Designed il-2 variants

Abstract

To provide variant IL-2 proteins and uses thereof.SOLUTION: Provided is a variant human IL-2 protein comprising one or more amino acid changes. The IL-2 variant protein has greater potency for activation of IL-2 signaling pathways in cells lacking CD25 expression, relative to wild-type IL-2.SELECTED DRAWING: None

Description

[Background technology]

[0001] Interleukin-2 (IL-2) is a type I alpha helical cytokine that functions as a multilineage lymphocyte growth factor. In activated lymphocytes and Treg cells, IL-2 signals through a high affinity (10 pM) heterotrimeric receptor complex consisting of the IL-2Rα (CD25), IL-2Rβ (CD122), and γc (CD132) chains. In resting lymphocytes, it signals through an intermediate affinity (1 nM) heterodimeric receptor complex consisting of the IL-2Rβ and γc chains.

[0002] The IL2-Rα subunit is constitutively expressed at high levels on regulatory T cells (Treg) and at lower levels on natural killer (NK) cells and resting effector CD8+ T cells, resulting in differential IL-2 potency between different immune cell compartments. From a clinical perspective, both IL-2 agonism and antagonism are of considerable importance and have been used for more than 30 years in the immunotherapy of malignancies.

[0003] The plasticity of IL-2 is a key parameter in generating small molecules that bind to IL-2 and block signaling, engineering "superagonist" mutant versions of IL-2 with high affinity for IL-2Rβ, and isolating antibodies that bind IL-2 and localize its effects to different immune cell subsets. Indeed, many efforts are focused in part on improving its therapeutic potential by engineering its ability to selectively target specific cell types.

[0004] In one approach, a monoclonal antibody against IL-2 can change its properties by binding to several different conformational epitopes, thereby modifying the interaction of IL-2 with any of the IL-2R subunits, leading to the proliferation of either Treg or Teff cells. For example, wild-type mouse IL-2 (mIL-2) can be administered in complex with an anti-mouse IL-2 monoclonal antibody (JES6-1) and used to preferentially induce Treg cell proliferation.

[0005] IL-2 can induce T cell expansion to enhance adoptive immunotherapy and has been approved by the FDA for the treatment of melanoma and renal cell carcinoma, with complete remissions in a subset of patients. However, IL-2 can also promote pathological responses, and the therapeutic goal is to maintain the desired effects of this cytokine while simultaneously blocking undesired deleterious responses.

[0006] We previously used the structure of a high affinity IL-2-IL-2R complex to develop IL-2 "superkines" with enhanced action due to their enhanced binding affinity to IL-2Rβ, which eliminates the functional requirement for IL-2Rα. We also used the super-IL-2 platform to generate mutants that retain increased binding affinity to IL-2Rβ but exhibit reduced binding to γc, thereby having defective IL-2Rβ-γc heterodimerization and signaling.

[0007] Continued refinement and development of human IL-2 signaling variants is of great interest for clinical purposes, including the treatment of cancer and autoimmune diseases, and variant proteins for this purpose are provided herein. Summary of the Invention

[0008] Mutant IL-2 proteins and uses thereof are provided. In some embodiments, the IL The -2 mutant protein has a greater potency in activating the IL-2 signaling pathway in cells lacking CD25 expression than wild-type IL-2. Such proteins may be referred to herein as IL-2 superagonists. The EC50 of the superagonist on CD25- cells may be reduced to at least 2-fold compared to the wild-type protein, at least 3-fold compared to the wild-type protein, at least 4-fold compared to the wild-type protein, at least 5-fold compared to the wild-type protein, for example, an EC50 of less than about 2 nM, less than about 1.5 nM, less than about 1 nM, less than about 0.5 nM, etc.

[0009] In one embodiment, provided herein are IL-2 superagonist variants having increased binding affinity for CD122 compared to wild-type IL-2, the binding affinity having a Kd of less than about 10-9, less than about 10-10, or less than about 10-12. Such variants are exemplified, for example, by variants of SEQ ID NO:1, SEQ ID NO:7, and SEQ ID NO:8, or alternatively in a reverted sequence in which residues at positions 69 and 115 are reverted to wild-type valine residues. Other variants have greater potency with respect to activating the IL-2 signaling pathway in cells lacking CD25 expression, but do not have substantially increased binding affinity compared to the wild-type IL-2 protein. Such variants are exemplified, for example, by variants of SEQ ID NO:6, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, or alternatively in a reverted sequence in which residues at positions 69 and 115 are reverted to wild-type valine residues.

[0010] In other embodiments, the mutant IL-2 proteins have greater selectivity for CD25-expressing cells and have reduced potency for activating the IL-2 signaling pathway in cells lacking CD25 expression compared to wild-type IL-2. Such proteins may be referred to herein as CD25-selective agonists.

[0011] The IL-2 variants find use, for example, as IL-2 selective agonists and superagonists in applications where modulation of IL-2 signaling is desirable, for example, by upregulation of IL-2 signaling, such as for the treatment of cancer, and for selective activation of IL-2 signaling in autoimmune diseases. Nucleic acids encoding such IL-2 variants, methods of making such IL-2 variants, pharmaceutical compositions comprising such IL-2 variants, and methods of treatment using such IL-2 variants are also provided.

[0012] In some embodiments, the IL-2 variant comprises one or more amino acid substitutions that increase the potency of IL-2 in CD25- cells selected from substitutions at amino acid positions 27, 28, 31, 32, 35, 39, 52, 72, 74, 75, the region including residues 74-80, 85, 86, and 92, numbered according to wild-type hIL-2.

[0013] In some embodiments, the IL-2 variant comprises one or more of the following amino acid changes: G27M, I28L, Y31A, K32D / E / S, K35S, M39L, or alternatively M39I, E52S, or alternatively E52D, E52N, E52T, L72A / Q / D / L / H / T, loop: GDDPKTI, DSTDETV, DSTDERI, DSTDSRI, SKDQDKV, ADDKDTI, ADDQDKI, AQSKNFHL, SKDQKKV, SDDQDKV, L80V / I, R81Δ, P82Δ, L85V, or alternatively L85M or L85A, I86V, and I92F.

[0014] In one embodiment, an IL-2 variant having greater binding affinity for CD122 compared to wild-type human IL-2 comprises the following amino acid changes: G27M, I28L, K32D, M39L, E52S, V69A, L72A / Q / D / L / H / T, loop: GDDPKTI, L80I, R81Δ, P82Δ, L85V, I86V, I92F, and V115I (e.g., as shown in SEQ ID NO:1, or alternatively, the reverted sequence of SEQ ID NO:1 in which residues at positions 69 and 115 are changed back to wild-type valine residues).

[0015] In one embodiment, an IL-2 variant having greater binding affinity for CD122 compared to wild-type human IL-2 comprises the following amino acid changes: G27M, I28L, K32D, V69A, L72Q, loop: DSTDETV, L80V, R81Δ, P82Δ, and V115I (e.g., as shown in SEQ ID NO:7, or alternatively, the reverted sequence of SEQ ID NO:7 in which residues at positions 69 and 115 are changed back to wild-type valine residues).

[0016] In one embodiment, an IL-2 variant having greater binding affinity for CD122 compared to wild-type human IL-2 comprises the following amino acid changes: G27M, I28L, K32D, M39L, E52S, V69A, L72A, loop: DSTDERI, L80I, R81Δ, P82Δ, L85V, I86V, I92F, and V115I (e.g., as shown in SEQ ID NO:8, or alternatively, the reverted sequence of SEQ ID NO:8 in which residues at positions 69 and 115 are changed back to wild-type valine residues).

[0017] In some embodiments, the IL-2 variant is CD25 selective and includes one or more of the following amino acid changes: G27M, I28L, K32E, E52S, V69A, L72Q, loop: GDDPKTI, L80I, R81Δ, P82Δ, and V115I (e.g., as shown in SEQ ID NO: 6, or alternatively, the reverted sequence of SEQ ID NO: 6 in which residues at positions 69 and 115 are changed back to wild-type valine residues).

[0018] In some embodiments, the IL-2 variant has greater potency for CD25- cells in the absence of greater binding affinity for CD122 and includes one or more of the following amino acid changes: G27M, I28L, Y31A, K32D / E / S, K35S, M39L, E52S, V69A, L72A / Q / D / L / H / T, loop: AQSKNFHL, and V115I (e.g., as shown in SEQ ID NO: 15, or alternatively, the reverted sequence of SEQ ID NO: 15 in which residues at positions 69 and 115 are changed back to wild-type valine residues).

[0019] In some embodiments, the IL-2 variant has greater potency for CD25- cells in the absence of greater binding affinity for CD122 and includes one or more of the following amino acid changes (e.g., as shown in SEQ ID NO: 16, or alternatively, the reverted sequence of SEQ ID NO: 16 in which residues at positions 69 and 115 are changed back to wild-type valine residues): G27M, I28L, K32D, M39L, E52S, V69A, L72Q, loop: AQSKNFHL, and V115I.

[0020] In some embodiments, the IL-2 variant has greater potency for CD25- cells in the absence of greater binding affinity for CD122 and includes one or more of the following amino acid changes (e.g., as shown in SEQ ID NO: 17, or alternatively, the reverted sequence of SEQ ID NO: 17 in which residues at positions 69 and 115 are changed back to wild-type valine residues).

[0021] In certain embodiments, the subject IL-2 variants have an enhanced ability to stimulate STAT5 phosphorylation in CD122+ T cells compared to wild-type hIL-2. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the subject IL-2 variants have an enhanced ability to stimulate pERK1 / ERK2 signaling in CD122+ T cells compared to wild-type hIL-2.

[0022] In some embodiments, the amino acid substitutions described herein are further combined with amino acid substitutions F42A, E61R, R38D, Y45A, or combinations thereof that introduce mutations that affect CD25 binding, e.g., as described in Thanos et al. (2006) PNAS 103(42):15422-7 and Vazquez-Lombardi See, e.g., et al. (2017) 8:15373.

[0023] In another aspect, provided herein is an IL-2 variant fusion protein comprising any one of the IL-2 variants described herein linked to a human Fc antibody fragment.

[0024] In another aspect, provided herein is a pharmaceutical composition comprising any one of the IL-2 variants or IL-2 variant fusion proteins described herein and a pharma- ceutically acceptable carrier.

[0025] In some embodiments, methods are provided for the use of the IL-2 variants provided herein for the treatment of disease. Superagonist IL-2 proteins induce superior expansion of cytotoxic T cells, resulting in improved responses in vivo with less expansion of T regulatory cells and reduced pulmonary edema. Treatment modalities include, but are not limited to, treatment of cancer, treatment of immune deficiencies, treatment of infectious diseases (especially chronic or difficult to treat infectious diseases), use as vaccine adjuvants, and the like. In such embodiments, an effective dose of the IL-2 variant is administered to an individual.

[0026] In some embodiments, a method is provided for in silico design of cytokine ligands based on crystallographic structures. The design principle is based on the topology of the ligand-receptor interaction. By preferentially stabilizing the cytokine with the receptor according to its high-resolution crystal structure, the cytokine can be converted into a superagonist. The method identifies where packing can be improved to increase binding. Core positions are redesigned and tested in a computer model to identify positions where improvements can be introduced. The resulting sequences are analyzed and compared. A computer model is then used to generate a scaffold structure ensemble that allows the entire core to be redesigned while simultaneously monitoring residue changes in the identified regions. A subset of residues that improve packing is visually selected. The redesign is performed iteratively until the identity of the subset is maintained and the sequences converge. Upon identification of candidate sequences, coding sequences can be generated and expressed for testing.

[0027] In some embodiments, the disclosure provides mutant human IL-2 proteins that contain one or more amino acid changes. In some embodiments, the disclosure provides mutant human IL-2 proteins that have greater potency for activating the IL-2 signaling pathway in cells lacking CD25 expression than wild-type IL-2, which contain one or more amino acid substitutions compared to wild-type human IL-2.

[0028] In some embodiments, the one or more amino acid changes are selected from the group consisting of an amino acid substitution at position 27 to methionine (G27M), an amino acid substitution at position 28, an amino acid substitution at position 31 to alanine (Y31A), an amino acid substitution at position 32, an amino acid substitution at position 35 to serine (K35S), an amino acid substitution at position 39, an amino acid substitution at position 52, an amino acid deletion at position 82 (P82Δ), and an amino acid substitution at position 115 to isoleucine (V115I) relative to the amino acid sequence of the wild-type human IL-2 protein set forth in SEQ ID NO:19.

