percentage of Treg cells among 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 the 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 the PBS group at each time point. [Figure 14] Figure 14 shows the proliferation of effector T cells and NK cells in IL-2 variant 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) the percentage of Ki67-positive expression and B) cell counts in IL-2 mutant Fc fusion protein-treated and control-treated mice. Data were obtained by FACS 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 the 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-treated and reference-treated mice after a single subcutaneous injection. A) Foxp3 and B) CD25 expression levels were analyzed by FACS analysis and are expressed as mean fluorescence intensity (MFI). Data are presented as mean ± SEM. ****p<0.0001 compared to the PBS group at each time point. [Figure 17]Figure 17 shows the dose-dependent increase in Treg cell proliferation and proliferation in mice after a single injection of the IL-2 variant Fc fusion protein P-0511. Blood was collected at the indicated time points for lymphocyte phenotyping and measurement of the Ki67 proliferation marker. (A) Percentage of Treg cells positive for the proliferation marker Ki67; (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 the PBS group at each time point. [Figure 18] Figure 18 shows the dose-dependent effect of the 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 and lymphocyte phenotyping was performed. 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 the PBS group at each time point. [Figure 19] Figure 19 shows a dose-dependent increase in A) the ratio of Treg cells to Tconv cells, 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 expansion were 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). Blood was collected 3 days after the first and third injections for lymphocyte phenotyping and measurement of the proliferation marker Ki67. (A) Percentage of Ki67-positive Treg cells; (B) Percentage of Treg cells among the total CD4+ T cell population; (C) Percentage of Treg cells among 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 the respective PBS groups. [Figure 21] Figure 21 shows that the increase in Treg cell numbers was maintained in mice receiving repeated injections of the IL-2 variant Fc fusion proteins (P-0511 and P-0512), but not in the wild-type Fc fusion protein (P-0531) or control. Compounds were administered subcutaneously once every three days (Q3D). Blood was collected 3 days after the first and third injections for lymphocyte phenotyping and measurement of the proliferation marker Ki67. (A) Number of Treg cells per microliter of whole blood; (B) Fold change in Treg numbers compared to the PBS control group. Data were obtained by FACS analysis and are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. ****p<0.0001, ***p<0.001, **p<0.01 compared to the respective PBS groups. [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 in mice receiving wild-type Fc fusion protein (P-0531) or control. Compounds were administered subcutaneously once every three days (Q3D). Blood was collected 3 days after the first and third injections for Treg and Tconv cell phenotyping. The ratios were calculated based on the %Treg and %Tconv of 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] Figure 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. Starting on day -2, mice were treated with P-0511 every 3 or 5 days. The kinetics of the DTH response, measured using the change in ear thickness relative to baseline values ​​(Δear thickness) at various time points after KLH exposure, are shown for the A) every 3-day and B) every 5-day dosing schedules. 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 the respective PBS groups 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. Starting on day -2, mice were treated with the compounds every 5 days. The kinetics of the DTH response was shown using the change in ear thickness relative to baseline values ​​(Δear thickness) 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 groups at each time point. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention relates to polypeptides that share a primary sequence with human IL-2, except for one or more 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 the therapeutic use of such IL-2-selective agonists, used alone or in combination with proteins or peptides that target affected tissues, 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 cells or Tconv cells (although they may retain receptor binding activity), making the polypeptides 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 in 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) 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 terminal amino acid 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 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, such as, for example, (1) to reduce susceptibility to proteolysis, (2) to reduce susceptibility to oxidation, (3) to alter binding affinity for the purpose of protein complex formation, (4) to alter binding affinity, and (5) to impart or alter other physicochemical or functional properties.

[0043] As used herein, an amino acid "substitution" 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 in a polypeptide with a functionally similar amino acid. 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 consider 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 hydropathic amino acid index in conferring interactive biological function to a protein is understood in the art (see, e.g., Kyte et al., 1982, J. Mol. Biol., 157:105-131). It is known that substitution of a particular amino acid with another amino acid having a similar hydropathic index or hydropathic score can retain similar biological activity. When making changes based on the hydropathic index, various embodiments include substitutions of amino acids whose hydropathic indices are within ±2 of each other, in various embodiments within ±1, and in various embodiments within ±0.5.

[0046] It is also understood in the art that substitutions of similar amino acids can be made efficiently on the basis of hydrophilicity, particularly when the biologically functional proteins or peptides thus generated are intended for use in the immunological embodiments disclosed herein. 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). When making changes based on similar hydrophilicity values, in various embodiments, substitutions of amino acids with hydrophilicity values ​​within ±2 are included, in various embodiments, within ±1, and in various embodiments, within ±0.5 are included.

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

[0049] TIFF0007827324000001.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 where changes can be made without destroying activity by targeting regions 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 among 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 skilled 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 skilled in the art can predict the importance of amino acid residues in a polypeptide that correspond to amino acid residues important for the activity or structure of the similar polypeptide. One skilled in the art can select chemically similar amino acid substitutions for such predicted important amino acid residues.

[0052] Furthermore, one skilled in the art can analyze the three-dimensional structure and amino acid sequence in relation to the structures of similar polypeptides. In light of such information, one skilled in the art can predict the arrangement of amino acid residues of a polypeptide relative to its three-dimensional structure. In various embodiments, one skilled in the art can select amino acid residues predicted to be present on the surface of the polypeptide so as not to make fundamental changes to these residues, as these residues may be involved in important interactions with other molecules. Furthermore, one skilled in the art can generate test variants containing single amino acid substitutions at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants can be used to gather information about suitable variants. For example, if a change to a particular amino acid residue is found to destroy activity, undesirably reduce activity, or result in inappropriate activity, variants containing such changes can be avoided. In other words, based on the information gathered from such routine experiments, one skilled in the art can easily identify amino acids for which further substitutions, alone or in combination with other mutations, should be avoided.

[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, for example, by 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 interchangeably with the term "% identity" and refers to the amino acid sequence identity between two or more peptide sequences or the 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 specified algorithm, meaning that a given sequence has at least 80% identity to another sequence of a different 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 the 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 interchangeably with the term "% homology" and refers to the amino acid sequence identity between two or more peptide sequences or the 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 as measured by a defined algorithm, i.e., a homolog of a given sequence has greater 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 compared to 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, 90:5873-5787, 1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which indicates the probability that a match between two nucleotide sequences or two amino acid sequences will occur by chance. 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 within the interface between two immunoglobulin heavy chains in 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, pp. 617-621 (1996); and Carter, J Immunol Meth 248, pp. 7-15 (2001).

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

[0063] A "linker" refers to a molecule that links two other molecules covalently or through ionic, van der Waals, or hydrogen bonds. For example, 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 connected by the cleavable linker. Cleavable linkers are generally cleaved by enzymes, typically peptidases, proteases, nucleases, lipases, etc. Cleavable linkers can also be cleaved by environmental factors, such as changes in temperature, pH, or 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 are described herein. Suitable non-immunogenic linker peptides include a (G4S)n peptide linker, an (SG4)n peptide linker, or a G4(SG4)n peptide linker. "n" is usually a number from 1 to 10, typically a number from 2 to 4.

[0065] A "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 pharmaceutically acceptable carrier. A "pharmacologically effective amount" refers to an amount of agent effective to achieve the intended pharmacological result. A "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier, solvent, buffer, and excipient, such as phosphate-buffered saline, 5% aqueous dextrose, 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 ed., 2005, Mack Publishing Co., Easton. A "pharmaceutically acceptable salt" refers to a salt that can be incorporated into a compound for pharmaceutical use, such as a metal salt (e.g., sodium salt, potassium salt, magnesium salt, calcium salt), ammonium salt, or organic amine salt.

[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, improvement or mitigation of disease symptoms, and remission or improved 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 preventative 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, mitigating, and / or delaying one or more of its symptoms. With respect to cancer or other unwanted cell growth, 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, and preferably stop cancer cell invasion into peripheral organs; (iv) inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay tumor onset and / or recurrence; and / or (vii) alleviate to some extent one or more symptoms associated with cancer. An effective amount can be administered in one or more administrations.

[0068] The phrases "administering" or "cause to be administered" refer to the act of managing and / or authorizing the administration of the agent / compound in question to a patient by a medical professional (e.g., a physician) or a person managing the medical care of a patient. Administering can include diagnosing and / or determining an appropriate treatment regimen and / or prescribing a particular agent / compound to a patient. Such prescribing can include, for example, drafting a prescription form, annotating a medical record, etc. 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, a patient can be a human (e.g., an adult male, adult female, adolescent male, adolescent female, boy, girl) receiving treatment from a physician or other medical professional in a hospital, psychiatric facility, as an outpatient, or in other clinical settings. In various embodiments, a patient can be an immunocompromised patient or a patient with a weakened immune system, including, but not limited to, patients with primary immunodeficiency disorders, AIDS patients, cancer patients and transplant patients taking certain immunosuppressants, and patients with genetic diseases affecting 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 contains at least a portion of the constant region. This term encompasses native-sequence Fc regions and variant Fc regions. The IgG Fc region 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 "knob" (protrusion) 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 can be used to promote heterodimerization of two non-identical immunoglobulin heavy chains as described herein. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system.

