Il-2 superantagonist constructs, methods and uses thereof

Abstract

A human interleukin-2 (IL-2) mutant protein or a variant thereof is provided. Also provided are pharmaceutical compositions comprising such IL-2 muteins.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 375,675, filed on September 14, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] Interleukin 2 (IL-2) is a multipotent cytokine produced primarily by activated CD4+ T cells that plays a vital role in generating a normal immune response. IL-2 promotes the proliferation and expansion of activated T lymphocytes, enhances B cell growth, and activates monocytes and natural killer cells. It is for these activities that IL-2 has been tested and approved for cancer treatment (aldesleukin, In eukaryotic cells, human IL-2 is synthesized as a 153 amino acid precursor polypeptide from which 20 amino acids are removed to produce mature secreted IL-2 (Taniguchi 1983). Recombinant human IL-2 has been produced in Escherichia coli (E. coli) (Rosenberg 1984), insect cells (Smith 1985), and mammalian COS cells (Taniguchi 1983).

[0004] Interleukin 2 (IL-2) is a type I cytokine with four alpha-helical bundles that was originally identified as a T cell growth factor (Morgan et al., Science 193:1007 (1976)) but was subsequently shown to have a wide range of effects. IL-2 promotes the differentiation of T helper cells (Zhu et al., Annual review of immunology 28:445 (2010); Liao et al., Nat Immunol 9:1288 (2008); and Liao et al., Nat Immunol 12:551 (2011)) and the development of regulatory T (Treg) cells (Cheng et al., Immunol Rev 241:63 (2011)), induces the killing activity of natural killer cells and lymphokine activation (Liao et al., Immunity 38:13 (2013)), and mediates activation-induced cell death (AICD) (Lenardo et al., Nature 353:858 (1991)).

[0005] IL-2 exerts its effects by interacting with three different receptors: interleukin 2 receptor alpha (IL-2Rα; CD25), interleukin 2 receptor beta (IL-2Rβ; CD122), and interleukin 2 receptor gamma (IL-2Rγ; CD132; common gamma chain). The first receptor to be identified was IL-2Rα, a 55 kD polypeptide (p55) that appears upon T cell activation and was originally called Tac (for T activation) antigen. IL-2Rα is expressed in approximately 10 -8 M of K d Binds to IL-2, and is also called the "low affinity" IL-2 receptor. IL-2 does not cause any detectable biological response when it binds to cells that only express IL-2Rα. In most cases, IL-2 acts through three different receptors: IL-2Rα, IL-2Rβ, and IL-2Rγ. Most cells, such as resting T cells, do not respond to IL-2 because they only express IL-2Rβ and IL-2Rγ with low affinity for IL-2. After stimulation, resting T cells express the IL-2 receptor IL-2Rα with relatively high affinity. IL-2 binds to IL-2Rα, causing the receptor to engage IL-2Rβ and IL-2Rγ in turn, thereby causing T cell activation. IL-2 "superkine" has been previously developed, which has a reinforcing effect due to the enhanced binding affinity to IL-2Rβ (Levin et al., Nature 484:529 (2012)).

[0006] Despite the wealth of knowledge about IL-2, including IL-2 superantagonists, there remains a need in the art for better therapies for treating cancer, including new therapies comprising the IL-2 muteins provided herein. Summary of the invention

[0007] IL-2 has a wide range of effects on the immune system and plays a crucial role in regulating immune activation and homeostasis and can be used to treat cancer.

[0008] The present invention provides an IL-2 mutant protein, which comprises amino acid substitutions L18R, Q22E and Q126T numbered according to wild-type human IL-2 (hIL-2) (SEQ ID NO: 8), and further comprises a group of amino acid substitutions selected from the group consisting of: F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T and S130R.

[0009] In some embodiments, the IL-2 mutant protein comprises amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, and S130R, optionally wherein the IL-2 mutant protein comprises the amino acid sequence of SEQ ID NO: 1.

[0010] In some embodiments, the IL-2 mutant protein comprises amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A and E62A, optionally wherein the IL-2 mutant protein comprises the amino acid sequence of SEQ ID NO:2.

[0011] In some embodiments, the IL-2 mutant protein comprises amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R and F42A, optionally wherein the IL-2 mutant protein comprises the amino acid sequence of SEQ ID NO:3.

[0012] In some embodiments, the IL-2 mutant protein comprises amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A and Y45A, optionally wherein the IL-2 mutant protein comprises the amino acid sequence of SEQ ID NO:4.

[0013] In some embodiments, the IL-2 mutant protein comprises amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F and Q126T, optionally wherein the IL-2 mutant protein comprises the amino acid sequence of SEQ ID NO:5.

[0014] In some embodiments, the IL-2 mutant protein comprises amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, F42A and E62A, optionally wherein the IL-2 mutant protein comprises the amino acid sequence of SEQ ID NO:6.

[0015] In some embodiments, the IL-2 mutant protein comprises amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T and F42A, optionally wherein the IL-2 mutant protein comprises the amino acid sequence of SEQ ID NO:7.

[0016] In some embodiments, the IL-2 mutein is fused to an albumin molecule, an Fc molecule, and / or another mutein, optionally wherein the other mutein is an IL-13 mutein or an IL-4 mutein.

[0017] In some embodiments, the IL-2 mutein is fused to an albumin molecule.

[0018] In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:18.

[0019] In some embodiments, the IL-2 mutein is fused to an Fc molecule.

[0020] In some embodiments, the IL-2 mutein is fused to an Fc molecule and an IL-13 mutein, optionally wherein the IL-13 mutein comprises amino acid substitutions L10V, V18I, D87S, T88S, L101F, K104R, and K105T (A11) numbered according to wild-type human IL-13 (hIL-13).