[0029] In some embodiments, the one or more amino acid substitutions compared to wild-type human IL-2 are selected from the group consisting of an amino acid substitution at position 27 to methionine (G27M), an amino acid substitution at position 28, an amino acid substitution at position 31 to alanine (Y31A), an amino acid substitution at position 32, an amino acid substitution at position 35 to serine (K35S), an amino acid substitution at position 39, an amino acid substitution at position 52, and an amino acid substitution at position 115 to isoleucine (V115I).

[0030] In some embodiments, a mutant human IL-2 protein of the disclosure comprises an amino acid deletion at position 81 (R81Δ) or a substitution at position 81.

[0031] In some embodiments, a mutant human IL-2 protein of the disclosure comprises one or more amino acid substitutions at positions selected from the group consisting of 69, 72, the region encompassing amino acid residues 74-80, 85, 86, and 92.

[0032] In some embodiments, a mutant human IL-2 protein of the disclosure comprises an amino acid substitution at position 28. In some embodiments, the amino acid substitution at position 28 comprises a substitution to leucine (I28L).

[0033] In some embodiments, a mutant human IL-2 protein of the disclosure comprises an amino acid substitution at position 32. In some embodiments, the amino acid substitution at position 32 is selected from the group consisting of a substitution to aspartic acid (K32D), a substitution to glutamic acid (K32E), and a substitution to serine (K32S).

[0034] In some embodiments, a mutant human IL-2 protein of the disclosure comprises an amino acid substitution at position 39. In some embodiments, the amino acid substitution at position 39 is selected from the group consisting of a substitution to leucine (M39L) and a substitution to isoleucine (M39I).

[0035] In some embodiments, a mutant human IL-2 protein of the disclosure comprises an amino acid substitution at position 52. In some embodiments, the amino acid substitution at position 52 is selected from the group consisting of a substitution with serine (E52S), a substitution with aspartic acid (E52D), a substitution with asparagine (E52N), and a substitution with threonine (E52T).

[0036] In some embodiments, a mutant human IL-2 protein of the disclosure comprises an amino acid substitution at position 69. In some embodiments, the amino acid substitution at position 69 comprises a substitution to alanine (V69A).

[0037] In some embodiments, a mutant human IL-2 protein of the disclosure comprises an amino acid substitution at position 72. In some embodiments, the amino acid substitution at position 72 is selected from the group consisting of an alanine substitution (L72A), a glutamine substitution (L72Q), an aspartic acid substitution (L72D), a histidine substitution (L72H), and a threonine substitution (L72T).

[0038] In some embodiments, a mutant human IL-2 protein of the disclosure comprises an amino acid substitution at position 81. In some embodiments, the amino acid substitution at position 81 comprises a substitution to aspartic acid (R81D).

[0039] In some embodiments, a mutant human IL-2 protein of the disclosure comprises an amino acid substitution at position 85. In some embodiments, the amino acid substitution at position 85 is selected from the group consisting of a substitution with valine (L85V), a substitution with methionine (L85M), and a substitution with alanine (L85A).

[0040] In some embodiments, a mutant human IL-2 protein of the disclosure comprises an amino acid substitution at position 86. In some embodiments, the amino acid substitution at position 86 comprises a substitution to valine (I86V).

[0041] In some embodiments, the mutant human IL-2 protein of the present disclosure comprises an amino acid substitution at position 92. In some embodiments, the amino acid substitution at position 92 is phenylalanine. It contains a substitution (I92F) to

[0042] In some embodiments, a mutant human IL-2 protein of the disclosure comprises an amino acid substitution in a region encompassing amino acid residues 74 to 80. In some embodiments, the amino acid substitution in the region encompassing amino acid residues 74 to 80 comprises the amino acid sequence of GDDPKTI, DSTDETV, DSTDERI, DSTDSRI, SKDQDKV, SKDQKKV, SDDQDKV, ADDKDTI, or ADDQDKI.

[0043] In some embodiments, mutant human IL-2 proteins of the disclosure include the amino acid changes G27M, I28L, K32D, M39L, E52S, substitutions in the region encompassing amino acid residues 74-80 with GDDPKTI, R81Δ, P82Δ, and V115I.

[0044] In some embodiments, the mutant human IL-2 proteins of the disclosure comprise amino acid changes that include or consist of G27M, I28L, K32D, M39L, E52S, V69A, L72A, substitutions in the region encompassing amino acid residues 74-80 with GDDPKTI, R81Δ, P82Δ, L85V, and V115I.

[0045] In some embodiments, the mutant human IL-2 proteins of the disclosure comprise amino acid changes that comprise or consist of G27M, I28L, K32D, M39L, V69A, L72A, substitutions in the region encompassing amino acid residues 74-80 with GDDPKTI, R81Δ, P82Δ, and (V115I).

[0046] In some embodiments, the mutant human IL-2 proteins of the disclosure comprise amino acid changes that include or consist of G27M, I28L, K32D, M39L, E52S, V69A, L72A, substitutions in the region encompassing amino acid residues 74-80 with GDDPKTI, R81Δ, P82Δ, and V115I.

[0047] In some embodiments, the mutant human IL-2 proteins of the disclosure comprise amino acid changes that comprise or consist of G27M, I28L, K32D, M39L, E52S, V69A, L72Q, substitutions in the region encompassing amino acid residues 74-80 with GDDPKTI, R81Δ, P82Δ, and V115I.

[0048] In some embodiments, the mutant human IL-2 proteins of the disclosure comprise amino acid changes that include or consist of G27M, I28L, K32D, M39L, E52S, V69A, L72D, substitutions in the region encompassing amino acid residues 74-80 with GDDPKTI, R81Δ, P82Δ, and V115I.

[0049] In some embodiments, the mutant human IL-2 proteins of the disclosure comprise amino acid changes that include or consist of G27M, I28L, K32E, E52S, V69A, L72Q, substitutions in the region encompassing amino acid residues 74-80 with GDDPKTI, R81Δ, P82Δ, and V115I.

[0050] In some embodiments, the mutant human IL-2 proteins of the disclosure comprise amino acid changes that include or consist of G27M, I28L, K32D, V69A, L72Q, substitutions in the region encompassing amino acid residues 74-80 with DSTDETV, R81Δ, P82Δ, and V115I.

[0051] In some embodiments, the mutant human IL-2 proteins of the disclosure comprise amino acid changes, the amino acid changes being G27M, I28L, K32D, M39L, E52S, V69A, L72A, a substitution in the region encompassing amino acid residues 74-80 with DSTDERI, R 81Δ, P82Δ, L85V, I86V, I92F, and V115I.

[0052] In some embodiments, the mutant human IL-2 proteins of the disclosure comprise amino acid changes that comprise or consist of G27M, I28L, K32D, M39L, E52S, V69A, L72Q, substitutions in the region encompassing amino acid residues 74-80 with DSTDSRI, R81Δ, P82Δ, and V115I.

[0053] In some embodiments, the mutant human IL-2 proteins of the disclosure comprise amino acid changes that include or consist of substitutions in the region encompassing amino acid residues 74-80 with G27M, I28L, K32D, M39L, E52S, V69A, SKDQKKV, R81Δ, P82Δ, L85V, I86V, I92F, and V115I.

[0054] In some embodiments, the mutant human IL-2 proteins of the disclosure comprise amino acid changes that include or consist of substitutions in the region encompassing amino acid residues 74-80 with G27M, I28L, K32S, M39L, E52S, V69A, SDDQDKV, R81Δ, P82Δ, L85V, I86V, I92F, and V115I.

[0055] In some embodiments, the mutant human IL-2 proteins of the disclosure comprise amino acid changes that comprise or consist of G27M, I28L, K32D, V69A, L72H, substitutions in the region encompassing amino acid residues 74-80 with ADDKDTI, R81Δ, P82Δ, and V115I.

[0056] In some embodiments, the mutant human IL-2 proteins of the disclosure comprise amino acid changes that include or consist of G27M, I28L, Y31A, K32S, K35S, V69A, L72T, substitutions of the region encompassing amino acid residues 74-80 with ADDQDKI, R81Δ, P82Δ, and V115I.

[0057] In some embodiments, the mutant human IL-2 proteins of the disclosure comprise amino acid changes comprising or consisting of G27M, I28L, K32D, V69A, R81D, and V115I.

[0058] In some embodiments, the mutant human IL-2 proteins of the disclosure comprise amino acid changes comprising or consisting of G27M, I28L, K32D, V69A, L72Q, R81D, and V115I.

[0059] In some embodiments, the mutant human IL-2 proteins of the disclosure comprise amino acid changes comprising or consisting of G27M, I28L, K32D, M39L, E52S, V69A, R81D, and V115I.

[0060] In some embodiments, the mutant human IL-2 proteins of the disclosure comprise amino acid changes comprising or consisting of G27M, I28L, K32D, M39L, E52S, V69A, L72Q, R81D, and V115I.

[0061] In some embodiments, the mutant human IL-2 proteins of the disclosure comprise amino acid changes consisting of G27M, I28L, K32D, L72Q, and R81D.

[0062] In some embodiments, the mutant human IL-2 proteins of the present disclosure have greater potency for activating the IL-2 signaling pathway in cells lacking CD25 expression. In some embodiments, the mutant IL-2 proteins have an EC50 that is at least two-fold lower than the wild-type protein for activating the IL-2 signaling pathway in cells lacking CD25 expression. In some embodiments, the mutant IL-2 proteins have increased binding affinity for human CD122. In some embodiments, the mutant IL-2 proteins have greater potency for activating the IL-2 signaling pathway in cells lacking CD25 expression, but do not have a substantially increased binding affinity compared to the wild-type IL-2 protein.

[0063] In some embodiments, the mutant human IL-2 proteins of the disclosure are fused to a human IgG Fc domain. In some embodiments, the IgG Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc domain.

[0064] In some embodiments, the disclosure provides a nucleic acid encoding a mutant human IL-2 protein of the disclosure. In some embodiments, the disclosure provides a vector comprising a nucleic acid, the nucleic acid encoding a mutant human IL-2 protein of the disclosure. In some embodiments, the disclosure provides a recombinant cell comprising a nucleic acid, the nucleic acid encoding a mutant human IL-2 protein of the disclosure. In some embodiments, the disclosure provides a recombinant cell comprising a vector disclosed herein.

[0065] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a mutant human IL-2 protein disclosed herein and a pharma- ceutically acceptable carrier.

[0066] In some embodiments, the disclosure provides a method of treating a disease in a subject in need thereof, the method comprising administering to the patient a mutant human IL-2 protein disclosed herein, or a pharmaceutical composition disclosed herein.

[0067] In some embodiments, the present disclosure provides a method of treating a disease requiring expansion of T cells, the method comprising administering an effective amount of a mutant human IL-2 protein disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the disease is a hyperproliferative disease. In some embodiments, the disease is cancer.