[0071] The term "effector function," as used herein, refers to a biological activity attributable to the Fc region of an immunoglobulin and varies depending on the immunoglobulin isotype. Examples of immunoglobulin effector functions include complement-dependent cytotoxicity (CDC) via C1q binding, Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, 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 α 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 inducing and maintaining 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 detecting phosphorylated STAT5), induction of proliferation (e.g., as measured by detecting Ki-67), and / or upregulation of expression of activation markers (e.g., CD25), and an increase in cell number.

[0075] As used herein, "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an immunoglobulin to bind to a specific antigen can be measured by 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). Generally, the affinity of a molecule X for its partner Y can be expressed as a dissociation constant (KD), which is the ratio of the dissociation rate constant to 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 specified, 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] A "polynucleotide" refers to a polymer composed of nucleotide units. Polynucleotides encompass not only naturally occurring nucleic acids such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), but also nucleic acid analogs. Nucleic acid analogs include those containing unnatural bases, which are nucleotides joined with other nucleotides via bonds other than natural phosphodiester bonds, and those containing bases added through bonds other than phosphodiester bonds. That is, nucleic acid analogs include, but are not limited to, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized using automated DNA synthesizers, etc. The term "nucleic acid" typically refers to large polynucleotides. The term "oligonucleotide" typically refers to short polynucleotides, usually less 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 a 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"; sequences 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 are referred to as "upstream sequences"; and sequences on the DNA strand having the same sequence as the RNA and located 3' to the 3' end of the coding RNA transcript are referred to as "downstream sequences."

[0081] "Complementary" refers to a topological compatibility, i.e., the identity of the interacting surfaces of two polynucleotides. That is, the two molecules can be described as complementary, and further, the contacting 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] "Specifically hybridizing to" or "specific hybridization" or "selectively hybridizing 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) of DNA or RNA. The term "stringent conditions" refers to conditions under which a probe will hybridize preferentially to its target and 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 guidance on 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, New York; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 3rd Edition, New York; and Ausubel et al. (eds.), Current Edition, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, New York.

[0083] Typically, highly stringent hybridization conditions and highly stringent wash conditions are selected to be approximately 5°C lower than the melting temperature (Tm) of a specific sequence at a defined ionic strength and pH. Tm is the temperature (under defined ionic strength and pH) at which 50% of a 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 between 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 at 42°C overnight. An example of highly stringent wash conditions is 0.15 M NaCl at 72°C for approximately 15 minutes. An example of stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes. For a description of SSC buffer, see Sambrook et al. A low stringency wash can be performed before a high stringency wash to remove background probe signal. For example, a moderately stringent wash for a duplex of more than about 100 nucleotides is 1x SSC at 45°C for 15 minutes. For example, a low stringency wash for a duplex of more than about 100 nucleotides is 4-6x 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] A "primer" refers to a polynucleotide capable of specifically hybridizing to a designated polynucleotide template and providing a point of initiation for the synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions conducive to synthesis, i.e., in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization, such as a DNA polymerase. Primers are typically single-stranded but can also be double-stranded. Primers are typically deoxyribonucleic acid, although a wide variety of synthetic and natural primers are useful for many applications. Primers are designed to be complementary to a template and hybridize to the template to serve as a site for the initiation of synthesis, but they need not precisely 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 target complementary polynucleotide, but need not exactly reflect the complementary sequence of the template. In such cases, specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. Probes can be labeled with chromogenic, radioactive, or fluorescent moieties and used as detectable moieties. A probe can also be a primer, where the probe provides a point of initiation for 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-deficient retroviruses, replication-deficient adenoviruses, and replication-deficient adeno-associated viruses), whose viral genome can contain 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 a host cell after introduction 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, CA, 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 a polynucleotide of the present disclosure. A host cell can be prokaryotic, e.g., E. coli, or eukaryotic, e.g., a unicellular eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., a human cell, monkey cell, hamster cell, rat cell, mouse cell, or 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 the desired level, unless control sequences operably linked to the nucleic acid have been introduced into the host cell. The term host cell is understood to refer 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, for example, a polypeptide or polynucleotide) refers to a molecule that, based on its origin or source of derivation, is (1) free from naturally occurring components that accompany it in its 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. That is, a molecule that is chemically synthesized or expressed in a cellular system different from the cell in which it naturally occurs would be "isolated" from its naturally occurring components. A molecule may be rendered substantially free of naturally occurring components by isolation using purification methods well known in the art. Molecular purity or homogeneity 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 achieved 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 typically comprises 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 polyacrylamide gel electrophoresis of a protein sample followed by visualization of a single polypeptide band by staining the gel using staining methods well known in the art. For certain purposes, higher resolution may be achieved 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 radiolabeled amino acid or the addition of a biotinyl moiety to the polypeptide that is detectable by marked avidin (e.g., streptavidin containing a fluorescent marker or an enzymatic activity that is 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 tags), magnetic agents such as gadolinium chelates, toxins such as pertussis toxin and 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 avoid potential steric hindrance.

[0092] The term "heterologous," as used herein, refers to a construct or state that is not native or does not exist in nature, e.g., a construct or state that can be achieved by replacing an existing native construct or state with a construct 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 encompass "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 surrounds 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 dictates 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. IL-2 regulation occurs through signaling pathway activation and transcription factors that act on the IL-2 promoter to initiate gene transcription de novo, as well as through regulation of IL-2 mRNA stability. IL-2 binds to a highly regulated multichain receptor containing α, β, and γ chains that mediates signal transduction through the Jak-STAT pathway. IL-2 delivers activation, proliferation, and differentiation signals to T cells, B cells, and NK cells. IL-2 is also important in mediating activation-induced T cell death, a function that provides an essential mechanism for terminating immune responses. A commercially available nonglycosylated human recombinant IL-2 product, aldesleukin (available from Prometheus Laboratories Inc., San Diego, CA, under the trade name PROLEUKIN®, des-alanyl-1, serine-125 human interleukin-2), has been approved for administration to patients with metastatic renal cell carcinoma and metastatic melanoma. IL-2 has also been suggested for use 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 potential toxicity limit the optimal dose of IL-2.

[0097] As used herein, the terms "native IL-2" and "native interleukin-2," in the context of a protein or polypeptide, refer to any naturally occurring mammalian interleukin-2 amino acid sequence, including immature or precursor forms and mature forms. 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), NP_001040595.1 (Macaca mulatta, immature), NP_000577.2 (human, precursor), CAA01199.1 (human, immature), AAD48509.1 (human, immature), 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 mature wild-type human IL-2 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 or more 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 the therapeutic use of such IL-2-selective agonists, used alone or in combination with proteins or peptides that target affected tissues, to treat 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, with a length of 100 to 500 amino acids, preferably 140 residues. These polypeptides retain a high sequence identity of greater than 90% with native IL-2. At these positions, these polypeptides are mutated to introduce amino acid residues different from those present at those positions in native IL-2.

[0101] The polypeptides of the present 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 receptor subunits β, γ, or βγ.

[0102] In various embodiments, an 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, an IL-2 variant comprises an amino acid sequence that differs from the native (or wild-type) IL-2 protein. In various embodiments, an 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 with 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, 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 for regulatory T cell (Treg) activation and proliferation.

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

[0104] TIFF0007827324000002.tif164170

[0105] TIFF0007827324000003.tif84170

[0106] TIFF0007827324000004.tif149170

[0107] TIFF0007827324000005.tif66170

[0108] TIFF0007827324000006.tif144170TIFF0007827324000007.tif156170

[0109] TIFF0007827324000008.tif147170

[0110] The present invention also encompasses additional modifications to the above-described IL-2 variant classes, particularly those listed in Tables 2A-2F. Those skilled in the art will appreciate that additional combinatorial mutations combining the preferred mutations listed in Tables 2A-2F may result in IL-2 agonists with enhanced Treg cell selectivity. Any additional combinatorial mutations, whether they increase the affinity for a specific 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 natures (phage libraries, libraries of gene expression in yeast or bacteria). In another aspect, the present invention relates to fusion proteins comprising any of the above-described immunomodulatory polypeptides conjugated to a carrier protein. The carrier protein can be albumin or the Fc region of a human immunoglobulin.

[0111] Fc domain IgG class immunoglobulins are the most abundant proteins in human blood. Their circulating half-life can reach as long as 21 days. Fusion proteins combining the Fc region of IgG with domains from other proteins, such as various cytokines or receptors, have been reported (see, e.g., Capon et al., Nature, 337:525-531, 1989; Chamow et al., Trends Biotechnol., 14:52-60, 1996; U.S. Patent Nos. 5,116,964 and 5,541,087). The prototype of a fusion protein is a homodimeric protein, in which the heavy chain variable region and CH1 domain are linked via cysteine ​​residues in the hinge region of the IgG Fc, resulting in a molecule similar to an IgG molecule lacking the light chain. The dimeric nature of fusion proteins containing the Fc domain can be advantageous for 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 same isotype.