[0021] In some embodiments, the IL-2 mutant protein is fused to an Fc molecule and an IL-4 mutant protein, optionally wherein the IL-4 mutant protein comprises amino acid substitutions R121K, Y124F, and S125R (KFR) or K117R, T118V, R121Q, E122S, Y124W, S125F, S128G, and S129A (RGA) numbered according to wild-type human IL-4 (hIL-4).

[0022] In some embodiments, the fusion protein comprises the amino acid sequence of one of SEQ ID NOs: 19-22.

[0023] In some embodiments, the IL-2 mutein has increased binding to CD122 compared to wild-type IL-2.

[0024] In some embodiments, the IL-2 mutein has reduced binding to CD25 compared to wild-type IL-2.

[0025] In some embodiments, the IL-2 mutein has inhibitory activity as determined using a HEKBlue IL-2 and / or CTLL2 assay.

[0026] In some embodiments, the IL-2 mutein inhibits IL-2-induced pSTAT5 signaling in human PBMCs.

[0027] In some embodiments, the IL-2 mutein is non-toxic to mice as determined using a maximum tolerated dose (MTD) assay.

[0028] In some embodiments, the IL-2 mutein reduces disease score as determined using an experimental autoimmune encephalomyelitis (EAE) assay.

[0029] The present invention also provides nucleic acids encoding the IL-2 mutant proteins described herein.

[0030] The present invention also provides a vector comprising a nucleic acid described herein.

[0031] The invention also provides host cells comprising a nucleic acid or a vector described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 :Schematic diagram of MDNA209, an IL-2 / IL-15 antagonist with a unique mechanism of action

[0034] Figures 2A-2D : IL-2 antagonist construct binds to IL-2Rβγc complex ( Figure 2A ) and binding to human CD25 and CD122 ( Figure 2B -D) SPR sensorgram.

[0035] Figures 3A-3C .HEK-Blue TM Representative dose response curves of various MDNA209 antagonist molecules in the IL-2 reporter gene assay. Compounds were tested as agonists (A, C) and antagonists (B). Figure 3A In the IL-2 activated EC 80 Values ​​and background levels are shown as upper and lower dashed lines, respectively.

[0036] Figure 4 : Representative graph of the dose response of IL-2-Fc used alone or in the presence of 30 nM MDNA209-Fc in the CTLL2 proliferation assay. The four-parameter logistic curve fit is represented by the solid line. The error bars represent the standard error of the mean of duplicate wells.

[0037] Figure 5A-5B : At 67pM rhIL-2 ( Figure 5A ) or 670pM rhIL-2 ( Figure 5B ) Representative dose response curve of the percentage of pSTAT5 in response to antagonist in the presence of .

[0038] Figure 6 .Effects of MDNA209FEAA-Fc-A11 (30 mg / kg) and A11-Fc (21 mg / kg and 11 mg / kg) on ​​the body weight of C57BL / 6J mice.

[0039] Figure 7 EAE scores (mean ± SEM) of mice treated with PBS, MDNA209FA-Fc or MDNA209-Fc at peak disease. Mice were treated on days 8, 12 and 15 after immunization. * indicates significant difference (t-test, p = 0.016, n = 7, mean ± SEM).

[0040] Figure 8 .EAE scores and body weights of mice treated with PBS, MDNA209FEAA-Fc-A11 (20 mg / kg), or a combination of MDNA209-Fc and A11-Fc (15.15 mg / kg and 14.1 mg / kg). Mice were treated on days 8, 12, and 15 after immunization. Data are shown as mean ± SEM, and * indicates significant difference, p < 0.05, t-test. The combination of MDNA209-Fc and A11-Fc had n = 7 on days 6-10, n = 6 on day 12, and n = 1 after day 15; for MDNA209FEAA-Fc-A11, n = 7 on days 6-14 and n = 4 after day 15. The scores of dead mice were used continuously.

[0041] Fig. 9 . EAE scores of mice treated with PBS or MDNA209FEAA-Fc-A11 according to the treatment dose schedule. Mice received 20 mg / kg and 5 mg / kg treatment on days 12, 16 and 19 after immunization as shown (dashed lines). Data are shown as mean ± SEM, PBS treatment group n = 7 and MDNA209FEAA-Fc-A11 treatment group n = 5.

[0042] Fig.10 IFNγ levels in PBMCs treated with increasing concentrations of MDNA209-Fc in the presence of indicated concentrations of rhIL-2 for 48 hours were quantified by ELISA.

[0043] Fig.11 MDNA209-Fc induces a dose-dependent inhibition of IFNγ production in human PBMCs stimulated with 0.3 μg / ml rhIL-2 from four different donors (shown as a percentage of the maximum value).

[0044] Figures 12A-12D Body weight of animals receiving PBS or MDNA209-Fc ( Figures 12A-12B ) and total lymphocyte count ( Figures 12C-12D ).

[0045] Fig.13. MDNA209-Fc detection ELISA was performed on plasma collected 5 minutes, 24 hours or 72 hours after injection of MDNA-Fc (10 mg / kg and 20 mg / kg) (N=3 / group).

[0046] Fig.14 .MDNA209-Fc inhibits proliferation of PBMCs in MLR assay. Representative non-linear fitting curves of stimulation index (SI) of MDNA209-Fc, MDNA209-albumin and MDNA209FEAA-Fc plotted for the corresponding tested dose ranges.

[0047] Figures 15A-15B .Sensorgrams of IL-2 antagonists binding to human IL2Rα (CD25) and IL2Rβ (CD122). DETAILED DESCRIPTION

[0048] To make the present disclosure more readily understood, certain terms and phrases are defined below and throughout the specification.

[0049] definition

[0050] All references cited herein are incorporated by reference in their entirety as if fully incorporated herein. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd Edition, J. Wiley & Sons (New York, NY 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5th Edition, J. Wiley & Sons (New York, NY 2001); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3rd Edition, 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 including the use of commercially available kits and reagents are generally performed according to the protocols and / or parameters specified by the manufacturer unless otherwise stated.