[0068] In some embodiments, the disclosure provides a method for in silico design of cytokines based on the crystallographic topology of ligand-receptor interactions, the method comprising identifying positions where packing can be improved to increase binding, redesigning and testing core positions in a computer model to identify positions where improvements can be introduced, generating an ensemble of scaffold structures and allowing the entire core to be redesigned while simultaneously monitoring residue changes in the identified regions, visually selecting a subset of entities that improve packing, retaining the identity of the subset and iteratively performing the redesign until the sequence converges, and generating a coding sequence for the in silico designed cytokine. [Brief description of the drawings]

[0069] [Figure 1A] FIG. 1 shows exemplary IL-2 variants expressed as fusion proteins to mouse serum albumin (MSA). [Figure 1B] FIG. 1 shows a representative gel filtration elution profile of MSA-IL-2 wild-type (WT) fusion protein. [Figure 1C] FIG. 1 shows a representative gel filtration elution profile of MSA-H9-superagonist IL-2 (Super 2) fusion protein. [Figure 1D] FIG. 1 shows a representative gel filtration elution profile of IL-2 mutant protein SEQ ID NO:3 (mutant seq-3). [Figure 1E] FIG. 1C shows a Coomassie stained SDS-PAGE gel of peak fractions 12 to 16 from gel filtration of MSA-H9-Super2 fusion protein, and FIG. 1B shows peak fractions 12 to 16 from gel filtration of MSA-IL-2WT. [Figure 1F] FIG. 1C shows a Coomassie stained SDS PAGE gel of peak fractions 12-16 from gel filtration of IL-2 mutant protein SEQ ID NO:3 (mutant seq3) of FIG. 1D. [Figure 2A]FIG. 1 illustrates design principle I showing the core design of four subsections that can potentially be improved, showing the packing between helix 1 and loop 3 of IL-2. [Figure 2B] FIG. 1 illustrates Design Principle I showing a core design of four subsections that can potentially be improved, showing the packing between helix 2 and helix 3 of IL-2. [Figure 2C] FIG. 1 illustrates Design Principle I showing a core design of four subsections that can potentially be improved, showing the packing between helix 1 and helix 2 of IL-2. [Figure 2D] FIG. 1 illustrates Design Principle I showing a four subsection core design that can potentially be improved, showing the packing between helix 1 and helix 3. [Figure 3A] FIG. 1 illustrates design principle II showing restructuring and stabilizing mutations of loop 3. A shortened, idealized loop 3 showing the two clusters that were experimentally tested. [Figure 3B] FIG. 1 illustrates design principle II showing restructuring and stabilizing mutations of loop 3, resampling and attempts to improve the native IL-2 loop by design. [Figure 4] 1 is a graph showing normalized thermal melting curves of selected in silico designed IL-2 variants monitored by circular dichroism. The abbreviations used are: wild type IL-2 (WT), mutant IL-2 protein of SEQ ID NO: 1 (seq1), mutant IL-2 protein of SEQ ID NO: 14 (seq14), mutant IL-2 protein of SEQ ID NO: 15 (seq15), mutant IL-2 protein of SEQ ID NO: 16 (seq16), mutant IL-2 protein of SEQ ID NO: 17 (seq17), mutant IL-2 protein of SEQ ID NO: 6 (seq6), mutant IL-2 protein of SEQ ID NO: 7 (seq7), mutant IL-2 protein of SEQ ID NO: 20 (Super2). [Figure 5A]FIG. 1 shows YT-1 CD25− cells, IL-2 H9 superagonist IL-2 (Super 2)-MSA fusion protein, and IL-2 WT-MSA fusion protein, as well as an exemplary in silico designed IL-2 mutant-MSA fusion protein. [Figure 5B] FIG. 1 is a graph showing dose response curves of STAT5 phosphorylation in CD25-YT-1 cells treated with in silico designed IL-2 mutant MSA fusion proteins. [Figure 6A] FIG. 1 shows YT-1 CD25+ cells, IL-2-H9 superagonist IL-2 (Super 2)-MSA fusion protein, and IL-2 WT-MSA fusion protein, as well as an exemplary in silico designed IL-2 mutant-MSA fusion protein. [Figure 6B] FIG. 1 is a graph showing dose response curves of STAT5 phosphorylation in CD25+ YT-1 cells treated with in silico designed IL-2 mutant MSA fusion proteins. [Figure 7A] FIG. 13 is a graph showing dose response curves of STAT5 phosphorylation in CD25-YT-1 cells treated with a selection of in silico designed IL-2 mutant MSA fusion proteins. [Figure 7B] FIG. 13 is a graph showing dose response curves of STAT5 phosphorylation in CD25+ YT-1 cells treated with a selection of in silico designed IL-2 mutant MSA fusion proteins. [Figure 8-1]Figure 1. Comparison of the EC50 values ​​of STAT 5 phosphorylation in CD25+ and CD25- YT-1 cells with WT IL-2 and the corresponding EC50 values ​​of STAT 5 phosphorylation in CD25+ and CD25- YT-1 cells with the designed IL-2 variants (Super2: variant IL-2 protein of SEQ ID NO:20, Seq1: variant IL-2 protein of SEQ ID NO:1, Seq2: variant IL-2 protein of SEQ ID NO:2, Seq3: variant IL-2 protein of SEQ ID NO:3, Seq4: variant IL-2 protein of SEQ ID NO:4, Seq5: variant IL-2 protein of SEQ ID NO:5, Seq6: variant IL-2 protein of SEQ ID NO:6, Seq7: variant IL-2 protein of SEQ ID NO:7, Seq8: variant IL-2 protein of SEQ ID NO:8, Seq9: variant IL-2 protein of SEQ ID NO:9). 15: mutant IL-2 protein of SEQ ID NO: 15; Seq16: mutant IL-2 protein of SEQ ID NO: 16; Seq17: mutant IL-2 protein of SEQ ID NO: 17). [Figure 8-2]Figure 1. Comparison of the EC50 values ​​of STAT 5 phosphorylation in CD25+ and CD25- YT-1 cells with WT IL-2 and the corresponding EC50 values ​​of STAT 5 phosphorylation in CD25+ and CD25- YT-1 cells with the designed IL-2 variants (Super2: variant IL-2 protein of SEQ ID NO:20, Seq1: variant IL-2 protein of SEQ ID NO:1, Seq2: variant IL-2 protein of SEQ ID NO:2, Seq3: variant IL-2 protein of SEQ ID NO:3, Seq4: variant IL-2 protein of SEQ ID NO:4, Seq5: variant IL-2 protein of SEQ ID NO:5, Seq6: variant IL-2 protein of SEQ ID NO:6, Seq7: variant IL-2 protein of SEQ ID NO:7, Seq8: variant IL-2 protein of SEQ ID NO:8, Seq9: variant IL-2 protein of SEQ ID NO:9). 15: mutant IL-2 protein of SEQ ID NO: 15; Seq16: mutant IL-2 protein of SEQ ID NO: 16; Seq17: mutant IL-2 protein of SEQ ID NO: 17). [Figure 9-1] FIG. 1 shows the amino acid sequences of in silico designed IL-2 mutants (SEQ ID NO: 1 to SEQ ID NO: 17), wild-type IL-2 (SEQ ID NO: 19), and Super-2 (SEQ ID NO: 20). [Figure 9-2] FIG. 1 shows the amino acid sequences of in silico designed IL-2 mutants (SEQ ID NO: 1 to SEQ ID NO: 17), wild-type IL-2 (SEQ ID NO: 19), and Super-2 (SEQ ID NO: 20). [Figure 10A] FIG. 1 shows the amino acid sequences of WT IL-2 (SEQ ID NO: 19), Super2 (SEQ ID NO: 20), and the in silico designed IL-2 variants of SEQ ID NO: 15 (SEQ ID NO: 15) and SEQ ID NO: 16 (SEQ ID NO: 16). [Figure 10B]1 is a graph showing amino acid substitutions in IL-2 variants SEQ ID NO: 15 (Seq15) and SEQ ID NO: 16 (Seq16) compared to WT IL-2 and the corresponding EC50 values ​​for STAT5 phosphorylation in CD25-YT-1 cells and CD25+YT-1 cells. [Figure 10C] 1 is a graph showing amino acid substitutions in Super-2 (SEQ ID NO: 20) compared to WT IL-2 and the corresponding EC50 values ​​for STAT5 phosphorylation in CD25- and CD25+ YT-1 cells. [Figure 11A] 1 is a chart showing the amino acid sequences of WT IL-2 (SEQ ID NO: 19), the in silico designed IL-2 variant SEQ ID NO: 15 (Seq15), and the IL-2 variant SEQ ID NO: 15 (A69 and I115 were reverted back to WT valine residues) (Seq15 rev), and the corresponding EC50 values ​​for STAT5 phosphorylation in CD25-YT-1 cells. [Figure 11B] 1 is a chart showing the amino acid sequences of WT IL-2 (SEQ ID NO: 19), in silico designed IL-2 variant SEQ ID NO: 1 (Seq1), and IL-2 variant SEQ ID NO: 1 (A69 and I115 were reverted back to WT valine residues) (Seq1 rev), and the corresponding EC50 values ​​for STAT5 phosphorylation in CD25-YT-1 cells. [Figure 11C] 13 is a graph showing dose response curves for STAT5 phosphorylation in CD25-YT-1 cells treated with MSA-IL-2 variant SEQ ID NO:1 (MSA-IL2 seq1), MSA-IL-2 variant SEQ ID NO:1 (A69 and I115 have been changed back to WT valine residues (MSA-IL-2 seq1 rev)), MSA-IL-2 variant SEQ ID NO:15 (MSA-IL2 seq15), MSA-IL-2 variant SEQ ID NO:15 (A69 and I115 have been changed back to WT valine residues (MSA-IL2 seq15 rev)), and MSA-IL-2 WT. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0070] In order to make this disclosure more readily understandable, certain terms and phrases are defined below and throughout the specification.

[0071] All references cited herein are incorporated by reference in their entirety as if fully set forth. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., J.Wiley&Sons (New York, NY 2001), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5th ed., J.Wiley&Sons (New York, NY 2001), and Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring harbor Laboratory Press (Cold Spring Harbor, NY 2001) provide those skilled in the art with a general guide to many of the terms used in this disclosure. Where appropriate, procedures involving the use of commercially available kits and reagents are generally carried out according to manufacturer-defined protocols and / or parameters unless otherwise specified.

[0072] As used herein, "IL-2" refers to wild-type IL-2, whether natural or recombinant. Mature human IL-2 occurs as a 133 amino acid sequence (minus a signal peptide of an additional 20 N-terminal amino acids) as described in Fujita, et. al., PNAS USA, 80, 7437-7441 (1983). The amino acid sequence of human IL-2 (SEQ ID NO: 18) is found in Genbank under accession locator NP_000577.2. The amino acid sequence of mature human IL-2 is shown in SEQ ID NO: 19. SEQ ID NO:18 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT SEQ ID NO:19 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0073] As used herein, "IL-2 variant" refers to a polypeptide in which specific substitutions have been made to the interleukin-2 protein. For example, FIG. 8 lists 17 IL-2 variant sequences. Their corresponding relative binding affinities to IL-2Rβ are shown in Table 1. IL-2 variants also have substitutions at other residues of the native IL-2 polypeptide chain. The variants may be characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites of the group or at other residues. According to the present disclosure, any such insertions, deletions, substitutions, and modifications result in IL-2 variants that retain the biological activity of IL-2. Exemplary variants may include substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.

[0074] The variants also include conservative modifications and substitutions at other positions of IL-2 (i.e., positions that have minimal effect on the secondary or tertiary structure of the variant). Such conservative substitutions include those described by Dayhoff in The Atlas of Protein Sequence and Structure 5 (1978) and Argos in EMBO J., 8:779-785 (1989). For example, amino acid substitutions falling within one of the following groups represent conservative changes: Group I: ala, pro, gly, gln, asn, ser, thr; Group II: cys, ser, tyr, thr; Group III: val, ile, leu, met, ala, phe; Group IV: lys, arg, his; Group V: phe, tyr, trp, his; and Group VI: asp, glu.

[0075] "Numbered according to IL-2" means that the selected amino acid is identified by reference to the position at which the amino acid normally occurs in the mature sequence of wild-type IL-2, e.g., G27 refers to the 27th amino acid that occurs in SEQ ID NO:19.

[0076] The term "cell type having an IL-2Rαβγ receptor" refers to cells known to have this receptor type, i.e., T cells, activated T cells, B cells, activated monocytes, and activated NK cells. The term "cell type having an IL-2Rβγ receptor" refers to cells known to have that receptor type, i.e., B cells, resting monocytes, and resting NK cells.

[0077] The term "identity" as used herein with respect to polypeptide or DNA sequences refers to the subunit sequence identity between two molecules. If a subunit position in both molecules is occupied by the same monomeric subunit (i.e., the same amino acid residue or nucleotide), then the molecules are identical at that position. The similarity between two amino acid or two nucleotide sequences is a linear function of the number of identical positions. Generally, sequences are aligned to obtain the highest order match. If necessary, calculations can be performed using published techniques and widely available computer programs such as the GCS program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990). Sequence identity can be measured using sequence analysis software, such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, using default parameters.

[0078] The terms "polypeptide," "protein," or "peptide" refer to any chain of amino acid residues, regardless of its length or post-translational modification (e.g., glycosylation or phosphorylation). When mutant IL-2 polypeptides of the disclosure are "substantially pure," they can be polypeptides that contain at least about 60% by weight (dry weight) of the polypeptide of interest, e.g., a mutant IL-2 amino acid sequence. For example, the polypeptide can be at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% by weight. Purity can be measured by any appropriate standard method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0079] "Operably linked" is intended to mean that the nucleotide sequence of interest (i.e., the sequence encoding the IL-2 variant) is linked to a control sequence in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). "Control sequences" include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). See, e.g., Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.). Control sequences include those that direct constitutive expression of a nucleotide sequence in many host cell types and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific control sequences). Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression constructs of the invention can be introduced into host cells to thereby produce the human IL-2 variants disclosed herein, or to produce biologically active variants thereof.

[0080] The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur over time, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still within the scope of the terms as used herein.

[0081] As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection, particle gun, or electroporation.