[0112] The term "Fc" refers to a molecule or sequence containing the sequence of a non-antigen-binding fragment of a full-length antibody, which may be in a monomeric or multimeric form. The original immunoglobulin source of native Fc is preferably human and may be any immunoglobulin, with IgG1 and IgG2 being preferred. Native Fc is composed of monomeric polypeptides that can be linked by covalent (i.e., disulfide) and non-covalent bonds into dimeric or multimeric forms. The number of intermolecular disulfide bonds between the monomeric subunits of native Fc molecules ranges from one to four, 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 the monomeric, dimeric, and multimeric forms of the Fc domain, which 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 their 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, the native Fc contains sites that may be removed because they confer structural features or biological activity not required for the fusion molecules of the 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 salvage receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0114] The term "Fc domain" encompasses native Fc and Fc variant molecules and sequences, as defined above. Similar to Fc variants and native Fc, the term "Fc domain" encompasses molecules in monomeric or multimeric form, either digested from a full-length antibody 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), typically 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 Fc domain monomer of the Fc domain contains an amino acid substitution in the CH2 antibody constant domain to reduce interaction or binding between the Fc domain and Fcγ receptors. 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, two Fc domain monomers of an Fc domain each contain an amino acid substitution that promotes heterodimerization of the two monomers. In various other embodiments, heterodimerization of Fc domain monomers can be promoted by introducing different but compatible substitutions (e.g., a "knob-into-hole" pair of residues) 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 stabilizing disulfide bridges (S354C on the "knob" side and Y349C on the "hole" side) have been introduced. The use of a heterodimeric Fc allows for the generation of monovalent IL-2 variants.

[0116] In various embodiments, the Fc domain may be mutated to further extend 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. Patent 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. Patent 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. Patent No. 8,546,543. In various embodiments, half-life extending mutations can be combined with amino acid substitutions that reduce binding to activating Fc receptors and / or effector function.

[0117] In various embodiments, the Fc domain sequence used to generate the IL-2 variant Fc fusion is the human IgG1-Fc domain sequence with reduced / eliminated effector function 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 set forth in SEQ ID NO: 46 below: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 46)

[0120] SEQ ID NO: 46 contains amino acid substitutions (underlined) that disrupt 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 disrupt 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 Knob-Fc domain with extended in vivo half-life 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 and C1q binding (underlined) and amino acid substitutions that 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 and C1q binding (underlined) and amino acid substitutions that 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 known in the art, peptide linkers contain flexible amino acid residues such as glycine and serine. In various embodiments, peptide linkers can contain 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 other embodiments, 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 other embodiments, the peptide linker may be a simple chemical bond, such as an amide bond (e.g., via PEG chemical attachment).

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

[0129] TIFF0007827324000009.tif123170

[0130] Polynucleotides In another aspect, the present disclosure provides isolated nucleic acid molecules comprising polynucleotides encoding the IL-2, IL-2 variants, IL-2 fusion proteins, or IL-2 variant fusion proteins of the present disclosure. The subject nucleic acids may be single-stranded or double-stranded. Such nucleic acids may be DNA or RNA molecules. Examples of DNA include 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 chemically synthesizing overlapping oligonucleotide fragments and then assembling the fragments to reconstitute part or all of the coding region and flanking sequences. RNA can be obtained using 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 libraries prepared from mRNA isolated from various tissues that express IL-2. DNA molecules of the present disclosure encompass not only full-length genes but also polynucleotides and fragments thereof. Full-length genes may also include sequences encoding an N-terminal signal sequence. Such nucleic acids can be used in methods such as for making novel IL-2 variants.

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

[0132] In various embodiments, the recombinant nucleic acid of the present disclosure may be operably linked to one or more regulatory nucleotide sequences within an expression construct. Regulatory sequences are art-recognized and selected to direct expression of the IL-2 variant. Thus, the term regulatory sequence encompasses promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990). Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, a promoter sequence, a leader or signal sequence, a ribosomal binding site, transcription start and stop sequences, translation start and stop sequences, and an enhancer or activator sequence. 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 elements of two or more promoters. 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 encoding an IL-2 variant operably linked to at least one regulatory sequence. The term "expression vector" refers to a plasmid, phage, virus, or vector for expressing a polypeptide from a polynucleotide sequence. Suitable vectors for expression in host cells are readily available, and insertion of nucleic acid molecules into the vector is accomplished using standard recombinant DNA techniques. Such vectors can contain a variety of expression regulatory sequences that can be used in these vectors to express DNA sequences encoding IL-2 variants, which regulate expression of the DNA sequence when operably linked to the vector. Useful expression control sequences include, for example, the SV40 early and late promoters, the tet promoter, 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 region of lambda phage, the regulatory region for the fd coat protein, promoters 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 of baculovirus systems, and other sequences known to regulate the expression of genes in prokaryotic or eukaryotic cells or their viruses, as well as 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 vector's copy number, the ability to control 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 acids 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 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 papillomavirus (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 discussion of gene therapy delivery systems below. Various methods used in preparing plasmids and transforming 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 Chapters 16 and 17 of Molecular Cloning: A Laboratory Manual, 2nd ed., Sambrook, Fritsch, and Maniatis (eds.) (Cold Spring Harbor Laboratory Press, 1989). 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 the Pcmv-Script vector (Stratagene, La Jolla, CA), the pcDNA4 vector (Invitrogen, Carlsbad, CA), and the pCI-neo vector (Promega, Madison, WI). 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 host cells 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 can be any prokaryotic or eukaryotic cell. For example, the IL-2 variants of the present disclosure can 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 for 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 and lysed, and the protein isolated. A cell culture medium includes host cells, 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 of skill in the art. These methods include, at some level, crude fractionation into 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 terms "isolated polypeptide" or "purified polypeptide," as used herein, are intended to refer to a composition isolatable from other components, where the polypeptide has been 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. Generally, "purified" refers to a polypeptide composition that has undergone fractionation 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 the majority of the composition, e.g., comprises 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 purification are well known to those of skill in the art. These methods include, for example, precipitation using ammonium sulfate, PEG, antibodies (immunoprecipitation), or heat denaturation, followed by centrifugation; chromatography, such as affinity chromatography (protein A column), ion exchange chromatography, gel filtration chromatography, reverse-phase chromatography, hydroxyapatite chromatography, and hydrophobic interaction chromatography; isoelectric focusing; gel electrophoresis; and combinations of these methods. As is well known in the art, it is contemplated that the order in which the various purification steps are performed, or certain steps are omitted, may still be suitable for preparing a substantially purified polypeptide.

[0141] Pharmaceutical Composition In another aspect, the present disclosure provides a pharmaceutical composition comprising an IL-2 variant or IL-2 variant fusion protein in admixture with a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are well known and understood by those skilled in the art and have been extensively described (see, e.g., Remington's Pharmaceutical Sciences, 18th ed., A.R. Gennaro (ed.), Mack Publishing Company, 1990). Pharmaceutically acceptable carriers may be included to alter, maintain, or preserve 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 pharmaceutically 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 borate, bicarbonate, Tris-HCl, citrate, phosphate, 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, PEG, sorbitan esters, polysorbates, e.g., polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stabilizers (sucrose or sorbitol); tonicity enhancing agents (such as 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 distilled water for injection, physiological saline, or artificial cerebrospinal fluid, to which other substances typically contained in compositions for parenteral administration may be added. Further exemplary solvents include neutral buffered saline or normal 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 contain sorbitol or a suitable substitute. In one embodiment of the present disclosure, a composition may be prepared for storage in the form of a lyophilized cake or aqueous solution by mixing a selected composition having the desired purity with any formulation agent (Remington's Pharmaceutical Sciences, supra). Additionally, therapeutic compositions may be formulated as lyophilized products using appropriate pharmaceutical excipients, such as sucrose. The optimal pharmaceutical composition can be determined by one of skill in the art based on the intended route of administration, delivery mode, and desired dose.

[0143] If 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 pharmaceutically acceptable solvent. A particularly suitable solvent for parenteral injection is sterile distilled water, in which the polypeptide is formulated as a sterile, isotonic solution, properly preserved. In various embodiments, pharmaceutical preparations suitable for injection 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 compound, thereby allowing for the preparation of highly concentrated solutions.