[0051] As used herein, "IL-2" means wild-type IL-2, whether natural or recombinant. As described in Fujita et al., PNAS USA, 80, 7437-7441 (1983), mature human IL-2 is present as a sequence having 133 amino acids (minus the signal peptide, consisting of an additional 20 N-terminal amino acids). The amino acid sequence of human IL-2 (SEQ ID NO: 11; full length) is found in Genbank at accession locator NP_000577.2. The amino acid sequence of mature human IL-2 is depicted in SEQ ID NO: 8 (human wild-type mature sequence; the position numbering of substitutions is based on this sequence). The mouse (Mus musculus) IL-2 amino acid sequence is found in Genbank at accession locator (SEQ ID NO: 13). The amino acid sequence of mature mouse IL-2 is depicted in SEQ ID NO: 12.

[0052] SEQ ID NO:11

[0053] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0054] SEQ ID NO:8

[0055] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0056] SEQ ID NO:13

[0057] MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ

[0058] SEQ ID NO:12

[0059] APTSSSTSSSTAEAQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ

[0060] As used herein, "IL-2 mutant protein" means an IL-2 polypeptide in which specific substitutions have been made to the interleukin-2 protein. The IL-2 mutant protein is characterized by amino acid insertions, deletions, substitutions and modifications at one or more sites within or at other residues of the native IL-2 polypeptide chain. According to the present disclosure, any such insertions, deletions, substitutions and modifications produce IL-2 mutant proteins that retain IL-2Rβ binding activity. Exemplary mutant proteins may include substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids.

[0061] Mutants also include conservative modifications and substitutions at other positions of IL-2 (i.e., those that have minimal effect on the secondary or tertiary structure of the mutant protein). 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 acids belonging to one of the following categories represent conservative changes: Class I: ala, pro, gly, gln, asn, ser, thr; Class II: cys, ser, tyr, thr; Class III: val, ile, leu, met, ala, phe; Class IV: lys, arg, his; Class V: phe, tyr, trp, his; and Class VI: asp, glu.

[0062] "According to IL-2 numbering" 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, for example, R81 refers to the eighty-first amino acid occurring in SEQ ID NO:8, i.e., arginine. L80 refers to the eighty-first amino acid occurring in SEQ ID NO:8, i.e., leucine. L85 refers to the eighty-fifth amino acid occurring in SEQ ID NO:8, i.e., leucine. I86 refers to the eighty-sixth amino acid occurring in SEQ ID NO:8, i.e., isoleucine. I92 refers to the ninety-second amino acid occurring in SEQ ID NO:8, i.e., isoleucine. F42 refers to the forty-second amino acid occurring in SEQ ID NO:8, i.e., phenylalanine. K43 refers to the forty-third amino acid occurring in SEQ ID NO:8, i.e., lysine. L18 refers to the eighteenth amino acid occurring in SEQ ID NO:8, i.e., leucine. Q22 refers to the twenty-second amino acid occurring in SEQ ID NO:8, i.e., glutamine. Q126 refers to the 126th amino acid in SEQ ID NO:8, i.e., glutamine. S130 refers to the 130th amino acid in SEQ ID NO:8, i.e., serine. E62 refers to the 62nd amino acid in SEQ ID NO:8, i.e., glutamic acid. Y45 refers to the 45th amino acid in SEQ ID NO:8, i.e., tyrosine.

[0063] As used herein, the abbreviations for the genetically encoded L-enantiomeric amino acids used in the disclosed methods are conventional and are shown in Table 1 below.

[0064] Table 1: Amino Acid Abbreviations

[0065]

[0066]

[0067] "Hydrophilic amino acids" refer to amino acids that exhibit a hydrophobicity less than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol. 179: 125-142. Genetically encoded hydrophilic amino acids include Thr (T), Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K) and Arg (R).

[0068] The term "cell type with IL-2Rαβγ receptor" means 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 with IL-2Rβγ receptor" means cells known to have this receptor type, i.e., B cells, resting monocytes, and resting NK cells.

[0069] As used herein with respect to polypeptides or DNA sequences, the term "identity" refers to the subunit sequence identity between two molecules. When a subunit position in two molecules is occupied by the same monomer subunit (i.e., the same amino acid residue or nucleotide), the molecules are identical at that position. The similarity between two amino acid or two nucleotide sequences is a direct function of the number of identical positions. In general, sequences are aligned so as to obtain the highest order match. If necessary, publicly available techniques and widely available computer programs such as the GCS package (Devereux et al., Nucleic Acids Res. 12: 387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J. Molecular Biol. 215: 403, 1990) can be used to calculate identity. 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) with its default parameters.

[0070] The terms "polypeptide," "protein," or "peptide" refer to any chain of amino acid residues, regardless of its length or post-translational modifications (eg, glycosylation or phosphorylation).

[0071] If the mutant IL-2 polypeptides of the present disclosure are "substantially pure," they can be at least about 60% by weight (dry weight) of the polypeptide of interest (e.g., a polypeptide containing 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 of the polypeptide of interest. Purity can be measured by any suitable standard method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0072] An "agonist" is a compound that interacts with a target to cause or promote increased activation of the target.

[0073] A "partial agonist" is a compound that interacts with the same target as an agonist, but does not produce the same magnitude of biochemical and / or physiological effects as the agonist, even at increasing doses.

[0074] A "superagonist" (also called a "super factor") is an agonist that is capable of producing a maximal response that is greater than the endogenous agonist of the target receptor and therefore has an efficacy exceeding 100%.