[0082] As used herein, the term "pharmaceutically acceptable carrier" includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics) can also be incorporated into the compositions.

[0083] As used herein, the terms "cancer" (or "cancerous"), "hyperproliferative", and "neoplastic" may be used to refer to cells capable of autonomous growth (i.e., an abnormal stage or condition characterized by rapidly proliferating cell growth). Hyperproliferative and neoplastic disease stages may be classified as pathological stages (i.e., characterizing or constituting a disease stage) or they may be classified as non-pathological stages (i.e., as a deviation from a normal stage but not associated with a disease stage). These terms are meant to include all types of cancerous growth or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasion. "Pathological hyperproliferative" cells occur in disease stages characterized by malignant tumor growth. Examples of non-pathological hyperproliferative cells include the proliferation of cells associated with wound repair. The terms "cancer" or "neoplasm" are used to refer to malignant tumors of various organ systems, including those affecting the lung, breast, thyroid, lymphatic glands and tissues, gastrointestinal organs, and urinary tract, as well as adenocarcinomas, which are generally considered to include malignant tumors such as most colon, renal cell, prostate and / or testicular cancers, non-small cell carcinoma of the lung, small intestine, and esophageal cancer.

[0084] The term "carcinoma" is art-recognized and refers to malignant tumors of epithelial or endocrine tissues, including cancers of the respiratory system, gastrointestinal system, genitourinary system, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, and melanoma. "Adenocarcinoma" refers to carcinomas originating from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0085] As used herein, the term "hematopoietic neoplastic disorder" includes diseases involving hyperplastic / neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid, or erythroid lineages, or their precursors. Preferably, the disease arises from poorly differentiated acute leukemias (e.g., erythroblastic leukemia and acute megakaryoblastic leukemia). Further exemplary myeloid disorders include, but are not limited to, acute promyelocytic leukemia (APML), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol. / Hemotol. 11:267-97). Lymphoid malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL) (including B-lineage ALL and T-lineage ALL), chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL), and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphoma include, but are not limited to, non-Hodgkin's lymphoma and its variants, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease, and Reid-Stenberg's disease.

[0086] IL-2 mutant In various embodiments, the present disclosure provides IL-2 polypeptides, which may be substantially purified, but are not necessarily so, that perform one or more of the biological activities of IL-2 (e.g., stimulation of cell proliferation) and function as agonists of wild-type IL-2R. IL-2 has been characterized as a T cell growth factor that induces proliferation of antigen-activated T cells and stimulation of NK cells.

[0087] Exemplary variant IL-2 polypeptides include an amino acid sequence that is at least about 80% identical to SEQ ID NO: 19 and can bind to IL-2Rβ with greater affinity than the polypeptide represented by SEQ ID NO: 19 binds to IL-2Rβ. Other IL-2 variants have enhanced activity in stimulating signaling through CD122 in the absence of increased binding affinity. For example, variant IL-2 polypeptides can have at least one mutation (e.g., deletion, addition, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) compared to wild-type IL-2 and have greater potency for activating the IL-2 signaling pathway in cells lacking CD25 expression compared to wild-type IL-2. The EC50 of the superagonist on CD25- cells can be reduced by at least 2-fold compared to the wild-type protein, at least 3-fold compared to the wild-type protein, at least 4-fold compared to the wild-type protein, or at least 5-fold compared to the wild-type protein, for example, an EC50 of less than about 2 nM, less than about 1.5 nM, less than about 1 nM, less than about 0.5 nM, less than about 0.3 nM, less than about 0.25 nM, less than about 0.2 nM, less than about 0.15 nM, less than about 0.1 nM, or less than about 0.05 nM.

[0088] Exemplary variant IL-2 polypeptides can be at least about 50%, at least about 65%, at least about 70%, at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identical to wild-type IL-2. Mutations can consist of changes in the number or content of amino acid residues. For example, variant IL-2 can have a greater or lesser number of amino acid residues than wild-type IL-2. Alternatively, or in addition, exemplary variant polypeptides can be The polypeptide may contain a substitution or deletion of one or more amino acid residues present in wild-type IL-2.

[0089] By way of illustration, a polypeptide comprising an amino acid sequence that is at least 95% identical to a reference amino acid sequence of SEQ ID NO: 19 is a polypeptide comprising a sequence that is identical to the reference sequence, except that it comprises up to five modifications of the reference amino acids of SEQ ID NO: 19. For example, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These modifications of the reference sequence may occur at the amino (N-) or carboxy (C-) terminal positions of the reference amino acid sequence, or anywhere between these terminal positions, and may be interspersed either individually among the residues of the reference sequence, or in one or more contiguous groups within the reference sequence.

[0090] In some embodiments, the disclosure provides a mutant human IL-2 protein comprising one or more amino acid changes. In some embodiments, the amino acid changes comprise amino acid substitutions. In some embodiments, the amino acid changes comprise amino acid deletions.

[0091] The substituted amino acid residues may, but are not necessarily, conservative substitutions and typically include substitutions within the group of glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, and phenylalanine, tyrosine. These mutations may be in amino acid residues that contact IL-2Rβ and / or IL-2Rγ.

[0092] The IL-2 variants find use, for example, as IL-2 selective agonists and superagonists in applications where modulation of IL-2 signaling is desirable, for example, by upregulation of IL-2 signaling, for the treatment of cancer, for selective activation of IL-2 signaling in autoimmune diseases, etc. Nucleic acids encoding such IL-2 variants, methods of making such IL-2 variants, pharmaceutical compositions comprising such IL-2 variants, and methods of treatment using such IL-2 variants are also provided.

[0093] In some embodiments, the IL-2 variant comprises one or more amino acid substitutions that increase the potency of IL-2 in CD25- cells selected from amino acid positions 27, 28, 31, 32, 35, 39, 52, 69, 72, 74, 75, the region including residues 74-80, 85, 86, 92, and 115, numbered according to wild-type hIL-2.

[0094] In one embodiment, a minimal set of positions for substitutions that provide enhanced IL-2 potency in CD25 cells may include amino acid substitutions at positions 27, 28, 32, 72, and the region including residues 74-83. Positions 27 and 28, which have hydrophobic residues, contribute most significantly to structural integrity. Position 72 affects signaling (Q>L). Positions 32 (originally K), 81 (originally R), and 52 (originally E) are substituted with consensus helix cap residues D, N, S, or T.

[0095] Loop 3 of IL-2 can be wild type or replaced with a redesigned loop. While the native loop is only three residues long, the redesigned structure includes residues before and after the helix such that all of the wild type residue positions 72-84 are reconstructed. The redesigned loop 3 sequence shares the same structural feature at the third position, e.g., GDDPKTI. As shown in the alignment of SEQ ID NOs: 1-13, it is preferred to have the loop one or two amino acids shorter in length than the native region from residues 72-83.

[0096] In some embodiments, the amino acid substitution at position 27 is a substitution to G or M. In some embodiments, the amino acid substitution at position 28 is a substitution to I or L. In embodiments, the amino acid substitution at position 31 is to A, L, M, or Y. In some embodiments, the amino acid substitution at position 39 is to L, I, or M. In some embodiments, the amino acid substitution at position 85 is to M, V, A, or L. In some embodiments, the amino acid substitution at position 86 is to I or V. In some embodiments, the amino acid substitution at position 92 is to F or I.

[0097] Reverting to the wild-type residues at positions 69 and 115 in the exemplary designed polypeptides was found to have no effect on activity, and therefore the sequences disclosed herein can have the wild-type sequences at those positions.

[0098] In some embodiments, the IL-2 variant comprises one or more amino acid changes selected from the group consisting of an amino acid substitution at position 27 to methionine (G27M), an amino acid substitution at position 28, an amino acid substitution at position 31 to alanine (Y31A), an amino acid substitution at position 32, an amino acid substitution at position 35 to serine (K35S), an amino acid substitution at position 39, an amino acid substitution at position 52, an amino acid deletion at position 82 (P82Δ), and an amino acid substitution at position 115 to isoleucine (V115I) compared to the amino acid sequence of a wild-type human IL-2 protein set forth in SEQ ID NO: 19.

[0099] In some embodiments, the IL-2 variant comprises one or more amino acid substitutions compared to wild-type human IL-2 selected from the group consisting of an amino acid substitution at position 27 to methionine (G27M), an amino acid substitution at position 28, an amino acid substitution at position 31 to alanine (Y31A), an amino acid substitution at position 32, an amino acid substitution at position 35 to serine (K35S), an amino acid substitution at position 39, an amino acid substitution at position 52, and an amino acid substitution at position 115 to isoleucine (V115I).

[0100] In some embodiments, the IL-2 variant comprises an amino acid deletion at position 81 (R81Δ) or a substitution at position 81.

[0101] In some embodiments, the mutant human IL-2 protein comprises one or more amino acid substitutions at positions selected from the group consisting of 69, 72, the region encompassing amino acid residues 74-80, 85, 86, and 92.

[0102] In some embodiments, the mutant human IL-2 protein comprises amino acid substitutions at positions 28, 32, 39, 52, 69, 72, 81, 85, 86, and 92.

[0103] In some embodiments, the amino acid substitution at position 28 comprises a substitution to leucine (I28L).

[0104] In some embodiments, the amino substitution at position 32 is selected from the group consisting of an aspartic acid substitution (K32D), a glutamic acid substitution (K32E), and a serine substitution (K32S).

[0105] In some embodiments, the amino acid substitution at position 39 is selected from the group consisting of a substitution to leucine (M39L) and a substitution to isoleucine (M39I).

[0106] In some embodiments, the amino acid substitution at position 52 is selected from the group consisting of a substitution to serine (E52S), a substitution to aspartic acid (E52D), a substitution to asparagine (E52N), and a substitution to threonine (E52T).

[0107] In some embodiments, the amino acid substitution at position 69 comprises a substitution to alanine (V69A).

[0108] In some embodiments, the amino acid substitution at position 72 is selected from the group consisting of an alanine substitution (L72A), a glutamine substitution (L72Q), an aspartic acid substitution (L72D), a histidine substitution (L72H), and a threonine substitution (L72T).

[0109] In some embodiments, the amino acid substitution at position 81 comprises a substitution to aspartic acid (R81D).

[0110] In some embodiments, the amino acid substitution at position 85 is selected from the group consisting of a substitution with valine (L85V), a substitution with methionine (L85M), and a substitution with alanine (L85A).

[0111] In some embodiments, the amino acid substitution at position 86 comprises a substitution to valine (I86V).

[0112] In some embodiments, the amino acid substitution at position 92 comprises a substitution to phenylalanine (I92F).

[0113] In some embodiments, the mutant human IL-2 protein comprises a substitution in a region encompassing amino acid residues 74 to 80. In some embodiments, the amino acid substitution in the region encompassing amino acid residues 74 to 80 comprises the amino acid sequence GDDPKTI, DSTDETV, DSTDERI, DSTDSRI, SKDQDKV, SKDQKKV, SDDQDKV, ADDKDTI, or ADDQDKI.

[0114] In some embodiments, the mutant human IL-2 protein comprises the amino acid changes G27M, I28L, K32D, M39L, E52S, a substitution in the region encompassing amino acid residues 74-80 with GDDPKTI, R81Δ, P82Δ, and V115I.

[0115] In some embodiments, the mutant human IL-2 protein comprises amino acid changes that include or consist of G27M, I28L, K32D, M39L, E52S, V69A, L72A, substitutions of the region encompassing amino acid residues 74-80 with GDDPKTI, R81Δ, P82Δ, L85V, and V115I.

[0116] In some embodiments, the mutant human IL-2 protein comprises amino acid changes that include or consist of G27M, I28L, K32D, M39L, V69A, L72A, a substitution of the region encompassing amino acid residues 74-80 with GDDPKTI, R81Δ, P82Δ, and V115I.

[0117] In some embodiments, the mutant human IL-2 protein comprises amino acid changes that include or consist of G27M, I28L, K32D, M39L, E52S, V69A, L72A, substitutions of the region encompassing amino acid residues 74-80 with GDDPKTI, R81Δ, P82Δ, and V115I.

[0118] In some embodiments, the mutant human IL-2 protein comprises amino acid changes that include or consist of G27M, I28L, K32D, M39L, E52S, V69A, L72Q, substitutions of the region encompassing amino acid residues 74-80 with GDDPKTI, R81Δ, P82Δ, and V115I.

[0119] In some embodiments, the mutant human IL-2 protein comprises amino acid changes that include or consist of G27M, I28L, K32D, M39L, E52S, V69A, L72D, substitutions of the region encompassing amino acid residues 74-80 with GDDPKTI, R81Δ, P82Δ, and V115I.