[0144] In various embodiments, therapeutic pharmaceutical compositions 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-specificity, 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 customarily used in the compounding of solid dosage forms such as tablets and capsules. In solid dosage forms for oral administration (such as capsules, tablets, pills, dragees, powders, granules, and the like), one or more therapeutic compounds of the present disclosure may be mixed with one or more pharmaceutically 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; and (5) solution retarding agents, such as paraffin. (6) absorption enhancers, such as quaternary ammonium compounds; (7) wetting agents, such as 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 glycol, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. For capsules, tablets, and pills, the pharmaceutical compositions may also contain buffering agents. Similar types of solid compositions may also be used as fillers in soft and hard gelatin capsules, employing excipients such as lactose or milk sugar and high molecular weight polyethylene glycols. Liquid dosage forms for oral administration include pharmaceutically 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 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, the pharmaceutical composition is intended for topical administration to the skin or mucosa. Topical formulations may further include one or more of a variety of agents known to be effective as skin or stratum corneum penetration enhancers. Examples include 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methyl alcohol, isopropyl alcohol, dimethyl sulfoxide, and azone. Additional agents may be included to make the formulation cosmetically acceptable. Examples include fats, waxes, oils, dyes, fragrances, preservatives, stabilizers, and surfactants. Keratolytic agents, such as those known in the art, may also be included. 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 pharmaceutically acceptable carrier, and, if required, any preservatives, buffers, or propellants. Ointments, pastes, creams, and gels may contain, in addition to a compound of the presently disclosed subject matter (e.g., an IL-2 variant), pharmaceutical excipients such as animal fats, vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0147] Additional pharmaceutical compositions contemplated for use herein include formulations comprising polypeptides 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 a pharmaceutical composition used therapeutically will vary depending on the type and purpose of the treatment. As will be understood by those skilled in the art, appropriate therapeutic dosage levels 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 patient's size (weight, body surface, or organ size) and condition (age and overall health). Clinicians can therefore adjust the dosage and route of administration to achieve optimal therapeutic efficacy. Typical dosages can range from about 0.001 mg / kg to about 100 mg / kg or more, depending on the factors discussed above. Preferably, the polypeptide composition may be injected or administered intravenously. Long-release pharmaceutical compositions may be administered every 3 to 4 days, weekly, 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 that achieves the desired effect is reached. The composition may therefore be administered as a single dose, or as multiple doses over time (at the same or different concentrations per dose), or as a continuous infusion. Further refinement of the appropriate dosage is performed periodically. Appropriate doses may be confirmed using appropriate dose-response data.

[0149] The pharmaceutical composition may be administered by any known route, such as oral, intravenous, intraperitoneal, intracerebral (intracerebroparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional injection, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, or intraperitoneal route, or intratumoral; as well as intranasal, enteral, topical, sublingual, transurethral, ​​vaginal, or rectal administration, sustained-release systems, or implanted devices. If desired, the composition may be administered by bolus injection, continuous infusion, or implanted device. Alternatively or additionally, the composition may be administered locally by implantation of a membrane, sponge, or other suitable material to which the molecule of interest is adsorbed or encapsulated. Where an implantation device is used, the device may be implanted in 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, the method 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 pharmaceutically acceptable carrier. An autoimmune disease according to the present invention is a disease or disorder arising from, directed against, or a co-segregate or manifestation of, or a condition resulting therefrom, an individual's own tissues. In various embodiments, autoimmune diseases include 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, and the like. These include, but are not limited to, autoimmune diseases such as rheumatoid arthritis, rheumatoid arthritis, rheumatic fever ...

[0151] The present disclosure provides a method for 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 IL-2 variant fusion protein of the present disclosure in a pharmaceutically acceptable carrier. "Inflammatory disease" encompasses all diseases involving acute inflammation or various forms of inflammation. Acute inflammation is the body's initial response to harmful stimuli and is caused by increased movement of plasma and leukocytes (e.g., granulocytes) from the blood to the injured tissue. Several biochemical events, involving the local vasculature, immune system, and various cells within the injured tissue, propagate and mature the inflammatory response. Prolonged inflammation, referred to as various forms of inflammation, is characterized by gradual changes in the types of cells present at the site of inflammation and by simultaneous tissue destruction and recovery from the inflammatory process. In another aspect, the present disclosure provides a method for treating an inflammatory disease in a subject, comprising administering to the subject 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 erythematosus, nephritis, Parkinson's disease, ulcerative colitis, collagen-related 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 present disclosure provides a method for organ transplantation or related graft-versus-host disease in a subject, the method comprising administering a therapeutically effective amount of a pharmaceutical composition of the present invention to a subject in need thereof. 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, intestinal organ transplantation, and thymus organ transplantation, or bone tissue transplantation, tendon tissue transplantation, corneal tissue transplantation, skin tissue transplantation, heart valve tissue transplantation, nerve tissue transplantation, and venous tissue transplantation. As used herein, the term "graft-versus-host disease" or "GvHD" refers to conditions, including acute and chronic conditions, caused by the effects of transplanted (graft) cells on host cells and tissues resulting from GVH. In other words, donor immune cells infused into the graft or donor immune cells developed from stem cells may recognize patient (host) cells as foreign and mount an immune response against them. Acute graft-versus-host disease (GvHD) is a disorder caused by donor immune cells, specifically in patients who have received an allogeneic bone marrow or blood cell transplant. 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 GvHD.

[0153] First, the E.C. 50 The therapeutically effective amount can be assessed from cell culture assays by measuring the EC 20 measured in cell culture medium in animal models. 50 The dose can be formulated to achieve a circulating plasma concentration range including: 100 mg / kg / day, ...

[0154] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, or several divided doses (multiple or repeated or maintenance doses) may be administered over time, with the dose being proportionally increased or decreased as required by the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form. As used herein, dosage unit form refers to physically discrete units suited 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 combination with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present disclosure are determined primarily 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, dosages and administration regimens can be adjusted according to methods well known in the therapeutic field. That is, the maximum tolerated dose can be easily established, and the effective amount for providing a detectable therapeutic effect to a subject can be determined, as can the time requirements for administering each agent so as to provide a detectable therapeutic effect to a subject. Thus, although certain dosages and administration regimens are exemplified herein, these examples are not intended to limit the dosages and administration regimens that can be administered to a subject in the practice of the present disclosure.

[0156] Dosage values ​​vary depending on the type and severity of the condition to be improved and may include single or repeated doses. It is further understood that for any particular subject, specific dosing regimens will need to be adjusted over time according to the individual needs and the 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. Furthermore, dosing regimens using the compositions of the present disclosure may be based on a variety of factors, including the type of disease, the subject's age, weight, sex, medical condition, severity of the condition, route of administration, and the particular antibody used. Thus, 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. Thus, the present disclosure encompasses intra-subject dose escalation as required by those skilled in the art. It is understood that determining appropriate dosages and dosing regimens is well known in the relevant art and would be accomplished by one of ordinary skill in the art given the teachings disclosed herein.

[0157] An exemplary, non-limiting daily dose range for a therapeutically or prophylactically effective amount 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. If administered repeatedly over several days or longer, depending on the condition, treatment will usually be sustained until a desired suppression of disease symptoms occurs.In various embodiments, the dosage is, based on the above figures, approximately 0.0001 to 100 mg / kg body weight, 0.0001 to 90 mg / kg body weight, 0.0001 to 80 mg / kg body weight, 0.0001 to 70 mg / kg body weight, 0.0001 to 60 mg / kg body weight, 0.0001 to 50 mg / kg body weight, 0.0001 to 40 mg / kg body weight, 0.0001 to 30 mg / kg body weight, 0.0001 to 20 mg / kg body weight, 0.0001 to 10 mg / kg body weight, 0.0001 to 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 The dose ranges may be within the ranges of 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 a patient. Note that dosage values ​​may vary depending on the type and severity of the condition to be improved.It is further understood that for any particular subject, specific dosage regimens will be adjusted over time according to the individual need and the 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 The LD (the dose that is therapeutically effective in 50% of a population) can be determined by standard pharmaceutical techniques in cell culture or experimental animals. 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 an IL-2 variant or IL-2 variant fusion protein pharmaceutical composition will depend on the treatment method and the nature of the particular disease being treated. Subjects 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 weekly; once every two weeks; once every three weeks; once weekly for two weeks, then monthly; once weekly for three weeks, then monthly; once monthly; once every two months; once every three months; once every four months; once every five months; or once every six months, or once yearly.

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

[0161] In another aspect, the present disclosure provides a method for treating an autoimmune disease in a subject, the method 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, e.g., corticosteroids, cyclosporine, cyclophosphamide, prednisone, azathioprine, methotrexate, rapamycin, tacrolimus, biologic agents, e.g., TNF-α blockers or antagonists, IL-10 agonists, or long-term half-life extended IL-10 (PEGylated, antibody- or Fc-fused IL-10); immunosuppressants, e.g., immunosuppressants that inhibit other lymphocyte surface markers (e.g., IL-10 agonists); antibodies against CD40, alpha-4 integrin, or 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., cyclosporin 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 agent / Cox-2 inhibitor,Hydroxychloroquine, sulfasalazopryine, 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, e.g., 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 the pharmaceutical composition of the present invention in combination with a second therapeutic agent that can inhibit or suppress the differentiation of Th1 cells, Th17 cells, Th22 cells, and / or other cells that secrete, or cause other cells to secrete, proinflammatory 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; or inhibit or suppress the activity of Th1 cells, Th17 cells, Th22 cells, and / or other cells that secrete, or cause other cells to secrete, proinflammatory 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. therapies 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, proinflammatory 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; and therapies 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, proinflammatory 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, or sudoxicam; a salicylate, such as aspirin, disalcid, benorylate, trilisate, safapryn, solprin, diflunisal, or fendosal; an acetic acid derivative, such as diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac, furofenac, tiopinac, zidometacin, acemetacin, fentiazac, zomepirac, climdanac, oxepinac, felbinac, or ketorolac; or a steroid derivative, such as mefepirac, ciclofen ... Nonsteroidal anti-inflammatory drugs 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, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen, and tiaprofenic acid; and pyrazoles such as phenylbutazone, oxyphenbutazone, feprazone, azapropazone, and trimethazone. Mixtures of these nonsteroidal anti-inflammatory drugs 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, desoximetasone, 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), fluradrenolone 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 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 administration of the second pharmaceutical composition. In various embodiments, the administration of the second pharmaceutical composition is terminated before the administration of the IL-2 variant composition. [Example]