[0075] "Operably linked" is intended to mean that the target nucleotide sequence (i.e., the sequence encoding the IL-2 mutant protein) is linked to the regulatory sequence in a manner that allows the nucleotide sequence to be expressed (e.g., expressed in an in vitro transcription / translation system or expressed in a host cell when the vector is introduced into the host cell). "Regulatory sequences" include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). See, for example, Goeddel (1990), Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.). Regulatory sequences include those that direct constitutive expression of nucleotide sequences in many types of host cells and regulatory sequences that direct expression of nucleotide sequences only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will recognize that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the expression level of the desired protein, and the like. The expression construct of the present invention can be introduced into a host cell to produce the human IL-2 mutant protein disclosed herein or to produce its biologically active variant.

[0076] The terms "host cell" and "recombinant host cell" are used interchangeably herein. It should be 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 in the next generation due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0077] As used herein, the terms "transformation" and "transfection" refer to various techniques recognized in the art for introducing exogenous nucleic acid (e.g., DNA) into host cells, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, particle gun or electroporation.

[0078] 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., compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics) can also be incorporated into the composition.

[0079] As used herein, the terms "cancer" (or "cancerous"), "hyperproliferative" and "neoplastic" refer to cells with autonomous growth capacity (i.e., an abnormal state or condition characterized by rapidly proliferative cell growth). Hyperproliferative and neoplastic disease states can be classified as pathological (i.e., characterizing or constituting a disease state), or they can be classified as non-pathological (i.e., a deviation from the normal state but not associated with a disease state). These terms are intended to include all types of cancerous growths or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or invasive stage. "Pathological hyperproliferative" cells occur in disease states characterized by malignant tumor growth. Examples of non-pathological hyperproliferative cells include cell proliferation associated with wound repair. The term "cancer" or "neoplasm" is used to refer to malignancies of various organ systems, including those affecting the lungs, breasts, thyroid glands, lymph glands and lymphatic tissues, reproductive system, gastrointestinal organs, and genitourinary tract, and to adenocarcinomas, which are generally considered to include malignancies such as most colon cancers, renal cell carcinomas, prostate cancers and / or testicular tumors, non-small cell lung cancers, small intestine cancers, and esophageal cancers. Cancers generally may include prostate cancer, ovarian cancer, breast cancer, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer including small cell lung cancer, kidney cancer, colorectal cancer, pancreatic cancer, stomach cancer, and brain cancer.

[0080] The term "carcinoma" is art-recognized and refers to malignancies of epithelial or endocrine tissue, including cancers of the respiratory system, gastrointestinal system, genitourinary system, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, and melanoma. "Adenocarcinoma" refers to a cancer that originates from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0081] As used herein, the term "hematopoietic neoplastic disorder" refers to a disease involving hyperplastic / neoplastic cells of hematopoietic origin, such as a disease caused by a bone marrow, lymphoid or erythroid cell lineage or its precursor cell. Preferably, the disease is caused by a poorly differentiated acute leukemia (e.g., erythroblastic leukemia and acute megakaryoblastic leukemia). Other exemplary bone marrow disorders include, but are not limited to, acute promyelocytic leukemia (APML), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) (reviewed in Aickus, L. (1991) Crit Rev. in Oncol. / Hemotol. 11: 267-97); lymphoid malignancies, including but 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). Other forms of malignant lymphoma include, but are not limited to, non-Hodgkin 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 Reed-Stemberg disease.

[0082] As used herein, the term "treatment" and the like refers to obtaining a desired pharmacological and / or physiological effect. This effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. As used herein, "treatment" covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject susceptible to the disease or at risk of acquiring the disease but not yet diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its development; and (c) alleviating the disease, i.e., causing the disease to regress. A therapeutically effective amount may be an amount that reduces the number of tumors, reduces the size of tumors, and / or increases survival.

[0083] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to mammals, including but not limited to humans and non-human primates, including monkeys and humans; mammalian sport animals (e.g., horses); mammalian livestock (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0084] The terms "pharmaceutically acceptable" and "physiologically acceptable" mean a biologically acceptable formulation, its gaseous, liquid or solid state, or a mixture thereof, suitable for one or more routes of administration, in vivo delivery or contact. A "pharmaceutically acceptable" or "physiologically acceptable" composition is a material that is not biologically or otherwise undesirable, for example, the material can be administered to a subject without causing substantial undesirable biological effects. Therefore, such pharmaceutical compositions can be used, for example, to administer IL-2 mutant proteins to a subject. In some embodiments, the administered IL-2 mutant protein further comprises a substitution at position F42A. In some embodiments, the administered IL-2 mutant protein further comprises a substitution at position K43N.

[0085] As used herein, the phrase "unit dosage form" refers to a physically discrete unit suitable as a unit dose for a subject to be treated; each unit contains a predetermined amount, optionally in combination with a pharmaceutical carrier (excipient, diluent, vehicle, or filler), which, when administered in one or more doses, produces a desired effect (e.g., a prophylactic or therapeutic effect). In some embodiments, the therapeutic effect is a reduction in the number of tumors. In some embodiments, the therapeutic effect is a reduction in tumor size. In some embodiments, the therapeutic effect is an increase in survival.

[0086] In some embodiments, the unit dosage form can be in, for example, ampoules and vials, including liquid compositions, or compositions in a freeze-dried or lyophilized state; for example, a sterile liquid carrier can be added prior to administration or in vivo delivery. Single unit dosage forms can be included in a multi-dose kit or container. IL-2 mutant proteins and pharmaceutical compositions thereof can be packaged in single or multiple unit dosage forms for ease of administration and uniformity of dosage.

[0087] A "therapeutically effective amount" will fall within a relatively broad range that can be determined experimentally and / or clinically. For example, for in vivo injection, for example, directly into a subject's tissue or vascular system (e.g., liver tissue or vein). Other effective doses can be readily determined by one of ordinary skill in the art through routine experiments establishing dose-response curves.

[0088] An "effective amount" or "sufficient amount" refers to an amount that provides a detectable response of any duration (long or short), any measurable or detectable degree to a subject, or an expected or desired result or benefit of any duration (e.g., minutes, hours, days, months, years, or a cure), either alone or in combination with one or more other compositions (therapeutics, such as drugs), treatments, regimens, or therapeutic regimen agents (including, for example, vaccine regimens), in single or multiple doses.