[0120] In some embodiments, the mutant human IL-2 protein comprises amino acid changes that include or consist of G27M, I28L, K32E, E52S, V69A, L72Q, substitutions of the region encompassing amino acid residues 74-80 with GDDPKTI, R81Δ, P82Δ, and V115I.

[0121] In some embodiments, the mutant human IL-2 protein comprises amino acid changes that include or consist of G27M, I28L, K32D, V69A, L72Q, a substitution of the region encompassing amino acid residues 74-80 with DSTDETV, R81Δ, P82Δ, and V115I.

[0122] In some embodiments, the mutant human IL-2 protein comprises amino acid changes that include or consist of G27M, I28L, K32D, M39L, E52S, V69A, L72A, a substitution in the region encompassing amino acid residues 74-80 with DSTDERI, R81Δ, P82Δ, L85V, I86V, I92F, and V115I.

[0123] In some embodiments, the mutant human IL-2 protein comprises amino acid changes that include or consist of G27M, I28L, K32D, M39L, E52S, V69A, L72Q, a substitution in the region encompassing amino acid residues 74-80 with DSTDSRI, R81Δ, P82Δ, and V115I.

[0124] In some embodiments, the mutant human IL-2 protein comprises amino acid changes that include or consist of substitutions in the region encompassing amino acid residues 74-80 with G27M, I28L, K32D, M39L, E52S, V69A, SKDQKKV, R81Δ, P82Δ, L85V, I86V, I92F, and V115I.

[0125] In some embodiments, the mutant human IL-2 protein comprises amino acid changes that include or consist of substitutions in the region encompassing amino acid residues 74-80 with G27M, I28L, K32S, M39L, E52S, V69A, SDDQDKV, R81Δ, P82Δ, L85V, I86V, I92F, and V115I.

[0126] In some embodiments, the mutant human IL-2 protein comprises amino acid changes that include or consist of G27M, I28L, K32D, V69A, L72H, substitutions in the region encompassing amino acid residues 74-80 with ADDKDTI, R81Δ, P82Δ, and V115I.

[0127] In some embodiments, the mutant human IL-2 protein comprises amino acid changes that include or consist of G27M, I28L, Y31A, K32S, K35S, V69A, L72T, substitutions of the region encompassing amino acid residues 74-80 with ADDQDKI, R81Δ, P82Δ, and V115I.

[0128] In some embodiments, the mutant human IL-2 protein comprises amino acid changes comprising or consisting of G27M, I28L, K32D, V69A, R81D, and V115I.

[0129] In some embodiments, the mutant human IL-2 protein comprises amino acid changes comprising or consisting of G27M, I28L, K32D, V69A, L72Q, R81D, and V115I.

[0130] In some embodiments, the mutant human IL-2 protein comprises amino acid changes comprising or consisting of G27M, I28L, K32D, M39L, E52S, V69A, R81D, and V115I.

[0131] In some embodiments, the mutant human IL-2 protein comprises amino acid changes comprising or consisting of G27M, I28L, K32D, M39L, E52S, V69A, L72Q, R81D, and V115I.

[0132] In some embodiments, the mutant human IL-2 protein comprises amino acid changes consisting of G27M, I28L, K32D, L72Q, and R81D.

[0133] In some embodiments, the IL-2 variant comprises one or more of the following amino acid changes: G27M, I28L, Y31A, K32D / E / S, K35S, M39L, E52S, V69A, L72A / Q / D / L / H / T, loop: GDDPKTI, DSTDETV, DSTDERI, SKDQDKV, ADDKDTI, ADDQDKI, AQSKNFHL, L80V / I, R81D, L85V, I86V, I92F, and V115I.

[0134] In one embodiment, an IL-2 variant having greater binding affinity for CD122 compared to wild-type human IL-2 comprises the amino acid changes G27M, I28L, K32D, M39L, E52S, V69A, L72A / Q / D / L / H / T, loop: GDDPKTI, L80I, R81Δ, P82Δ, L85V, I86V, I92F, and V115I (e.g., as shown in SEQ ID NO:1, or alternatively, the reverted SEQ ID NO:1 in which residues at positions 69 and 115 are changed back to wild-type valine residues).

[0135] In one embodiment, an IL-2 variant having greater binding affinity for CD122 compared to wild-type human IL-2 comprises the following amino acid changes: G27M, I28L, K32D, V69A, L72Q, loop: DSTDETV, L80V, R81Δ, P82Δ, and V115I (e.g., as shown in SEQ ID NO:7, or alternatively, reverted SEQ ID NO:7 in which residues 69 and 115 are reverted back to wild-type valine residues).

[0136] In one embodiment, an IL-2 variant having greater binding affinity for CD122 compared to wild-type human IL-2 comprises the following amino acid changes: G27M, I28L, K32D, M39L, E52S, V69A, L72A, loop: DSTDERI, L80I, R81Δ, P82Δ, L85V, I86V, I92F, and V115I (e.g., as shown in SEQ ID NO:8, or alternatively, reverted SEQ ID NO:8 in which residues 69 and 115 are reverted back to wild-type valine residues).

[0137] In some embodiments, the IL-2 variant comprises one or more of the following amino acid changes: G27M, I28L, K32E, E52S, V69A, L72Q, loop: GDDPKTI, L80I, R81Δ, P82Δ, and V115I (e.g., as shown in SEQ ID NO:6, or alternatively, reverted SEQ ID NO:6 in which residues 69 and 115 are changed back to wild-type valine residues).

[0138] In some embodiments, the IL-2 variants have greater potency for CD25- cells in the absence of greater binding affinity to CD122, and are characterized by the following: G27M, I28L, Y31A , K32D / E / S, K35S, M39L, E52S, V69A, L72A / Q / D / L / H / T, loop: AQSKNFHL, and V115I (e.g., as shown in SEQ ID NO:15, or alternatively, reverted SEQ ID NO:15 in which residues 69 and 115 are changed back to wild-type valine residues).

[0139] In some embodiments, the IL-2 variant has greater potency for CD25- cells in the absence of greater binding affinity to CD122 and includes one or more of the following amino acid changes: G27M, I28L, K32D, M39L, E52S, V69A, L72Q, loop: AQSKNFHL, and V115I (e.g., as shown in SEQ ID NO: 16, or alternatively, reverted SEQ ID NO: 16 in which residues at positions 69 and 115 are changed back to wild-type valine residues).

[0140] In some embodiments, the IL-2 variant has greater potency for CD25- cells in the absence of greater binding affinity for CD122 and includes one or more of the following amino acid changes: G27M, I28L, K32D, M39L, E52S, V69A, loop: AQSKNFHL, and V115I (e.g., as shown in SEQ ID NO: 17, or alternatively, reverted SEQ ID NO: 17 in which residues at positions 69 and 115 are reverted to wild-type valine residues).

[0141] An "agonist" is a compound that interacts with a target to cause or promote increased activation of the target.

[0142] A "partial agonist" is a compound that interacts with the same target as an agonist, but increasing dosages of the partial agonist do not produce as great a biochemical and / or physiological effect as an agonist.

[0143] A "super agonist," "super-agonist," or "superagonist" is a type of agonist that can produce a maximal response that is greater than the endogenous agonist of the target receptor, and thus has an efficacy greater than 100%.

[0144] An "antagonist" is a compound that opposes the action of an agonist, e.g., by preventing, reducing, inhibiting, or neutralizing the activity of the agonist. An "antagonist" can also prevent, inhibit, or reduce constitutive activity of a target, e.g., a target receptor, even in the absence of a specified agonist.

[0145] In certain embodiments, the IL-2 variants have an enhanced ability to stimulate one or more signaling pathways that depend on IL-2Rβ / IL-2Rγc heterodimerization. In some embodiments, the subject IL-2 variants have an enhanced ability to stimulate STAT5 phosphorylation in IL-2Rβ+ cells compared to wild-type hIL-2. In some embodiments, the IL-2 variants stimulate STAT5 phosphorylation in IL-2Rβ+ cells at levels 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater than the level at which wild-type IL-2 stimulates STAT5 phosphorylation in the same cells, and may increase STAT5 phosphorylation by 1-fold, 2-fold, 3-fold, 4-fold or greater. In some embodiments, the IL-2 variant stimulates STAT5 phosphorylation in IL-2Rβ+ cells to a level that is about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% greater than the level at which wild-type IL-2 stimulates STAT5 phosphorylation in the same cells. In some embodiments, STAT5 phosphorylation in IL-2Rβ+ cells is increased by about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, or about 100-fold. In some embodiments, the IL-2R+ cells are T cells. In certain embodiments, T cells are CD8+ T cells. In some embodiments, the CD8+ T cells are freshly isolated CD8+ T cells. In other embodiments, the CD8+ T cells are activated CD8+ T cells. In other embodiments, the IL-2Rβ+ cells are natural killer (NK) cells.

[0146] In some embodiments, the variants have an enhanced ability to stimulate ERK1 / ERK2 signaling in IL-2Rβ+ cells compared to wild-type hIL-2. In some embodiments, the IL-2 variants stimulate pERK1 / ERK2 signaling in IL-2Rβ+ cells at levels 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater than the level at which wild-type IL-2 stimulates pERK1 / ERK2 signaling in the same cells, and may increase pERK1 / ERK2 signaling by 1-fold, 2-fold, 3-fold, 4-fold or greater. In some embodiments, the IL-2 variant stimulates pERK1 / ERK2 signaling in IL-2Rβ+ cells at a level that is about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% greater than the level at which wild-type IL-2 stimulates pERK1 / ERK2 signaling in the same cells. In some embodiments, the IL-2 variant stimulates pERK1 / ERK2 signaling by about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, or about 100-fold. In some embodiments, the IL-2Rβ+ cells are T cells. In certain embodiments, the T cells are CD8+ T cells. In some embodiments, the CD8+ T cells are freshly isolated CD8+ T cells. In other embodiments, the CD8+ T cells are activated CD8+ T cells. In other embodiments, the IL-2Rβ+ cells are natural killer (NK) cells.

[0147] STAT5 and ERK / 2 signaling can be measured, for example, by phosphorylation of STAT5 and ERK1 / 2 using any suitable method known in the art. For example, STAT5 and ERK1 / 2 phosphorylation can be measured using antibodies specific for phosphorylated versions of these molecules in combination with flow cytometry analysis as described herein.

[0148] In certain embodiments, the IL-2 variant has enhanced ability to induce lymphocyte proliferation compared to wild-type IL-2. In some embodiments, the lymphocyte is a T cell. In certain embodiments, the lymphocyte is a primary CD8+ T cell. In other embodiments, the lymphocyte is an activated CD8+ T cell. Cell proliferation can be measured using any suitable method known in the art. For example, lymphocyte proliferation can be measured using the carboxyfluorescein diacetate succinimidyl diester (CFSE) dilution assay described herein or by [3H]-thymidine incorporation. In some embodiments, the IL-2 variant induces lymphocyte proliferation at 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the level at which wild-type IL-2 induces lymphocyte proliferation. In some embodiments, the IL-2 variant induces lymphocyte proliferation at a level that is about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% greater than the level at which wild-type IL-2 induces lymphocyte proliferation.

[0149] Recombinant expression of IL-2 variants, expression vectors and host cells In various embodiments, the polypeptides used in the practice of the invention are synthetic or produced by expression of recombinant nucleic acid molecules. The polypeptides may be chimeric (e.g., fusion proteins containing at least a mutant IL-2 polypeptide and a heterologous polypeptide). It may be encoded by a hybrid nucleic acid molecule containing one sequence encoding all or a portion of the variant IL-2 and a second sequence encoding all or a portion of a heterologous polypeptide. For example, the subject IL-2 variants described herein may be fused to a hexa-histidine tag to facilitate purification of bacterially expressed proteins, or to a hemagglutinin tag to facilitate purification of eukaryotically expressed proteins.

[0150] Methods for constructing DNA sequences encoding IL-2 variants and expressing those sequences in a suitable transformed host include, but are not limited to, using PCR-assisted mutagenesis. Mutations consisting of deletions or additions of amino acid residues to the IL-2 polypeptide can also be made by standard recombinant techniques. For deletions or additions, the nucleic acid molecule encoding IL-2 is optionally digested with an appropriate restriction endonuclease. The resulting fragment can either be expressed directly or further manipulated, for example, by ligating it to a second fragment. Ligation can be facilitated if both ends of the nucleic acid molecule contain complementary nucleotides that overlap each other, although blunt-ended fragments can also be ligated. PCR-generated nucleic acids can also be used to generate various variant sequences.