[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 attenuate the affinity of IL-2 for the IL-2Rβ and / or γc receptor subunits. In situations where IL-2Rβγ interaction is weakened, the enhanced IL-2 sensitivity of Tregs conferred by IL-2Rα expression may confer a distinct growth advantage to this cell subset. 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 variant panel was created containing one to three mutations (introducing conservative and non-conservative amino acid substitutions) at residues located 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-bonding network with receptor subunit side chains at the IL-2Rβ interface. Similarly, N88 is a potential hotspot for IL-2 / IL-2Rβ interaction and participates in a critical hydrogen bond with the receptor chain. Q126 is essential for γc interaction, and Q22 is also located at the γc interface. We hypothesized that mutations at or near these residues might impair 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. Consequently, substitution of residue D20 of the critical toxicity motif, or the adjacent residues L19 and L21, with non-aliphatic residues, was expected to eliminate the toxicity motif, suppress endothelial cell injury, and significantly reduce VLS.

[0168] In the present invention, the present invention relates to a nucleotide sequence having one to three of the following 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 as C-terminal fusions to the Fc homodimer via a "GGGSGGGS" linker (SEQ ID NO: 55). IL-2 variants with the amino acid substitutions D20I, D20I / N88G, D20E, or L19N were expressed as N-terminal fusions 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: 73-107, 147-189, and 198-211. 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 highly expressing IL-2Rα, prioritizing the proliferation of Treg cells over other lymphocytes, including CD4+ conventional T cells, CD8+ T cells, and NK cells. Furthermore, mutations at positions 19, 20, or 21 are expected to remove toxic motifs involved in vascular toxicity, allowing the resulting molecules to possess two beneficial properties: enhanced selectivity for Treg activation and reduced endothelial cell damage. However, optimal mutations or combinations of mutations are crucial for maximizing the window for selective targeting of Treg subsets by adjusting the level of damage to effector T and NK cell activation while maintaining sufficiently high potency for Treg activation.

[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 Treg cell proliferation advantage.

[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 was driven by a CMV promoter, and a synthetic SV40 poly(A) signal sequence was present at the 3' end of the CDS. A leader sequence was added to 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 two or more expression vectors were present, the vectors were transfected at a 1:1 ratio. For transfection, HEK293 cells were cultured in serum-free FreeStyle™ 293 Expression Medium (ThermoFisher Scientific). For production in 1000 ml shake flasks (working volume 330 ml), 0.8 × 10 HEK293 cells were transfected 24 hours prior to transfection. 6 Cells were seeded at a density of 1000 cells / ml. A total of 330 μg of DNA expression vector was mixed with 16.7 ml of Opti-MEM medium (Thermo Fisher Scientific). 0.33 mg of PEI diluted in 16.7 ml of Opti-MeM medium was added, and the mixture was vortexed for 15 seconds and then left at room temperature for 10 minutes. The DNA / PEI solution was then added to the cells and incubated at 37°C in an 8% CO2 incubator. Sodium butyrate (Millipore Sigma) was added to the cells at a final concentration of 2 mg / L on day 4 to help maintain protein expression. After 6 days of culture, the supernatant was collected and purified by centrifugation at 2200 rpm for 20 minutes. The solution was sterile filtered (0.22 μm filter, Corning). Secreted proteins were purified from the cell culture supernatant using protein A affinity chromatography.

[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 protein was eluted with 25 mM sodium citrate, 25 mM sodium chloride (pH 3.2). The protein solution was neutralized by adding 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 (BioRad). The target protein was concentrated using an Amicon® Ultra-15 Concentrator 10 KDa NMWC (Merck Millipore).

[0174] The purity and molecular weight of the purified constructs were analyzed by SDS-PAGE with and without reducing agents and Coomassie staining (Imperial™ Stain). The NuPAGE® Pre-Cast Gel System (4-12% or 8-16% Bis-Tris, Thermo Fisher Scientific) was used according to the manufacturer's instructions. The protein concentration of the purified protein samples was determined by measuring the UV absorbance at 280 nm (Nanodrop spectrophotometer, Thermo Fisher Scientific) and dividing by the molar extinction coefficient calculated based on the amino acid sequence. The amount of aggregation of the constructs was analyzed using an Agilent 1200 high-performance liquid chromatography (HPLC) system. The samples were injected onto an AdvanceBio size-exclusion column (300 Å, 4.6 x 150 mm, 2.7 μm, LC column, Agilent) at 25°C using 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 development suitability of fusion compounds The design approach to find mutation combinations that result in variant proteins with desired biological properties faced significant 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 by the SEC chromatogram shown in Figure 1A, in which wild-type IL-2 Fc fusion protein (P-0250) was transiently expressed at low levels (approximately 3 mg / L in HEK-293F cells), revealing a high tendency for aggregation. Painstaking efforts to engineer amino acid substitutions in IL-2 targeted for desired biological activity generally resulted in mutant proteins with even lower stability. A significant proportion of the IL-2 variants in this study were expressed at extremely low levels, and some variants exhibited a significantly increased tendency for aggregation, as exemplified by the SEC chromatogram of P-0318 (SEQ ID NO: 95) shown in Figure 1B. This poses challenges for therapeutic manufacturing and storage.

[0177] We also observed significant differences in the expression profiles and aggregation propensity of IL-2 variant fusions 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 the D20I and N88R amino acid substitutions, while P-0318 contains the D20I and N88I mutations. Both variant fusions expressed at similarly low levels. As can be seen in Figure 1B, P-0318 was highly aggregation-prone, with 65% of the peaks being high-molecular-weight species, which resulted in fewer expected species in the chromatogram (indicated by the arrow). In contrast, P-0317 was relatively pure, with 7.5% aggregates (Figure 1C). It may be inferred that the N88R mutation may reduce the aggregation tendency of the resulting fusion protein. However, fusion proteins P-0254 (SEQ ID NO: 71) and P-0324 (SEQ ID NO: 96) derived from IL-2 with a single N88R mutation or a double D20T / N88R mutation, respectively, were prone to aggregation, with aggregates ranging from 30 to 40%. Therefore, the contribution of individual amino acid substitutions to protein stability appears to be context-dependent.

[0178] The fact that amino acid substitutions in IL-2 often reduce protein stability is further complicated by the unpredictable contribution of various 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 adjust the selectivity of IL-2 because this residue is located close to Q126, which is essential for γc interaction. Native IL-2 contains an unpaired cysteine ​​at position 125, which was replaced with serine in Proleukin; however, in the present invention, S125 is considered the wild-type IL-2 residue. IL-2 containing an alanine substitution at position 125 is also widely used. Because 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) in order to interfere with the interaction of Q126 with γc to alter biological activity. All of the resulting fusion molecules, except for P-0471 (SEQ ID NO: 178), could not be characterized due to low expression levels. P-0471, in contrast, was expressed at significantly higher levels (titer of 19.3 mg / L versus 4.0 mg / L) and had a greatly reduced tendency to aggregate (1% aggregation versus 21.7%) when compared to its S125 counterpart (P-0372). Prompted by the impressive improvements in development suitability, particularly in product purity, we assessed whether such enhancements by isoleucine substitution at position 125 could be reproduced under different mutational conditions.

[0180] Therefore, the S125I substitution was introduced into several IL-2 variant Fc fusion molecules. Constructs with the Ile-125 substitution in IL-2 were expressed using the same vector and under the same culture conditions as their Ser-125 counterparts and purified using MabSelectSure. Table 4 summarizes the expression levels (mg / L) and purity (aggregation rate) of exemplary molecules, as assessed by SEC chromatography. Two molecules in the same row in Table 4 share identical amino acid substitutions, differing only in whether residue 125 is serine or 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 Figures 1D and 1E. As can be seen from Table 4, the isoleucine substitution at position 125 resulted in 4- to 11-fold enhanced expression levels and uniformly lower aggregation propensity.