[0089] An "effective amount" or "sufficient amount" for treatment (e.g., to improve or provide a therapeutic benefit or improvement) is generally effective to provide a response to one, multiple or all adverse symptoms, consequences or complications of the disease, one or more adverse symptoms, conditions, illnesses, pathologies or complications caused by or associated with the disease to a measurable extent, but reducing, reducing, inhibiting, suppressing, limiting or controlling disease progression or deterioration is also a satisfactory result. In some embodiments, an effective amount is an amount sufficient to reduce the number of tumors. In some embodiments, an effective amount is an amount sufficient to reduce the size of a tumor. In some embodiments, an effective amount is an amount sufficient to increase survival.

[0090] "Prevention" and its grammatical variants mean a method of contacting, administering or delivering in vivo to a subject before a disease. Administration or in vivo delivery to a subject can be performed before the development of adverse symptoms, symptoms, complications, etc. caused by or associated with the disease. For example, screening (e.g., genetic screening) can be used to identify such subjects as candidates for the methods and uses, but the subject may not show the disease. Therefore, such subjects include those who screen positive for insufficient or lacking amounts of functional gene products (proteins), or for producing abnormal, partially functional or non-functional gene products (proteins) that cause the disease; and subjects who screen positive for abnormal or defective (mutated) gene products (proteins) that cause the disease, even if such subjects do not show symptoms of the disease.

[0091] I. Detailed description

[0092] IL-2 mutant proteins are described herein, comprising amino acid substitutions L18R, Q22E and Q126T numbered according to wild-type human IL-2 (hIL-2) (SEQ ID NO: 8), and further comprising a set of amino acid substitutions selected from the group consisting of F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T and S130R. Such IL-2 mutant proteins can be used, for example, to treat cancer. Also provided are nucleic acids encoding such IL-2 mutant proteins, methods for preparing such IL-2 mutant proteins, pharmaceutical compositions comprising such IL-2 mutant proteins, and methods of treatment using such IL-2 mutant proteins.

[0093] A. IL-2 mutant protein

[0094] The substituted amino acid residues may be (but not necessarily) conservative substitutions, generally including substitutions within the following categories: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. These mutations may be at amino acid residues that contact IL-2Rβ and / or IL-2Rγ.

[0095] More specifically, mutations (whether conservative or non-conservative, by addition or deletion) may be generated at one or more positions. For example, the mutations may be L18R, Q22E and Q126T, and may also include a group of mutations selected from the group consisting of F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T and S130R.

[0096] For example, the mutations may be: L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A, and E62A.

[0097] For example, the mutations may be: L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, and F42A.

[0098] For example, the mutations may be: L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A, and Y45A.

[0099] For example, the mutations may be: L18R, Q22E, L80F, R81D, L85V, I86V, I92F and Q126T.

[0100] For example, the mutations may be: L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, F42A, and E62A.

[0101] For example, the mutations may be: L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, and F42A.

[0102] Table 2: List of MDNA209 variants analyzed

[0103]

[0104]

[0105] In some embodiments, according to the wild-type human IL-2 numbering of SEQ ID NO: 8, the substitution in the IL-2 mutant protein comprises L18R, Q22E and Q126T, and further comprises a set of amino acid substitutions selected from the group consisting of: F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T and S130R. In some embodiments, according to the wild-type human IL-2 numbering of SEQ ID NO: 8, the substitution in the IL-2 mutant protein comprises L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T and S130R. In some embodiments, the substitutions in the IL-2 mutant protein comprise L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A and E62A according to the wild-type human IL-2 numbering of SEQ ID NO: 8. In some embodiments, the substitutions in the IL-2 mutant protein comprise L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R and F42A according to the wild-type human IL-2 numbering of SEQ ID NO: 8. In some embodiments, according to the wild-type human IL-2 numbering of SEQ ID NO: 8, the substitution in the IL-2 mutant protein comprises L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A and Y45A. In some embodiments, according to the wild-type human IL-2 numbering of SEQ ID NO: 8, the substitution in the IL-2 mutant protein comprises L18R, Q22E, L80F, R81D, L85V, I86V, I92F and Q126T. In some embodiments, according to the wild-type human IL-2 numbering of SEQ ID NO: 8, the substitution in the IL-2 mutant protein comprises L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, F42A and E62A. In some embodiments, according to the wild-type human IL-2 numbering of SEQ ID NO: 8, the substitutions in the IL-2 mutant protein include L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T and F42A.

[0106] In some embodiments, the substitutions in the IL-2 mutant protein that result in increased and / or enhanced IL-2Rβ binding include L18R, Q22E, L80F, R81D, L85V, I86V, I92F, and Q126T according to the numbering of wild-type human IL-2 of SEQ ID NO: 8. In some embodiments, the IL-2 mutant protein used in the present invention comprises L18R, Q22E, L80F, R81D, L85V, I86V, I92F, and Q126T, and exhibits increased IL-2Rβ binding. In some embodiments, the IL-2 mutant protein used in the present invention further comprises a substitution at position S130R. In some embodiments, the IL-2 mutant protein used in the present invention further comprises a substitution at position S130R. In some embodiments, the IL-2 mutant protein used in the present invention further comprises a substitution at position F42A. In some embodiments, the IL-2 mutant protein used in the present invention further comprises a substitution at position E62A. In some embodiments, the IL-2 mutant protein used in the present invention further comprises a substitution at position Y45A. In some embodiments, according to the wild-type human IL-2 numbering of SEQ ID NO: 8, the substitution in the IL-2 mutant protein comprises L18R, Q22E and Q126T, and further comprises a group of amino acid substitutions selected from the group consisting of: F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T and S130R. In some embodiments, according to the wild-type human IL-2 numbering of SEQ ID NO: 8, the IL-2 mutant protein comprises substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T and S130R. In some embodiments, the IL-2 mutant protein comprises substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A and E62A according to the wild-type human IL-2 numbering of SEQ ID NO: 8. In some embodiments, the IL-2 mutant protein comprises substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R and F42A according to the wild-type human IL-2 numbering of SEQ ID NO: 8. In some embodiments, the IL-2 mutant protein comprises substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A and Y45A according to the wild-type human IL-2 numbering of SEQ ID NO: 8. In some embodiments, the IL-2 mutant protein comprises substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, and Q126T, based on the wild-type human IL-2 numbering of SEQ ID NO:8.In some embodiments, the IL-2 mutant protein comprises substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, F42A and E62A according to the wild-type human IL-2 numbering of SEQ ID NO: 8. In some embodiments, the IL-2 mutant protein comprises substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T and F42A according to the wild-type human IL-2 numbering of SEQ ID NO: 8.