[0151] The complete amino acid sequence can be used to construct a reverse-translated gene. A DNA oligomer containing a nucleotide sequence encoding an IL-2 variant can be synthesized. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. Each oligonucleotide typically contains a 5' or 3' overhang for complementary assembly.

[0152] In addition to producing variant polypeptides through expression of nucleic acid molecules modified by recombinant molecular biology techniques, the subject IL-2 variants can be chemically synthesized. Chemically synthesized polypeptides are routinely produced by one of skill in the art.

[0153] Once assembled (by synthesis, site-directed mutagenesis, or otherwise), the DNA sequence encoding the IL-2 variant is inserted into an expression vector and operably linked to appropriate expression control sequences for expression of the IL-2 variant in the desired transformed host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operably linked to transcriptional and translational expression control sequences that are functional in the selected expression host.

[0154] The DNA sequence encoding the IL-2 variant, whether prepared by site-directed mutagenesis, chemical synthesis, or other methods, may also include a DNA sequence encoding a signal sequence. Such a signal sequence, if present, should be a sequence recognized by the cell selected for expression of the IL-2 variant. It may be prokaryotic, eukaryotic, or a combination of the two. It may be the signal sequence of native IL-2. The inclusion of a signal sequence depends on whether it is desired to secrete the IL-2 variant from the recombinant cell in which it is made. If the selected cell is a prokaryotic cell, it is generally preferred that the DNA sequence does not encode a signal sequence. If the selected cell is a eukaryotic cell, it is generally preferred that a signal sequence is encoded, most preferably using the wild-type IL-2 signal sequence.

[0155] IL-2 mutant fusion proteins As discussed above, exemplary subject IL-2 variants include those comprising a subject IL-2 variant and a heterologous polyclonal antibody. The heterologous polypeptides can be prepared as fusion or chimeric polypeptides comprising a peptide (i.e., a non-IL-2 polypeptide or variant thereof) (see, e.g., U.S. Pat. No. 6,451,308). Exemplary heterologous polypeptides can increase the circulating half-life of the chimeric polypeptide in vivo, thus further enhancing the properties of the variant IL-2 polypeptide. In various embodiments, the polypeptide that increases the circulating half-life can be a serum albumin, such as human serum albumin (HAS) or mouse serum albumin (MSA), or an Fc region of an IgG subclass of antibody lacking an IgG heavy chain variable region. An exemplary Fc region can contain mutations that inhibit complement fixation and Fc receptor binding, or it can be lytic, i.e., capable of binding complement or lysing cells via another mechanism, such as antibody-dependent complement lysis.

[0156] An "Fc region" can be a naturally occurring or synthetic polypeptide that is homologous to the IgG C-terminal domain produced by digesting IgG with papain. IgG Fc has a molecular weight of about 50 kDa. A variant IL-2 polypeptide can include the entire Fc region, or a smaller portion that retains the ability to extend the circulating half-life of the chimeric polypeptide of which it is a part. In addition, full-length or fragmented Fc regions can be variants of the wild-type molecule. That is, they can contain mutations that may or may not affect the function of the polypeptide, and native activity, as further described below, is not necessary or desired in all cases. In certain embodiments, an IL-2 variant fusion protein (e.g., an IL-2 partial agonist or antagonist described herein) comprises an IgG1, IgG2, IgG3, or IgG4 Fc region.

[0157] In some embodiments, the mutant human IL-2 protein is fused to a human IgG Fc domain. In some embodiments, the IgG Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc domain.

[0158] Fc regions can be "lytic" or "nonlytic", but are typically nonlytic. Nonlytic Fc regions typically lack high affinity Fc receptor binding sites and C1q binding sites. The high affinity Fc receptor binding site of mouse IgG Fc contains a Leu residue at position 235 of IgG Fc. Thus, the Fc receptor binding site can be disrupted by mutating or deleting Leu235. For example, substitution of Glu for Leu235 inhibits the ability of the Fc region to bind to the high affinity Fc receptor. The mouse C'1q binding site can be functionally disrupted by mutating or deleting Glu318, Lys320, and Lys322 residues of IgG. For example, substitution of Ala residues for Glu318, Lys320, and Lys322 renders IgG Fc unable to direct antibody-dependent complement lysis. In contrast, soluble IgG Fc regions have a high affinity Fc receptor binding site and a C'1q binding site. The high affinity Fc receptor binding site includes the Leu residue at position 235 of IgG Fc, and the C'1q binding site includes the Glu318, Lys320, and Lys322 residues of IgG1. Soluble IgG Fc has wild-type residues or conservative amino acid substitutions at these sites. Soluble IgG Fc can target cells for antibody-dependent cellular cytotoxicity or complement-directed cytolysis (CDC). Mutations suitable for human IgG are also known.

[0159] In other embodiments, the chimeric polypeptide can include a subject IL-2 variant and a polypeptide that functions as an antigen tag, such as a FLAG sequence, which is recognized by a highly specific anti-FLAG antibody that is biotinylated as described herein (see also Blanar et al., Science 256:1014, 1992; LeClair et al., Proc. Natl. Acad. Sci. USA 89:8145, 1992). In some embodiments, the chimeric polypeptide further comprises a C-terminal c-myc epitope tag.

[0160] In other embodiments, the chimeric polypeptide comprises a mutant IL-2 polypeptide and a heterologous polypeptide that functions to enhance expression of the mutant IL-2 polypeptide or direct the cellular localization of the mutant IL-2 polypeptide, such as the Aga2p agglutinin subunit (see, e.g., Boder and Wittrup, Nature Biotechnol. 15:553-7, 1997).

[0161] In other embodiments, chimeric polypeptides can be produced that include mutant IL-2 and an antibody or antigen-binding portion thereof. The antibody or antigen-binding component of the chimeric protein can serve as a targeting moiety. For example, it can be used to localize the chimeric protein to a specific subset of cells or target molecules. Methods for producing cytokine-antibody chimeric polypeptides are described, for example, in U.S. Patent No. 6,617,135.

[0162] Nucleic acid molecules encoding mutant IL-2 In some embodiments, the subject IL-2 variants can be obtained by expression of a nucleic acid molecule, such as those described above, either alone or as part of a chimeric polypeptide. Just as IL-2 variants can be described in terms of their identity to a wild-type IL-2 polypeptide, the nucleic acid molecules encoding them necessarily have a certain identity to the molecule encoding wild-type IL-2. In some embodiments, the nucleic acid molecule encoding a subject IL-2 variant can be at least 50%, at least 65%, at least 75%, at least 85%, or at least 95%, at least 99% identical to the nucleic acid encoding wild-type IL-2 (e.g., SEQ ID NO: 19).

[0163] The nucleic acid molecules provided can contain naturally occurring sequences or sequences that differ from naturally occurring sequences, but due to the degeneracy of the genetic code, encode the same polypeptide. These nucleic acid molecules can be composed of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, e.g., those produced by phosphoramidite-based synthesis), or combinations or modifications of nucleotides within these types of nucleic acids. In addition, the nucleic acid molecules can be double-stranded or single-stranded (i.e., either the sense or antisense strand).

[0164] Nucleic acid molecules are not limited to sequences that code for a polypeptide, but may also include some or all of the non-coding sequences located upstream or downstream of a coding sequence (e.g., the coding sequence for IL-2). Those skilled in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. These can be generated, for example, by treating genomic DNA with restriction endonucleases or by performing polymerase chain reaction (PCR). If the nucleic acid molecule is a ribonucleic acid (RNA), the molecule can be produced, for example, by in vitro transcription. In some embodiments, the present disclosure provides nucleic acids that code for the mutant human IL-2 proteins of the present disclosure.

[0165] Exemplary isolated nucleic acid molecules of the disclosure can include fragments not found in nature, etc. Thus, the disclosure encompasses recombinant molecules, such as molecules in which a nucleic acid sequence (e.g., a sequence encoding a mutant IL-2) is incorporated into a vector (e.g., a plasmid or viral vector) or into the genome of a heterologous cell (or the genome of a homologous cell, at a location other than the natural chromosomal location).

[0166] As discussed above, the subject IL-2 variants can be present as part of a chimeric polypeptide. In addition to, or in place of, the heterologous polypeptides discussed above, the subject nucleic acid molecules can contain sequences encoding a "marker" or "reporter." Marker or Reporter Examples of genes include f-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo.sup.r, G418.sup.r), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacz (encoding .beta.-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT). Those skilled in the art will recognize additional sequences that can function as additional useful reagents, e.g., markers or reporters.

[0167] The subject nucleic acid molecules can be obtained by introducing mutations into IL-2-encoding DNA obtained from any living cell, such as a mammalian cell. Thus, the subject nucleic acids (and the polypeptides they encode) can be from a mouse, rat, guinea pig, cow, sheep, horse, pig, rabbit, monkey, baboon, dog, or cat. In one embodiment, the nucleic acid molecule is human.

[0168] Expression of mutant IL-2 gene products The above-described nucleic acid molecules can be contained within vectors, for example, that are capable of directing their expression in cells that have been transduced with the vector. Thus, in addition to the subject IL-2 variants, expression vectors containing nucleic acid molecules encoding the subject IL-2 variants and cells transfected with these vectors are included among the preferred embodiments.

[0169] Of course, it should be understood that not all vectors and expression control sequences function equally well to express the DNA sequences described herein. Nor do all hosts function equally well in the same expression system. However, one skilled in the art may make a selection among these vectors, expression control sequences, and hosts without undue experimentation. For example, when selecting a vector, the host should be considered since the vector must replicate therein. The copy number of the vector, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered. For example, vectors that may be used include those that allow the DNA encoding the IL-2 variant to be amplified in copy number.

[0170] In some embodiments, the human IL-2 variants of the present disclosure are expressed from a vector, preferably an expression vector. The vectors are either useful for autonomous replication in a host cell or can be integrated into the genome of the host cell upon introduction into the host cell, thereby replicating together with the host genome (e.g., non-episomal mammalian vectors). Expression vectors are capable of directing the expression of coding sequences to which they are operably linked. In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids (vectors). However, other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), are also included.

[0171] Exemplary recombinant expression vectors can include one or more control sequences, selected on the basis of the host cell to be used for expression, operably linked to the nucleic acid sequence to be expressed.

[0172] Expression constructs or vectors can be designed for expression of the IL-2 variant or variants thereof in prokaryotic or eukaryotic host cells.

[0173] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells are described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, 1999). (Harbor Laboratory Press, Plainview, NY) and other standard molecular biology laboratory manuals.

[0174] In some embodiments, the disclosure provides a vector comprising a nucleic acid, the nucleic acid encoding a mutant human IL-2 protein disclosed herein.

[0175] In some embodiments, recombinant IL-2 variants or biologically active variants thereof may also be made in eukaryotes, such as yeast or human cells. Suitable eukaryotic host cells include insect cells (examples of baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39)), yeast cells (examples of vectors for expression in the yeast S. cerevisiae include pYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, CA) and the like). Suitable expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187:195)), or mammalian cells (mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187:195)). Suitable mammalian cells include Chinese hamster ovary cells (CHO) or COS cells. In mammalian cells, the control functions of the expression vector are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other expression systems suitable for both prokaryotic and eukaryotic cells, see Chapters 16 and 17 of Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY).See Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.).

[0176] The sequences encoding the human IL-2 variants of the present disclosure can be optimized for expression in a host cell of interest. The GC content of the sequence can be adjusted to the average level for a given cell host, calculated with reference to known genes expressed in the host cell. Methods for codon optimization are well known in the art. The codons in the IL-2 variant coding sequence can be optimized to enhance expression in the host cell, such that about 1%, about 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons in the coding sequence are optimized for expression in a particular host cell.

[0177] In some embodiments, the nucleic acid insert encoding the subject IL-2 variants in such vectors may be operably linked to a promoter selected, for example, based on the cell type in which expression is desired. In selecting an expression control sequence, various factors should also be considered. These include, for example, the relative strength of the sequence, its controllability, and compatibility with the actual DNA sequence encoding the subject IL-2 variants, particularly with respect to potential secondary structures. Hosts should be selected based on their compatibility with the selected vector, the toxicity of the products encoded by the DNA sequences of the invention, their secretion properties, their ability to correctly fold the polypeptide, their The DNA sequence should be selected having regard to the fermentation or culture requirements of the host, and the ease of purification of the product encoded by the DNA sequence.