[0181] TIFF0007827324000010.tif83170

[0182] This study clearly demonstrates that an isoleucine substitution at position 125 results in a universal improvement in the suitability for development of IL-2 fusion constructs while fully retaining biological activity. Ile substitutions at position 125 of wild-type IL-2 and IL-2 variants with different mutational configurations in Fc fusion formats all resulted in 4- to 11-fold enhanced expression levels and uniformly reduced aggregation propensity. Table 4 summarizes the expression levels (mg / L) and purity (aggregation rate) of Protein A purified products, as assessed by SEC chromatography, of exemplary molecules. This discovery is particularly valuable because engineering IL-2 for favorable biological properties has been hindered by the fact that modifications to marginally stable wild-type IL-2 often result in less stable mutant proteins. Problems associated with engineering IL-2 can be alleviated by a single amino acid substitution at position 125 with isoleucine. In summary, the isoleucine substitution at position 125 of IL-2, i.e., the IL-2 variant, significantly improved 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 the amino acids interacting with the β, γ, or βγ receptor subunits. These substitutions were intended to reduce IL-2's ability 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 from buffy coats of healthy donors by Ficoll-Hypaque centrifugation. PBMCs were starved for 1 hour in serum-free MACS buffer at 4°C. 2 × 10 5 PBMCs were treated with serial dilutions of test compounds for 30 minutes at 37°C. Cells were fixed and permeabilized using the Foxp3 / Transcription Factor Staining Buffer Set (EBIO) by incubating with 1x Foxp3 Fixation / Permeabilization Working Solution for 30 minutes and then 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 minutes 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+ conventional T cell subsets, respectively), and data are expressed as the percentage of pStat5+ cells in the gated populations.

[0185] Figure 2 shows the dose-response effect of exemplary IL-2 variant Fc fusion proteins, compared with wild-type fusion protein, on STAT5 phosphorylation in CD4+ Treg and Tconv cells. The 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. The activity of wild-type IL-2 was approximately 250-fold greater in Treg cells than in CD4+ Tconv cells, consistent with the higher expression levels of the high-affinity trimeric receptor in Treg cells.

[0186] Various substitutions of the aspartic acid at position 20, P-0364 (D20E), P-0363 (D20T), P-0365 (D20N), P-0366 (D20Q), and P-0367 (D20S), demonstrated 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 potential Treg-biased IL-2 agents for activating Treg cells for the treatment of autoimmune diseases. Furthermore, mutation at D20, a key residue in the proposed toxin-like motif, is expected to remove the toxic motif and suppress endothelial cell damage. Therefore, these variants are expected to have Treg-selective activity and an improved safety profile for VLS. Furthermore, P-0368 did not exhibit 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 the 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 with 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 (Figures 4A and 4C). Variants P-0372, P-0374, P-0423, and P-0427 largely retained their biological activity in CD4+ T cells (Figures 4B and 4D), whereas variants P-0373 and P-0424 exhibited reduced activity in CD4+ T cells. The mutant P-0425(L19D) showed a slight reduction in its ability to induce STAT5 phosphorylation in Treg cells, but this activity was significantly impaired in CD4+ Tconv cells (Figures 4C and 4D). The selective activation of Treg cells by mutants P-0373, P-0424, and P-0425 over CD4+ Tconv cells, as demonstrated above, and in particular the wide window of selective targeting of Treg subsets by P-0373 and P-0425, make these mutants potential Treg-biased IL-2 drug candidates for activating Treg cells for the treatment of autoimmune diseases. Importantly, L19 is part of a 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 for differential selectivity for Treg lymphocytes c Targeted amino acid substitution combinations In Example 4, it was shown that specific mutations aimed at reducing the affinity of IL-2 for 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 for both the IL-2Rβ and γc receptor subunits through a combination of certain amino acid substitutions targeted at the β receptor and other substitutions targeted at the γ receptor can result in a window of favorable activity and selectivity for Treg lymphocytes.

[0190] Such rationale is illustrated 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, using the wild-type IL-2 fusion protein P-0250 as a control. Similarly, Figures 5C and 5D show the STAT5 phosphorylation activity of P-0303 (Q126E), which contains an amino acid substitution targeted to disrupt interaction with the γ receptor. The data indicated that each 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 variant. As shown in Figures 5E and 5F, the combination of the L19Y and Q126E mutations in P-0419 significantly widened the window for selective activation of Treg cells. P-0419 largely retained its Treg activation potential, and the activity profile of the P-0419 variant was very 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 targeting the γ receptor does not necessarily result in a favorable window of activity and selectivity. This requires the correct amount of activity modulation for each variant. The four IL-2 variants in Figures 6A and 6B share the same L19Y substitution targeting the β receptor, and the additional mutations designed to target the γ receptor are Q126E in P-0419, Q126K in P-0464, S125I in P-0471, and Q22K in P-0474. While all variants retained comparable activity in inducing STAT5 phosphorylation in Treg cells (Figure 6A), such activity varied significantly in CD4+ Tconv cells (Figure 6B), demonstrating differences in the ability to selectively modulate Treg activation via combinations of amino acid substitutions.

[0192] However, combining IL-2 variants that already exhibit biased specificity for the Treg subset with further receptor attenuation by combining the Q126E substitution can significantly attenuate or eliminate activity against Treg cells. As shown in Figures 6C and 6D, both variants P-0373 (L19N) and P-0363 (D20T) already exhibited some or significant bias in selectivity toward Treg cells (Figures 6C and 6D). Their respective counterparts, P-0417 and P-0322, which contain the additional Q126E substitution, exhibited significantly reduced Treg cell activation potential. Therefore, it is important to find the right combination of residue substitutions to tailor activity to favor activation potential and bias specificity toward Treg cells.

[0193] We evaluated 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), and their activity is shown in Figures 7A-7D. Compared with IL-2 variants P-0424 (L19H) and P-0303 (Q126E), each containing a single amino acid substitution, variant P-0447 (L19H, Q126E), which contains a combination of two amino acid substitutions, exhibited robust biological activity in stimulating STAT5 phosphorylation in Treg cells, but this activity was almost completely lost in Tconv cells (Figures 7A and 7B). In a separate study evaluating P-0419, P-0447, P-0448, and P-0449 against two benchmark compounds, all four variants significantly demonstrated the ability to induce STAT5 phosphorylation in Treg cells, but this activity was largely abolished in CD4+Tconv cells (Figures 7C and 7D). Consistent with Figures 5E and 5F, P-0419 had an activity profile comparable to that of benchmark-1, whereas 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 have the potential to be Treg-biased IL-2 agents for activating Treg cells for the treatment of autoimmune diseases. Furthermore, these variants are expected to have an improved safety profile and reduced VLS due to the removal of potentially 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 resulted in a general improvement in the development suitability of IL-2 fusion constructs. To make the S125I substitution a viable approach to alleviating the development suitability issue of IL-2 engineering, it will be important to demonstrate that such an amino acid substitution does not impair the biological activity of the resulting fusion protein compared to its Ser-125 counterpart.

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

[0197] TIFF0007827324000011.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 Figure 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 Figure 8. As shown in Figures 8A-8F, the S125I substitution slightly increased the activity of the three test compounds, did not alter Treg selectivity for P-0531 and P-0491, and further widened the window of Treg selectivity for P-0511.

[0199] The data thus demonstrate 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 resulted in universal developmental improvements (increased production yield, reduced aggregation, and decreased 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 for addressing challenges 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+ / high CD25 expression) and Tconv cells (CD4+ / Foxp3- / low CD25 expression), 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- / CD25+), CD8 cytotoxic effector T cells, and NK cells, in comparison with 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 significant proliferation advantage of these variants over CD4+ T cells is conferred by the constitutively high expression of IL-2Rα (CD25) on T cells. CD25 expression can be induced on CD4+ effector T cells after immune stimulation. Therefore, it is desirable to confirm that 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. Flow cytometry analysis was performed by gating the CD4+ / Foxp3+ / CD25+, CD4+ / Foxp3- / CD25+, and CD4+ / Foxp3- / CD25- populations to represent Treg cell subsets, CD4 effector cell subsets, and CD4 naive T cell subsets, respectively. Data are presented as the percentage of pSTAT5-positive cells in the gated populations and are shown in Figure 9. P-0512 had a comparable activity profile to Criterion-1 in all three T cell subsets, whereas P-511 outperformed both Criterion-2 and Criterion-3 in terms of activity and selectivity window for Treg cells relative to both Teff cells and naive CD4 T cells. Criterion-2 showed much weaker activation capacity for each of the three subsets. Figures 9A and 9B clearly demonstrate the preferential activation of Tregs over Teffs by IL-2 variants with attenuated IL-2Rβγ interaction, particularly P-0511, despite moderate to high levels of CD25 expression on Teffs.

[0203] Furthermore, P-0511 and P-0512 were tested for their ability to stimulate the proliferation of NK cells and CD8+ T cells in comparison with wild-type and reference molecules. The intracellular fluorescent label, carboxyfluorescein diacetate succinimidyl ester (CFSE), was used. Briefly, human PBMCs (1 × 10 5Cells (1000 cells / well) were labeled with CFSE, seeded into 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, followed by 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 CD8+ T cell and NK cell stimulatory capacities compared to the wild-type IL-2 fusion molecule, P-0250. Supporting the STAT5 phosphorylation assay (Figure 9), P-0512 had an activity profile comparable to that of 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] We compared the STAT5 phosphorylation activity of P-0511 with that of P-0531, the S125I counterpart of wild-type P-0250, in responder cells other than CD4+ T cell subsets, including CD8+ T cells and NK cells. Like P-0531, P-0511 exhibited significant activity in stimulating STAT5 phosphorylation in Treg cells (Figure 11A), whereas this 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 primarily a dimeric IL-2R, containing IL-2Rβ and γc. To confirm whether the significant attenuation of pSTAT5 signaling by P-0511 in CD8+ T cells and NK cells was due to impaired interaction with IL-2Rβ and γc, we developed an ELISA assay.