[0107] In some embodiments, the mutant protein comprises substitutions L18R, Q22E and Q126T, and one or more substitutions selected from the group consisting of: F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T and S130R, all compared to wild-type human IL-2 (SEQ ID NO: 8).

[0108] In some embodiments, amino acid substitutions that increase IL-2Rβ binding affinity include: L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A, E62A and / or Y45A. In some embodiments, amino acid substitutions that increase IL-2Rβ binding affinity include: L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A, E62A and / or Y45A.

[0109] In some embodiments, the subject IL-2 mutant protein having a stronger binding affinity to IL-2Rβ compared to wild-type human IL-2 comprises amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T and S130R. In some embodiments, the IL-2 mutant protein has the following amino acid sequence:

[0110]

[0111] In some embodiments, the subject IL-2 mutant protein having a stronger binding affinity to IL-2Rβ compared to wild-type human IL-2 comprises amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A and E62A. In some embodiments, the IL-2 mutant protein has the following amino acid sequence:

[0112] In some embodiments, the subject IL-2 mutant protein having a stronger binding affinity to IL-2Rβ compared to wild-type human IL-2 comprises amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R and F42A. In some embodiments, the IL-2 mutant protein has the following amino acid sequence:

[0113] In some embodiments, the subject IL-2 mutant protein having a stronger binding affinity to IL-2Rβ compared to wild-type human IL-2 comprises amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A and Y45A. In some embodiments, the IL-2 mutant protein has the following amino acid sequence:

[0114] In some embodiments, the subject IL-2 mutant protein having a stronger binding affinity to IL-2Rβ compared to wild-type human IL-2 comprises amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F and Q126T. In some embodiments, the IL-2 mutant protein has the following amino acid sequence:

[0115] In some embodiments, the subject IL-2 mutant protein having a stronger binding affinity to IL-2Rβ compared to wild-type human IL-2 comprises amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, F42A and E62A. In some embodiments, the IL-2 mutant protein has the following amino acid sequence:

[0116] In some embodiments, the subject IL-2 mutant protein having a stronger binding affinity to IL-2Rβ compared to wild-type human IL-2 comprises amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T and F42A. In some embodiments, the IL-2 mutant protein has the following amino acid sequence:

[0117] In some embodiments, the IL-2 mutein sequence is at least about 90% identical to any one of SEQ ID NO: 1 to SEQ ID NO: 7. In some embodiments, the IL-2 mutein sequence is at least about 95% identical to any one of SEQ ID NO: 1 to SEQ ID NO: 7. In some embodiments, the IL-2 mutein sequence is at least about 98% identical to any one of SEQ ID NO: 1 to SEQ ID NO: 7. In some embodiments, the IL-2 mutein sequence is at least about 99% identical to any one of SEQ ID NO: 1 to SEQ ID NO: 7.

[0118] B. IL-2 mutant protein fusion protein

[0119] IL-2 mutant proteins can be prepared in the form of fusion polypeptides or chimeric polypeptides, which include the subject IL-2 mutant protein and a heterologous polypeptide (i.e., a polypeptide that is not IL-2 or a mutant thereof) (see, e.g., U.S. Pat. No. 6,451,308). Exemplary heterologous polypeptides can increase the in vivo circulation half-life of the chimeric polypeptide, and thus can further enhance the properties of the mutant IL-2 polypeptide. In various embodiments, the polypeptide that increases the circulation half-life can be a serum albumin (such as human serum albumin), PEG, a PEG derivative, or an Fc region of an antibody IgG subclass lacking an IgG heavy chain variable region. Exemplary Fc regions can include mutations that inhibit complement fixation and Fc receptor binding, or they can be soluble, i.e., able to bind complement, or lyse cells via another mechanism such as antibody-dependent complement lysis (ADCC; USSN 08 / 355,502 filed December 12, 1994).

[0120] The "Fc region" can be a naturally occurring or synthetic polypeptide homologous to the C-terminal domain of IgG produced by papain digestion of IgG. The molecular weight of IgG Fc is approximately 50 kDa. The mutant 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, the full-length or fragmented Fc region can be a variant of the wild-type molecule. In some embodiments, the IL-2 mutant protein fusion protein (e.g., the IL-2 mutant protein described herein) includes an IgG1, IgG2, IgG3, or IgG4 Fc region (see, e.g., FIG. 2A to FIG. 2B In some embodiments, the Fc region comprises the substitution N297A.

[0121] In some embodiments, the IL-2 mutein is linked directly or indirectly to a heterologous fusion polypeptide.

[0122] In some embodiments, IL-2 mutant protein is directly connected to the Fc region. In some embodiments, IL-2 mutant protein is connected to the Fc region via a linker peptide such as GGGGS. In some embodiments, the linker is (GGGGS)n, wherein n is an integer between 1 and 10. In some embodiments, the linker is GGGGS. In some embodiments, the linker is GGGGSGGGGS (SEQ ID NO: 14). In some embodiments, the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 15). In some embodiments, the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 16). In some embodiments, the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 17).