[0178] In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, vectors can contain origins of replication and other genes that code for selection markers. For example, the neomycin resistance (neor) gene confers G418 resistance to cells in which it is expressed, thus allowing phenotypic selection of transfected cells. Those skilled in the art can readily determine whether a given regulatory element or selectable marker is suitable for use in a particular experimental context.

[0179] Viral vectors that can be used in the present invention include, for example, retrovirus, adenovirus and adeno-associated vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, e.g., Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, NY).

[0180] Prokaryotic or eukaryotic cells that contain and express a nucleic acid molecule encoding the IL-2 variants of the subject matter disclosed herein are also a feature of the invention. The cells of the invention are transfected cells, i.e., cells into which a nucleic acid molecule, e.g., a nucleic acid molecule encoding a variant IL-2 polypeptide, has been introduced by recombinant DNA techniques. The progeny of such cells are also considered to be within the scope of the invention.

[0181] The expressed polypeptides can be purified from the expression system using routine biochemical procedures and can be used, for example, as therapeutic agents as described herein.

[0182] In some embodiments, the resulting IL-2 variants are glycosylated or non-glycosylated depending on the host organism used to produce the variant. If bacteria are selected as the host, the IL-2 variants produced will be non-glycosylated. Eukaryotic cells, on the other hand, will glycosylate the IL-2 variants, but perhaps not in the same manner that native IL-2 is glycosylated. The IL-2 variants produced by the transformed host can be purified according to any suitable method. Various methods are known for purifying IL-2. See, for example, Current Protocols in Protein Science, Vol 2. Eds: John E. Coligan, Ben M. Dunn, Hidde L. Ploehg, David W. Speicher, Paul T. Wingfield, Unit 6.5 (Copyright 1997, John Wiley and Sons, Inc.). IL-2 variants can be isolated from inclusion bodies produced in E. coli, or from conditioned medium from either mammalian or yeast cultures producing a given variant using cation exchange, gel filtration, and / or reverse phase liquid chromatography.

[0183] The biological activity of the IL-2 variants can be assayed by any suitable method known in the art, including PHA blast proliferation and NK cell proliferation.

[0184] Treatment method In some embodiments, mutant IL-2 polypeptides and / or nucleic acids expressing them are administered to a subject to treat a proliferative or infectious disorder, such as cancer, chronic pathogenic infections, AIDS, etc. In treating such diseases, the disclosed IL-2 variants may have advantageous properties, such as increased efficacy, reduced toxicity, unexpected additive effects, and unexpected synergistic effects, and the combination of the variants with antibodies provides more than simple additive effects. Moreover, in certain embodiments where IL-2 variant treatment has been ineffective in treating aberrant apoptotic and proliferative / differentiative disorders, for example, a combination of an IL-2 variant with an anti-IL-2 antibody is effective.

[0185] Exemplary disorders include cancer (e.g., carcinoma, sarcoma, metastatic disorders, or hematopoietic neoplastic disorders such as leukemia). Metastatic tumors can arise from a number of primary tumor types, including, but not limited to, the carcinomas, hematopoietic malignancies, etc. described above. Compositions of the invention (e.g., mutant IL-2 polypeptides and / or nucleic acid molecules encoding them) can also be administered to patients with viral infections (e.g., AIDS or influenza).

[0186] Other examples of proliferative disorders include skin disorders. Skin disorders may involve abnormal activity of cells or groups of cells or layers in the skin, epidermis, or subcutaneous layers, or abnormalities in the dermal-epidermal junction. For example, skin disorders may involve abnormal activity of keratinocytes (e.g., hyperproliferative basal cells and immediate suprabasal keratinocytes), melanocytes, Langerhans cells, Merkel cells, immune cells, and other cells found in one or more of the epidermal layers, such as the basal layer (stratum germinativum), spinous layer, granular layer, lucid layer, or stratum corneum. In other embodiments, disorders may involve abnormal activity of skin cells, such as dermal endothelium, fibroblasts, immune cells (e.g., mast cells or macrophages), found in skin layers, such as the papillary layer or reticular layer. Examples of skin disorders include psoriasis, psoriatic arthritis, dermatitis (eczema), e.g., exfoliative dermatitis or atopic dermatitis, pityriasis rubra pilaris, pityriasis rosea, parapsoriasis, pityriasis lichenoidis, lichen planus, lichen nitidus, ichthyosis-like skin diseases, keratoderma, skin diseases, alopecia areata, pyoderma gangrenosum, vitiligo, pemphigoid (e.g., ophthalmic cicatricial pemphigoid or bullous pemphigoid), urticaria, porokeratosis, joint diseases involving hyperproliferation and inflammation of epithelium-associated cells lining the joint capsule. These include rheumatoid arthritis, dermatitis such as seborrheic dermatitis and photodermatitis, keratosis such as seborrheic keratosis, senile keratosis, actinic keratosis, photoinduced keratosis, and follicular keratosis, acne vulgaris, keloids and prophylaxis against keloid formation, nevus, warts including verruca, condyloma, or condyloma acuminata, and human papillomavirus (HPV) infections such as genital warts, leukoplakia, lichen planus, and keratitis. The skin disorder may be dermatitis, such as atopic dermatitis or allergic dermatitis, or psoriasis.

[0187] Patients suitable for treatment may also have psoriasis. The term "psoriasis" is intended to have its medical meaning, i.e., a disease that primarily affects the skin and results in raised, thickened, scaling, non-scarring lesions. The lesions are usually sharply demarcated erythematous papules covered with overlapping shiny scales. The scales are typically silvery or slightly milky white. Nail involvement is frequent, resulting in pitting, nail separation, thickening, and discoloration. Psoriasis may be associated with arthritis and may be severe. Hyperproliferation of keratinocytes, along with epidermal inflammation and reduced differentiation of keratinocytes, is a major feature of psoriatic epidermal hyperplasia. Multiple mechanisms have been invoked to explain the hyperproliferation of keratinocytes that characterizes psoriasis. Deregulated cellular immunity has also been implicated in the pathogenesis of psoriasis. Examples of psoriatic disorders include chronic constant psoriasis, plaque psoriasis, eruptive (guttate) psoriasis, erythrodermic psoriasis, generalized pustular psoriasis (Von Zumbusch type), circular pustular psoriasis, and localized pustular psoriasis.

[0188] In some embodiments, the disclosure provides a method of treating a disease in a subject in need thereof, the method comprising administering to the patient a mutant human IL-2 protein disclosed herein.

[0189] Alternatively, or in addition to administering directly to a patient, in some embodiments, the variant IL-2 polypeptides may be used in ex vivo methods, such as administering to cells (e.g., peripheral blood lymphocytes or purified lymphocytes isolated from a patient and placed or maintained in culture). The contacting step may include a step in which the cells are stimulated or treated with other agents, for example to stimulate proliferation or to expand the cell population that is responsive to the antigen of interest (e.g., a cancer antigen or a viral antigen). The cells are then administered to the patient after being treated.

[0190] In some embodiments, the present disclosure provides a method of treating a disease requiring expansion of T cells, the method comprising administering an effective amount of a mutant human IL-2 protein disclosed herein.

[0191] Pharmaceutical Compositions and Methods of Administration In some embodiments, the mutant IL-2 polypeptides and nucleic acids can be incorporated into compositions, including pharmaceutical compositions. Such compositions typically include a polypeptide or nucleic acid molecule and a pharma- ceutically acceptable carrier.

[0192] A pharmaceutical composition is formulated to be compatible with its intended route of administration. In one embodiment, the mutant IL-2 polypeptide is administered orally. In one embodiment, the mutant IL-2 polypeptide is administered via a parenteral route. Examples of parenteral routes of administration include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral applications may contain components of a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates, or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted (e.g., to a pH of about 7.2 to 7.8, e.g., 7.5) with an acid or base such as monobasic and / or dibasic sodium phosphate, hydrochloric acid, or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0193] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™, (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition should be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants, for example, sodium dodecyl sulfate. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include an isotonic agent in the composition, such as sugars, polyalcohols, such as mannitol, sorbitol, sodium chloride. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, such as aluminum monostearate and gelatin.

[0194] Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Dispersions can be prepared by the following method. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the other required ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which produces a powder of the active ingredient and any additional desired ingredients from its previously sterile-filtered solution.

[0195] Oral compositions, if used, generally include an inert diluent or edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of tablets, lozenges, or capsules, for example, gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. can contain binders such as microcrystalline cellulose, tragacanth gum, or gelatin, excipients such as starch or lactose, disintegrants such as alginic acid, Primogel™, or corn starch, lubricants such as magnesium stearate or Sterotes™, glidants such as colloidal silicon dioxide, sweeteners such as sucrose or saccharin, or flavoring ingredients such as peppermint, methyl salicylate, or orange flavor, or compounds of a similar nature.

[0196] In certain embodiments, for administration by inhalation, the mutant IL-2 polypeptides or nucleic acids encoding them may be delivered in the form of an aerosol spray from a pressured container or dispenser containing a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798. In other embodiments, inhalation is performed using pressurized, non-aerosol administration.

[0197] Systemic administration of variant IL-2 polypeptides or nucleic acids may also be by transmucosal or transdermal means. For transmucosal or nasal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compounds can be formulated into ointments, salves, gels, or creams, as generally known in the art.

[0198] In some embodiments, the disclosure provides a method of treating a disease in a subject in need thereof, the method comprising administering to the patient a pharmaceutical composition comprising a mutant human IL-2 protein disclosed herein.

[0199] In some embodiments, the present disclosure provides a method of treating a disease requiring T cell expansion, the method comprising administering an effective amount of a pharmaceutical composition comprising a mutant human IL-2 protein disclosed herein.

[0200] In some embodiments, the compounds (variant IL-2 polypeptides or nucleic acids) can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0201] In some embodiments, the compound (mutant IL-2 polypeptide or nucleic acid) may also be a compound that is capable of acting as a medicament for the treatment of various conditions, including, but not limited to, those described in McCaffrey et al. (Nature 41 8:6893, 2002), Xia et al. (Nature Biotechnol. 20:1006-1010, 2002), or Putnam (Am. J. Health Syst. Pharm. 53:151-160, 1996, erratum at Am. JH The antibodies can be administered by transfection or infection using methods known in the art, including those described in Health Syst. Pharm. 53:325, 1996.

[0202] In one embodiment, the variant IL-2 polypeptide or nucleic acid is prepared with a carrier that protects the variant IL-2 polypeptide against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations can be prepared using standard techniques. Materials can also be commercially obtained from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes that target infected cells with monoclonal antibodies against viral antigens) can also be used as pharma- ceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

[0203] Dosage, toxicity, and therapeutic efficacy of such mutant IL-2 polypeptides or nucleic acids can be determined according to standard pharmaceutical procedures in cell cultures or experimental animals, for example to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are preferred. Compounds that exhibit toxic side effects can be used, but care must be taken in designing delivery systems that target such compounds to the affected tissue site in order to minimize potential damage to non-infected cells, thereby reducing side effects.

[0204] Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that includes the ED50 with little or no toxicity. Dosages can vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods of the invention, a therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (e.g., the concentration of the test compound that achieves half-maximal inhibition of symptoms) determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0205] As defined herein, a therapeutically effective amount (e.g., effective dosage) of a mutant IL-2 polypeptide will depend on the polypeptide selected. For example, a single dose within the range of about 0.001-0.1 mg / kg of a mutant IL-2 polypeptide may be administered, and in some embodiments, about 0.005, 0.01, 0.05 mg / kg may be administered. In some embodiments, 600,000 IU / kg is administered (IU may be determined by lymphocyte proliferation bioassay and expressed in International Units (IU) established by the World Health Organization International Standard for Interleukin-2 (human)). Dosages may be similar to, but are expected to be less than, those prescribed for PROLEUKIN®. The compositions may be administered from once or more per day to once or more per week, including once every other day. One of ordinary skill in the art will appreciate that certain factors, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present, may influence the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of a mutant IL-2 polypeptide of the invention may include a single treatment or may include a series of treatments. In one embodiment, the composition is administered every 8 hours for 5 days, followed by a rest period of 2-14 days, e.g., 9 days, followed by administration every 8 hours. will be administered for an additional 5 days.