[0206] Briefly, noncovalent complexes of IL-2Rβ-ECD (NP_000869) and γc-ECD (NP_000197) via heterodimeric Fc chains were coated onto each well of a Nunc Maxisorp 96-well microplate at 2 μg / well. After overnight incubation at 4°C and blocking with SuperBlock (Thermo Fisher Scientific), 100 μl / well of IL-2 Fc fusion protein, diluted threefold starting at 100 nM or 270 nM, was added to each well. After 1 hour of incubation at room temperature, 1 μg / ml of biotin-mouse anti-human IL-2 antibody (BD Biosciences) was added to each well, followed by 1 hour of incubation with 1 μg / ml of HRP-avidin (Thermo Fisher Scientific). 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, and the plate was developed for 10 min in the dark at room temperature. 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 shown in Figure 11E.

[0207] As shown in Figure 11E, P-0531, a wild-type IL-2 fusion protein with improved development compatibility, binds to the IL-2 dimeric receptor complex with sub-nM affinity (EC 50 =0.06 nM); the reference 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, indicating that two IL-2 mutations in P-0511 at both the β and γc receptor subunit interfaces dramatically impaired its interaction with the complex. Surprisingly, despite virtually abolishing binding to the dimeric IL-2 receptor complex, P-0511's activity against Tregs was only slightly reduced compared to the wild-type IL-2 fusion. P-0511 exemplifies an IL-2 variant with a favorable window of activity and selectivity 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 with 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 against Treg cells but exhibited reduced or absent activity against Tconv cells and other lymphocyte subsets. Some variants had activity profiles similar to those of Criterion 1, while others resembled the activity characteristics of Criterion 2 or Criterion 3. Furthermore, most of these IL-2 variants had their proposed toxin-like motifs removed to reduce VLS. Importantly, the incorporation of the S125I amino acid substitution resulted in IL-2 variant fusions with improved development suitability profiles while retaining biological activity and selectivity in Treg cells. These variants are potently Treg-biased IL-2 agents for the treatment of autoimmune diseases with improved safety profiles.

[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 the ability of the fusion proteins to preferentially stimulate the proliferation and proliferation of regulatory T cells (CD4+CD25+FoxP3+ T cells) over effector T cells and NK cells was determined in vivo.

[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 administered vehicle (PBS), 0.3 mg / kg of each test compound, or IL-2 reference compound. Peripheral blood samples were collected in heparinized tubes on days 3, 5, and 7 post-treatment. Each group contained six mice, and baseline blood was collected two days prior to treatment (day -2). After red blood cell lysis, total viable mononuclear blood cells were counted using trypan blue dead cell exclusion, followed by intracellular staining for immune cell phenotypes and the Ki67 proliferation marker using flow cytometry analysis. Cells were separately stained 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 Treg cell proliferation and expansion, as demonstrated by increases in Ki67-positive Treg cells and the ratio of Treg cells to total CD4+ T cells or total lymphocytes (Figures 12A-C). After a single injection, this effect was observed 3 days after injection and persisted until days 5 or 7. In contrast to the ex vivo confirmation that benchmark-1 consistently exhibited the highest activity among IL-2 variants in inducing Treg phosphorylation, all three tested variants, P-0511, P-0512, and P-0514, demonstrated stronger in vivo efficacy in stimulating Treg cell proliferation and expansion than the benchmark molecules in mice. P-0511, P-0512, and P-0514 exhibited comparable activity. The relative in vivo activity ranking 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, while standard-2 was much weaker in stimulating Treg cell proliferation and proliferation (Figures 12A-C).

[0212] For effector T cells and NK cells, Standard-1 showed strong Ki67 stimulation of cytotoxic CD8+ T cells and NK cells, whereas Standard-2 and Standard-3 showed less potent Ki67 stimulation of CD8+ T cells and NK cells (Figures 13A-C). Variant P-0514 showed Ki67 stimulation of CD8+ T cells similar to Standard-1, while variants P-0511 and P-0512, like Standard-2 and Standard-3, showed mild Ki67 stimulation of CD8+ T cells and NK cells (Figures 13A-C). The data suggest that variants P-0511 and P-0512 exhibit superior Treg bioactivity and selectivity compared to Standard-1 and Standard-2. Standard-3 was ineffective in stimulating and expanding both Treg and effector cells.

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

[0214] Compared to the three references, 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 counts 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 superior Treg activation and functionality.

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

[0217] Collectively, these data demonstrated that variants P-0511, P-0512, and P-0514 exhibited 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 for combating 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 Female Balb / C mice (n = 5 per group) were subcutaneously administered a single dose of vehicle (PBS) or P-0511 (1 mg / kg, 0.3 mg / kg, 0.1 mg / kg, or 0.03 mg / kg) 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 their spleens were removed. Blood lymphocyte phenotypes, 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 weight or spleen weight in any of the treatment groups (data not shown).

[0220] As shown in Figure 17, a dose-dependent increase in Treg cell proliferation, reflected by an increase in the percentage of Ki67-positive cells (Figure 17A), was observed in mice treated with P-0511 at dose levels of 1 mg / kg, 0.3 mg / kg, or 0.1 mg / kg. Treatment with 0.03 mg / kg had the least effect. At the three highest dose levels, stimulation of Ki67 expression in Treg cells peaked on day 3, plateaued until day 5, and then declined. Consequently, P-0511 treatment dose-dependently increased the ratio of Tregs 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 to the proliferation / activation Ki67 marker (Figure 17), i.e., peaking on day 3 and continuing through day 5. Administration of 1 mg / kg stimulated Tregs to a greater extent and duration, with the signal persisting for up to 5 days.

[0221] Furthermore, P-0511 treatment resulted in a dose-dependent and statistically significant increase in the proportion of Tregs relative to total lymphocytes (Figure 18A), whereas 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). At peak, Tregs accounted for 4.5% of total lymphocytes after a single 1 mg / kg dose, compared with 3.1% after 0.3 mg / kg and 1.4% after 0.1 mg / kg. 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). This suggests that P-0511 preferentially expanded Treg cells over Tconv cells. Additionally, the expression of Treg cell functional markers, including CD25 (Figure 19B) and FoxP3 (Figure 19C), also increased in a dose-dependent manner. The increases in mean fluorescence intensity (MFI) of CD25 and FoxP3 peaked on day 3 and attenuated to lower levels by day 5.

[0223] Collectively, these data demonstrate that P-0511 potently and preferentially activates and expands Tregs in a dose-dependent manner. Careful consideration is needed to achieve an optimized dosing strategy aimed at maximizing its activity, promoting the activation, proliferation, and expansion of immunosuppressive Treg cells while sparing cytotoxic effector T cells and NK cells.

[0224] Example 10 Pharmacodynamic study in mice after repeated administration of IL-2 variant Fc fusion proteins Female Balb / C mice (7 weeks old) were acclimated in-house for 5–7 days before testing. Mice (n = 5 per group) were subcutaneously administered vehicle (PBS), 0.3 mg / kg of P-0511, P-0512, P-0531, or Reference-1 compound on days 0, 3, and 6. Peripheral blood samples were collected on day 3, 3 days after the first injection, and on day 9, 3 days after multiple (three) injections. Based on previous in vivo experiments, Treg cell activation, proliferation, and proliferation were expected to peak on day 3, so 3 days post-injection was selected for data collection and analysis. Changes in blood lymphocyte activation, proliferation, 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 proteins, nearly 90% of Treg cells in all test groups were positive for Ki67 expression, and these Ki67-positive cells remained significantly higher even after the third administration of all test compounds (Figure 20A). Interestingly, Treg cells, expressed as the percentage of Tregs relative to total CD4 T cells (%) or total lymphocytes (%), were dramatically reduced to near-control levels in P-0531- and Criterion-1-treated mice after three consecutive every-third treatments compared to a single treatment, whereas they remained significantly higher in P-0511- and P-0512-treated mice (Figures 20B-C). These data suggest that wild-type IL-2 or Criterion-1 may promote Treg cell exhaustion or induce Treg desensitization due to their stronger activity in Treg stimulation. A further explanation could be that differences in half-life or "receptor sinks" on non-lymphocytes could alter drug exposure, leading to little or no Treg selectivity for wild-type IL-2 or canonical-1.

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

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

[0228] Example 11 Suppression of antigen-driven inflammation by IL-2 variant Fc fusion proteins in a murine model of delayed-type hypersensitivity (DTH) The in vivo ability of IL-2 variant-induced Treg cells 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 for 7 days and randomly assigned to groups. Subcutaneous administration of vehicle (PBS), 0.1 mg / kg, or 0.3 mg / kg P-0511 was initiated 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 with 100 μg of keyhole limpet hemocyanin (KLH) in 200 μl saline. For every-3-day administration, two additional 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-day administration, one additional subcutaneous injection was administered. On day 3, mice received an injection of PBS, 0.1 mg / kg, or 0.3 mg / kg P-0511. On day 5, mice received intradermal KLH (5 μg / 10 μl saline) injection into the right ear. Right ear thickness was measured using a caliper before KLH administration on day 5 and every day from days 6 to 8, corresponding to 24, 48, and 72 hours after KLH administration. As a positive control, one group of mice received intraperitoneal injections of 5 mg / kg dexamethasone every day from days 5 to 8.