[0123] Fc regions can be "soluble" or "insoluble", but are typically insoluble. Insoluble Fc regions typically lack a high affinity Fc receptor binding site and a C'1q binding site. The high affinity Fc receptor binding site of mouse IgG Fc includes a Leu residue at position 235 of IgG Fc. Therefore, the Fc receptor binding site can be destroyed by mutating or deleting Leu 235. For example, replacing Leu 235 with Glu will inhibit the ability of the Fc region to bind to a high affinity Fc receptor. The mouse C'1q binding site can be functionally destroyed by mutating or deleting the Glu 318, Lys 320, and Lys 322 residues of IgG. For example, replacing Glu 318, Lys 320, and Lys 322 with Ala residues renders IgG1 Fc unable to direct antibody-dependent complement lysis. In contrast, a soluble IgG Fc region has 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 Glu 318, Lys 320 and Lys 322 residues of IgG1. Solubility IgG Fc has wild-type residues or conservative amino acid substitutions at these sites. Solubility IgG Fc can target cells to produce antibody-dependent cellular toxicity or complement-directed cell lysis (CDC). Suitable mutations of human IgG are also known (see, for example, Morrison et al., The Immunologist 2: 119-124, 1994; and Brekke et al., The Immunologist 2: 125, 1994).

[0124] In other embodiments, the chimeric polypeptide may include a subject IL-2 mutant protein and a polypeptide that acts as an antigen tag, such as a FLAG sequence. As described herein, the FLAG sequence is recognized by a biotinylated highly specific anti-FLAG antibody (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.

[0125] In other embodiments, the chimeric polypeptide includes a mutant IL-2 polypeptide and a heterologous polypeptide that serves to enhance expression or direct cellular localization of the mutant IL-2 polypeptide, such as an Aga2p lectin subunit (see, e.g., Boder and Wittrup, Nature Biotechnol. 15:553-7, 1997).

[0126] In other embodiments, chimeric polypeptides comprising a mutant IL-2 and an antibody or an antigen binding portion thereof can be produced. The antibody or antigen binding component of the chimeric protein can be used as a targeting moiety. For example, it can be used to localize the chimeric protein to a specific cell subset or target molecule. Methods for producing cytokine-antibody chimeric polypeptides are described, for example, in U.S. Patent No. 6,617,135.

[0127] In other embodiments, chimeric polypeptides comprising mutant IL-2 and IL-4 proteins can be produced. Any IL-4 sequence or variant thereof can be used in the form of a fusion with an IL-2 mutant or variant (including those described herein). In some embodiments, the mutant IL-2 is fused to an IL-4 mutant having a sequence of SEQ ID NO: 23, which is shown below:

[0128] SEQ ID NO:23 (KFR)

[0129] KCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMKEKFRKCSS

[0130] In some embodiments, the mutant IL-2 is fused to an IL-4 mutant having a sequence of SEQ ID NO: 24, which is shown below:

[0131] SEQ ID NO:24 (cpRGA)

[0132] MDTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLRVIMQSKWFKCGAGGNGGHKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAAS

[0133] In some embodiments, the mutant IL-2 is fused to an IL-4 mutant having a sequence of SEQ ID NO: 25, which is shown below:

[0134] SEQ ID NO:25 (RGA)

[0135] HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLRVIMQSKWFKCGA

[0136] In some embodiments, mutant IL-2 is fused to an IL-4 mutant having a sequence at least about 90% identical to SEQ ID NO: 23. In some embodiments, mutant IL-2 is fused to an IL-4 mutant having a sequence at least about 90% identical to SEQ ID NO: 24. In some embodiments, mutant IL-2 is fused to an IL-4 mutant having a sequence at least about 90% identical to SEQ ID NO: 25. In some embodiments, mutant IL-2 is fused to an IL-4 mutant having a sequence at least about 90% identical to SEQ ID NO: 23. In some embodiments, mutant IL-2 is fused to an IL-4 mutant having a sequence at least about 95% identical to SEQ ID NO: 24. In some embodiments, mutant IL-2 is fused to an IL-4 mutant having a sequence at least about 95% identical to SEQ ID NO: 25. In some embodiments, mutant IL-2 is fused to an IL-4 mutant having a sequence at least about 98% identical to SEQ ID NO: 23. In some embodiments, mutant IL-2 is fused to an IL-4 mutant having a sequence at least about 98% identical to SEQ ID NO: 24. In some embodiments, mutant IL-2 is fused to an IL-4 mutant having a sequence at least about 98% identical to SEQ ID NO: 25. In some embodiments, mutant IL-2 is fused to an IL-4 mutant having a sequence at least about 99% identical to SEQ ID NO: 23. In some embodiments, mutant IL-2 is fused to an IL-4 mutant having a sequence at least about 99% identical to SEQ ID NO: 24. In some embodiments, mutant IL-2 is fused to an IL-4 mutant having a sequence at least about 99% identical to SEQ ID NO: 25.

[0137] In some embodiments, SEQ ID NO: 23 is linked to IL-2 or an IL-2 mutant protein described herein. In some embodiments, SEQ ID NO: 24 is linked to IL-2 or an IL-2 mutant protein described herein. In some embodiments, SEQ ID NO: 25 is linked to IL-2 or an IL-2 mutant protein described herein.