[0206] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0207] The following examples are provided to illustrate certain embodiments of the invention provided herein and should not be construed as limiting. EXAMPLES

[0208] Example 1: Design, expression, and purification of IL-2 mutant proteins Sequence variants were designed and tested with the goal of creating better versions of IL-2 with higher affinity for IL-2 Rbeta or higher potency in CD25-negative cells. Computational predictions and designed variants were used to test IL-2 signaling in YT-1(CD25-) and YT-1(CD25+) cells. IL-2 variants containing wild-type and H9 super2 regulatory sequences were cloned into Expi293 expression vectors and transiently expressed from Expi293 cells. IL-2 sequence variants were expressed as MSA fusion proteins and purified by SEC. The effect of the proteins on pSAT5 signaling was tested in YT-1(CD25-) and YT-1(CD25+) cells.

[0209] The design principle is based on the topology associated with IL-2, which suggests inherent flexibility in the molecule based on the observation that the connections between the helices are non-trivial loops with disulfide bonds, and non-idealities in the structural elements: loop arrangement, packing geometry, and helical cap. Since structural dynamics can be related to biological function, we theorized that by preferentially stabilizing IL-2 with the receptor, following the high-resolution crystal structure, we could convert wild-type IL-2 into a superagonist. The four regions highlighted are where design efforts were made to improve packing.

[0210] RosettaRemodel, a module of the Rosetta modeling software suite, was used to test the quality of the native structure packing. Core positions were subjected to redesign using the normal Rosetta force field (Ref 2015) and also under a softened repulsion parameter setting (soft_rep_design) to identify positions where potential improvements could be introduced. The resulting sequences run under the two different settings were analyzed and compared. Visual inspection clustered the changes into distinct regions, which are highlighted in Figure 2A, Figure 2B, Figure 2C, and Figure 2D.

[0211] The standard Rosetta force field is then used to generate a scaffold ensemble that allows the entire core to be redesigned while simultaneously monitoring residue changes in four regions. A subset of residues that improve packing was visually selected. The identity of the subset was then retained and step 3 was carried out iteratively until the sequence converged. The third loop was reconstructed, as it is mostly disordered in the wild-type apoprotein structure and may be involved in improved binding of the evolved super-2 molecule. These steps were repeated for the loop variants.

[0212] The third loop in the wild-type IL-2 crystal structure is partially disordered. We tried to stabilize it by redesigning a new sequence of the original length and, alternatively, to idealize the connection by shortening and completely reconstructing the loop without interrupting the position of the helix it bridges. As seen in Figure 3A and Figure 3B, loop 3 also serves as a conduit between the different regions of the core that we try to optimize. Reconstruction of loop 3 should have the most significant changes in the intrinsic structural dynamics of IL-2. The thermodynamics of the overall stability are likely determined by the inter-helical packing. The loop modifications are mainly due to the formation of a tetramer structure, which is called the tetramer structure. This should change the loading and structural dynamics.

[0213] As shown in Figure 1A, IL-2 variants were expressed as fusion proteins with mouse serum albumin (MSA) for half-life extension and recombinant expression from Expi-293 cells. Figures 1B, 1C, and 1D show representative gel filtration traces (superdex75) of MSA fusion proteins of human IL-2WT (Figure 1B), super2 (H9-super2) (Figure 1C), and the in silico IL-2 variant SEQ ID NO:3 (variant seq-3) (Figure 1D).

[0214] Normalized thermal melting curves of selected in silico designed variants monitored by circular dichroism are shown in Figure 4. WT IL-2 and H9 Super 2 (Super 2) show folding transitions at about 62°C, while other variants selected in silico designed variants are more thermally stable at about 5°C. All of the in silico designed variants improved thermal stability compared to wild type. SEQ ID NO: 6 was also included, but appears to have the opposite effect in CD25+ / -YT-1 cells to the remaining clones.

[0215] Example 2: CD25 - and CD25 + Stimulation of natural killer (YT-1 cells) The in silico designed IL-2 variants were tested for dose-response curves of STAT5 phosphorylation on CD25-YT-1 cells. For comparison, Super2 (H9) and WT IL-2 were included. Most of the in silico designed variants exhibit left-shifted dose-response curves indicating enhanced potency compared to WT IL-2, as shown in Figure 5B. 2x10 5 CD25+ or CD25- YT-1 cells were washed with FACS buffer and resuspended in 200 μL of FACS buffer at the indicated concentration of IL-2 variant per well of a 96-well plate. Cells were stimulated for 20 min at room temperature and then fixed by adding formaldehyde to 1.5% and incubated for 10 min. Cells were permeabilized with 100% ice-cold methanol for 20 min on ice, followed by incubation at -80°C overnight. Fixed and permeabilized cells were washed with excess FACS buffer and incubated for 20 min with 50 μL of Alexa647-conjugated anti-STAT5 pY694 (BD Biosciences, San Jose, CA) diluted 1:20 in FACS buffer. Cells were washed twice in FACS buffer and mean cell fluorescence was determined using the FL-4 channel of an Accuri C6 flow cytometer. Dose-response curves and EC50 values ​​were calculated in GraphPad Prism after subtraction of the mean cell fluorescence of unstimulated cells.

[0216] The most potent in silico designed IL-2 variants tested for dose-response curves of STAT5 phosphorylation on CD25+ and CD25- YT-1 cells are shown for comparison. Super2 (H9) and wt IL-2, shown in Figure 6B, are included for comparison. Super2 (also called H9) is an IL-2 variant with the following mutations: L80F, R81D, L85V, I86V, and I92F. The amino acid sequence of Super2 is shown by SEQ ID NO:20. Sequence number 20 (Super 2) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHFDPRDVVSNINVFVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0217] 2×10 5 CD25+ or CD25- YT-1 cells were washed with FACS buffer and resuspended in 200 μL of FACS buffer at the indicated concentrations of IL-2 variants per well of a 96-well plate. Cells were stimulated for 20 min at room temperature and then fixed by adding formaldehyde to 1.5% and incubated for 10 min. Cells were permeabilized with 100% ice-cold methanol for 20 min on ice, followed by incubation at -80°C overnight. Fixed, permeabilized cells were washed with excess FACS buffer and incubated for 20 min with 50 μL of Alexa647-conjugated anti-STAT5 pY694 (BD Biosciences, San Jose, CA) diluted 1:20 in FACS buffer. Cells were washed twice in FACS buffer and mean cell fluorescence was determined using the FL-4 channel of an Accuri C6 flow cytometer. Dose-response curves and EC50 values ​​were calculated in GraphPad Prism after subtracting the mean cell fluorescence of unstimulated cells.

[0218] FIG. 8 is a table showing sequence differences between IL-2, Super2, and in silico IL-2 variants. The EC50 values ​​for STAT5 phosphorylation on CD25+ and CD25-YT-1 cells are shown in the right column. FIG. 9 provides the sequences of all of the in silico designed IL-2 variants. FIG. 10A shows the amino acid sequences of WT IL-2 (SEQ ID NO: 19), Super2 (SEQ ID NO: 20), and the in silico designed IL-2 variants SEQ ID NO: 15 (SEQ ID NO: 15) and SEQ ID NO: 16 (SEQ ID NO: 16). FIG. 10B is a table showing sequences comparing substitutions in IL-2 variants SEQ ID NO: 15 (SEQ ID NO: 15) and SEQ ID NO: 16 (SEQ ID NO: 16) compared to WT IL-2 (SEQ ID NO: 19) and the corresponding EC50 values ​​for STAT5 phosphorylation in CD25-YT-1 cells and CD25+YT-1 cells. FIG. 10C is a table showing sequence substitutions in Super-2 (SEQ ID NO: 20) compared to WT IL-2 and the corresponding EC50 values ​​for STAT5 phosphorylation on CD25-YT-+1 and CD25+YT-1 cells.

[0219] Example 3: Binding Affinity of IL-2 Variants

[0220] [Table 1]

[0221] Biacore measurements were performed on each of the variants for binding to IL-2Rβ. Many of the designed variants have higher affinity for IL-2Rβ than WT IL-2, especially sequences 1, 7, and 8. Sequence 6 has lower affinity for IL-2Rβ but is highly selective for CD25. Sequences 15, 16, and 17 have similar binding affinity for IL-2Rβ as WT IL-2, but maintain very potent EC50 for CD25-YT-1 cells. Variants with enhanced potency for CD25-YT-1 cells include, for example, sequences 1, 7, 8, 11, 12, 13, 15, 16, and 17.

[0222] To perform surface plasmon resonance (SPR) analysis, test mutant proteins were generated in Expi293 as described previously. IL-2Rβ was biotinylated and immobilized on the chip, and IL-2 mutants were flowed over the chip. Experiments were performed on a BIAcore® T100 (GE Healthcare, Piscataway, NJ).

[0223] The specific IL-2 mutant residues were reverted back to the wild-type amino acids at positions 69 and 115 (A69V and I115V, respectively) numbered relative to wild-type IL2 (SEQ ID NO: 19).

[0224] Example 4: NK cytotoxicity assay The effect of in silico designed IL-2 variants on NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) is assessed using an endothelial growth factor receptor (EGFR)-expressing squamous cell tumor cell line (SCC6) and an EGFR-specific monoclonal antibody.

[0225] Primary NK cells were collected and isolated from healthy donors and cell purity was assessed. SCC6 target cells were cultured at 1 × 10 6 Label with 150 μCi 51Cr per cell for 2 hours. Lysis rates are measured after 5 hours of co-culture of NK cells at various effector:target cell ratios (e.g., 0:1, 1:1, 2:1, 5:1) with 51Cr-labeled SCC6 cells in medium alone or in medium containing IL-2, an in silico designed IL-2 variant of an EGFR-specific monoclonal antibody, or a combination thereof.

[0226] Example 5: Memory T cells and T regulatory (T reg ) Effects of IL-2 variants on cell populations The effect of the in silico designed IL-2 variants on T cell populations in vivo can be assessed in C57Bl / 6 mice receiving IL-2, the in silico designed IL-2 variants, or IL-2 / anti-IL-2 monoclonal antibody complexes. Spleen cell suspensions are prepared from the spleens of treated mice, and total numbers of splenic CD3+, CD4+, and CD44 high memory T cells are assessed by flow cytometry using standard techniques familiar to those of skill in the art.

[0227] Example 6: Evaluation of in vivo toxicity of IL-2 variants The in vivo toxicity of the in silico designed IL-2 variants can be compared to WT IL-2 in an acute pulmonary edema model. C57Bl / 6 mice are injected intraperitoneally daily for 5 days with IL-2, in silico designed IL-2 variants, or IL-2 / anti-IL-2 monoclonal antibody conjugates. Six days after the first treatment, lungs are removed and weighed before and after drying overnight at 58° C. under vacuum. Lung wet weight is calculated by subtracting the initial lung weight from the lung weight after dehydration.

[0228] Example 7: In vivo antitumor activity of IL-2 variants The antitumor activity of IL-2 variants can be tested in an in vivo mouse model. Bl6F10 melanoma cells are injected into the dorsal dermis of C57Bl / 6 mice. Tumor nodules grow to approximately 15 mm 2 After reaching a tumor size of 100 μg / mL, mice are injected daily for 5 days with IL-2, in silico designed IL-2 variants, or IL-2 / anti-IL-2 monoclonal antibody conjugates. After treatment, serum and tumor lysates are prepared and analyzed for standard tumor markers.

[0229] Although the present invention has been described with its detailed description, it should be understood that the foregoing description is intended to illustrate, but not to limit, the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. For example, although IL-2 is referred to throughout this specification, one of skill in the art will understand that the methods and compositions described herein are equally applicable to other cytokines, such as granulocyte-macrophage colony stimulating factor (GM-CSF), IL-2, IL-3, IL-5, IL-6, or IL-15, which have this property. Thus, the present invention also includes variants of GM-CSF, IL-2, IL-3, IL-5, IL-6, and IL-15 that have increased binding affinity to their respective receptors compared to the wild type, and methods for identifying and using these variants.

[0230] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 985,694, filed March 5, 2020, the entire disclosure of which is incorporated herein by reference in its entirety for all purposes. is incorporated herein.

[0231] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on Mar. 4, 2021, is named STAN-1701 WO Seq listing_ST25.txt, and is 24kb in size.

Claims

[Claim 1] 1. A mutant human IL-2 protein comprising one or more amino acid changes, The one or more amino acid changes are selected from the group consisting of an amino acid substitution at position 27 to methionine (G27M), an amino acid substitution at position 28, an amino acid substitution at position 31 to alanine (Y31A), an amino acid substitution at position 32, an amino acid substitution at position 35 to serine (K35S), an amino acid substitution at position 39, an amino acid substitution at position 52, an amino acid deletion at position 82 (P82Δ), and an amino acid substitution at position 115 to isoleucine (V115I), compared to the amino acid sequence of a wild-type human IL-2 protein represented by SEQ ID NO:19.

Images