[0229] FIG. 23 shows the kinetics of the DTH response in terms of the change in ear thickness relative to baseline values ​​(Δ ear 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 ear pinna, and this ear swelling persisted for 72 hours in the PBS group. It was clear that the immunosuppressive steroid dexamethasone, administered four consecutive times daily at 5 mg / kg, potently inhibited KLH-induced inflammatory responses, 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 third or every fifth day 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-third and every-fifth-day administrations, 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 with that of Reference-1 (0.3 mg / kg, every 5 days). As shown in Figure 24, P-0511 demonstrated 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 0.1 mg / kg, respectively. Reference-1 demonstrated mild inhibition of ear swelling, with the effect of 0.3 mg / kg Reference-1 being similar to that achieved with 0.1 mg / kg P-0511 (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 even after KLH administration without repeated administration. This example highlights the importance of adjusting the administration regimen to achieve optimal efficacy.

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

[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. Peripheral blood Tregs, non-regulatory CD4 T cells, CD8 T cells, and NK cells, as well as naive and memory cells, will be immunophenotyped by FACS to determine their pharmacodynamic properties. Cell activation and proliferation will also be monitored by measuring CD69 and Ki67. A complete blood count (CBC) with a five-part differential count (neutrophils, lymphocytes, monocytes, eosinophils, and basophils) will also be performed.

[0235] The PK characteristics of P-0511 will be evaluated in cynomolgus monkey plasma samples by measuring full-length intact P-0511 using a mouse anti-human IL-2 monoclonal antibody (BD Pharmingen) to coat a 96-well plate for P-0511 capture. P-0511 will be detected using a goat anti-human Fc polyclonal-HRP (Thermo Fisher Scientific), and its plasma concentration will then be quantified. In addition to plasma samples collected on days -7, -3, 2, 4, 6, 8, 11, 14, 18, 21, 28, 32, 35, 43, 50, and 57, three additional plasma samples were collected 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 glutamyltransferase, 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. While the articles and methods of the present disclosure have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes may be made therein without departing from the spirit and scope of the present disclosure. All such modifications and equivalents, whether now existing or later developed, apparent to those 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 of those skilled 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 single-letter codes, as defined in 37 CFR §1.822. SEQ ID NO: 1 is the amino acid sequence of human IL-2 precursor. SEQ ID NO: 2 is the native amino acid sequence of mature 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 the reference Fc-IL-2 variant fusion proteins. SEQ ID NOs: 214 to 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 / eliminated 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 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 69) P-0305 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTAEAAAKEAAAKEAAAKACPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 70) P-0254 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 71) P-0363 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 72) P-0364 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLELQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 73) P-0365 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLNLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 74) P-0366 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLQLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 75) P-0367 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLSLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 76) P-0368 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLYLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 77) P-0252 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 78) P-0372 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 79) P-0373 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLNDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 80) P-0374 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLRDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 81) P-0253 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 82) P-0302 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISIINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 83) P-0375 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISQINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 84) P-0376 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISEINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 85) P-0377 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISTINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 86) P-0378 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISMINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 87) P-0303 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 88) P-0304 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSLSIISTLT (SEQ ID NO: 89) P-0369 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSNSIISTLT (SEQ ID NO: 90) P-0370 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSDSIISTLT (SEQ ID NO: 91) P-0371 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSMSIISTLT (SEQ ID NO: 92) P-0251 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISGINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 93) P-0317 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 94) P-0318 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLILQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISIINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 95) P-0324 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 96) P-0322 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 97) P-0323 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSLSIISTLT (SEQ ID NO: 98) P-0325 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 99) P-0326 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSLSIISTLT (SEQ ID NO: 100) P-0417 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLNDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 101) P-0418 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLRDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 102) P-0419 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (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 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 147) P-0424 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 148) P-0425 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLDDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 149) P-0426 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLPDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 150) P-0427 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLSDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 151) P-0428 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDSQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 152) P-0429 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDNQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 153) P-0430 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDRQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 154) P-0497 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSKSIISTLT (SEQ ID NO: 155) P-0498 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSHSIISTLT (SEQ ID NO: 156) P-0499 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSYSIISTLT (SEQ ID NO: 157) P-0500 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFEQSIISTLT (SEQ ID NO: 158) P-0501 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFKQSIISTLT (SEQ ID NO: 159) P-0502 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFHQSIISTLT (SEQ ID NO: 160) P-0503 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFWQSIISTLT (SEQ ID NO: 161) P-0531 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 162) P-0505 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLNMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 163) P-0506 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLHMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 164) P-0507 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLKMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 165) P-0508 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLYMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 166) P-0509 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLIMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 167) P-0447 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 168) P-0448 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 169) P-0449 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLSDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSESIISTLT (SEQ ID NO: 170) P-0464 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSKSIISTLT (SEQ ID NO: 171) P-0465 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSHSIISTLT (SEQ ID NO: 172) P-0466 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSYSIISTLT (SEQ ID NO: 173) P-0467 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFEQSIISTLT (SEQ ID NO: 174) P-0468 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFKQSIISTLT (SEQ ID NO: 175) P-0469 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFHQSIISTLT (SEQ ID NO: 176) P-0470 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFWQSIISTLT (SEQ ID NO: 177) P-0471 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 178) P-0472 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLNMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 179) P-0473 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLHMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 180) P-0474 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLKMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 181) P-0475 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLYMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 182) P-0476 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLIMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 183) P-0480 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSKSIISTLT (SEQ ID NO: 184) P-0491 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 185) P-0492 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLDDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 186) P-0493 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLELQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT (SEQ ID NO: 187) P-0494 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIISTLT(SEQ ID NO: 188) P-0495 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLYDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIESIISTLT(SEQ ID NO: 189) P-0496(Standard-2) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 190) Standard-1 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINKIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (SEQ ID NO: 191) standard-3 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT (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 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIESIISTLT (SEQ ID NO: 198) P-0512 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT (SEQ ID NO: 199) P-0513 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSKSIISTLT (SEQ ID NO: 200) P-0514 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIESIISTLT (SEQ ID NO: 201) P-0515 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLQDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT (SEQ ID NO: 202) P-0582 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLLTLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT (SEQ ID NO: 203) P-0583 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLNDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT (SEQ ID NO: 204) P-0584 DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLRDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIKSIISTLT (SEQ ID NO: 205) P-0585 DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGSAPTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIESIISTLT (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 APTSSSTKKTQLQLEHLLHDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIESIISTLTGGGGSGGGGSGGGGSGGGGSCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPG (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 fusion protein comprising: 1) an IL-2 variant polypeptide; and 2) an Fc domain, wherein the fusion protein is in a dimeric form, each of the IL-2 variant polypeptides of the fusion protein is fused to the Fc domain by a peptide linker, and wherein the IL-2 variant polypeptide comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 193 and 194.

2. 2. The isolated fusion protein of claim 1, wherein the IL-2 variant polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

193.

3. 2. The isolated fusion protein of claim 1, wherein the IL-2 variant polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

194.

4. 4. The isolated fusion protein of any one of claims 1 to 3, wherein stimulation with the IL-2 variant polypeptide results in a regulatory T cell (Treg) / conventional T cell (Tconv) ratio of at least 2.

5.

5. 4. The isolated fusion protein according to any one of claims 1 to 3, wherein stimulation with the IL-2 variant polypeptide results in greater stimulation of Treg than stimulation of Tconv.

6. 2. The isolated fusion protein of claim 1, wherein the fusion protein comprises: a fusion protein in which the IL-2 variant polypeptide is fused at its N-terminal amino acid to the C-terminal amino acid of the Fc domain; and a fusion protein in which the IL-2 variant polypeptide is fused at its C-terminal amino acid to the N-terminal amino acid of the Fc domain. An isolated fusion protein characterized in that it is selected from the group consisting of:

7. 1. An isolated fusion protein comprising an IL-2 variant polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 193 fused to an Fc domain comprising the amino acid sequence set forth in SEQ ID NO: 45 using a peptide linker having the amino acid sequence set forth in SEQ ID NO: 55, wherein the isolated fusion protein is in a dimeric form.

8. 1. An isolated fusion protein comprising 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 using a peptide linker having the amino acid sequence set forth in SEQ ID NO:55, wherein the isolated fusion protein is in a dimeric form.

9. 2. The isolated fusion protein of claim 1, comprising an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs: 198-199.

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

198.

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

199.

12. A pharmaceutical composition comprising the isolated fusion protein of any one of claims 1 to 11 in admixture with a pharmaceutically acceptable carrier.

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