[0138] In some embodiments, the IL-2 mutein sequence is at least about 90% identical to any one of SEQ ID NO: 1 to SEQ ID NO: 7 (e.g., any one of the IL-2 sequences provided herein). In some embodiments, the IL-2 mutein sequence is at least about 95% identical to any one of SEQ ID NO: 1 to SEQ ID NO: 7 (e.g., any one of the IL-2 sequences provided herein). In some embodiments, the IL-2 mutein sequence is at least about 98% identical to any one of SEQ ID NO: 1 to SEQ ID NO: 7 (e.g., any one of the IL-2 sequences provided herein). In some embodiments, the IL-2 mutein sequence is at least about 99% identical to any one of SEQ ID NO: 1 to SEQ ID NO: 7 (e.g., any one of the IL-2 sequences provided herein). In some embodiments, the IL-2 mutant protein further comprises an A11 mutant protein or a variant thereof, and / or is conjugated to an A11 mutant protein or a variant thereof, including, for example, Fc-A11 (1:2); version 1 (SEQ ID NO: 9) or Fc-A11 (1:2); version 2 (SEQ ID NO: 10).

[0139] In some embodiments, the IL-2 mutant fusion protein sequence is at least about 90% identical to any one of SEQ ID NO: 18 to SEQ ID NO: 22. In some embodiments, the IL-2 mutant fusion protein sequence is at least about 95% identical to any one of SEQ ID NO: 18 to SEQ ID NO: 22. In some embodiments, the IL-2 mutant fusion protein sequence is at least about 98% identical to any one of SEQ ID NO: 18 to SEQ ID NO: 22. In some embodiments, the IL-2 mutant fusion protein sequence is at least about 99% identical to any one of SEQ ID NO: 18 to SEQ ID NO: 22. In some embodiments, the IL-2 mutant fusion protein sequence comprises any one of SEQ ID NO: 18 to SEQ ID NO: 22.

[0140] Table 3: List of exemplary MDNA209 fusions

[0141]

[0142]

[0143]

[0144] C. Recombinant expression of IL-2 mutant proteins, expression vectors and host cells

[0145] In various embodiments, the polypeptides used in the practice of the present invention are synthesized or produced by expression of a recombinant nucleic acid molecule. If the polypeptide is a chimera (e.g., a fusion protein containing at least a mutant IL-2 polypeptide and a heterologous polypeptide), it can be encoded by a hybrid nucleic acid molecule containing a sequence encoding all or part of the IL-2 mutant protein and a second sequence encoding all or part of the heterologous polypeptide. For example, the subject IL-2 mutant protein described herein can be fused to a hexa-histidine tag to facilitate purification of bacterially expressed proteins, or to a hemagglutinin tag to facilitate purification of proteins expressed in eukaryotic cells.

[0146] Methods for constructing DNA sequences encoding IL-2 mutant proteins and expressing those sequences in appropriately transformed hosts include, but are not limited to, mutagenesis techniques assisted by PCR. Mutations consisting of deletions or additions of amino acid residues of the IL-2 polypeptide can also be performed using standard recombinant techniques. In the case of deletions or additions, the nucleic acid molecules encoding IL-2 are optionally digested with appropriate restriction endonucleases. The resulting fragments can be expressed directly or further manipulated by, for example, connecting them to a second fragment. If the two ends of the nucleic acid molecule contain complementary nucleotides that overlap each other, the connection can be facilitated, but blunt-ended fragments can also be connected. Nucleic acids produced by PCR can also be used to generate various mutant sequences.

[0147] The complete amino acid sequence can be used to construct a reverse translation gene. DNA oligomers containing the nucleotide sequence encoding the IL-2 mutant protein can be synthesized. For example, several small oligonucleotides encoding multiple parts of the desired polypeptide can be synthesized and then connected. Individual oligonucleotides usually contain 5' or 3' overhangs for complementary assembly.

[0148] In addition to producing mutant polypeptides by expressing nucleic acid molecules that have been altered by recombinant molecular biology techniques, the subject IL-2 mutant proteins can also be chemically synthesized. Chemically synthesized polypeptides are routinely generated by those skilled in the art.

[0149] Once assembled (by synthesis, site-directed mutagenesis or other methods), the DNA sequence encoding the IL-2 mutant protein is inserted into an expression vector and operably linked to expression control sequences suitable for expressing the IL-2 mutant protein in the desired transformed host. Correct assembly can be confirmed by nucleotide sequencing, restriction mapping and expression of the biologically active polypeptide in a suitable host. As is well known in the art, in order to obtain high expression levels of the transfected gene in the host, the gene must be operably linked to transcriptional and translational expression control sequences that are functional in the selected expression host.

[0150] Whether the DNA sequence encoding IL-2 mutant protein prepared by site-directed mutagenesis, chemical synthesis or other methods can also include a DNA sequence encoding a signal sequence. If present, such a signal sequence should be a signal sequence recognized by the cell selected for expressing the IL-2 mutant protein. It can be prokaryotic, eukaryotic or a combination of the two. It can also be a signal sequence of natural IL-2. The inclusion of the signal sequence depends on whether it is necessary to secrete the IL-2 mutant protein from the recombinant cell that produces it. If the selected cell is prokaryotic, it is generally preferred that the DNA sequence does not encode a signal sequence. If the selected cell is eukaryotic, it is generally preferred that a coding signal sequence and most preferably a wild-type IL-2 signal sequence are used.

[0151] D. Nucleic acid molecules encoding mutant IL-2

[0152] In some embodiments, the subject IL-2 mutant proteins, either alone or as part of a chimeric polypeptide (e.g., those described above), can be obtained by expression of a nucleic acid molecule. Just as IL-2 mutant proteins can be described for their identity to wild-type IL-2 polypeptides, nucleic acid molecules encoding them must also have a certain identity to nucleic acid molecules encoding wild-type IL-2. For example, nucleic acid molecules encoding subject IL-2 mutant proteins can be at least 50%, at least 65%, preferably at least 75%, more preferably at least 85%, and most preferably at least 95% (e.g., 99%) identical to nucleic acids encoding wild-type IL-2 (e.g., SEQ ID NO: 8).

[0153] The nucleic acid molecules provided may contain naturally occurring sequences, or sequences that are different from naturally occurring sequences but encode the same polypeptide due to the degeneracy of the genetic code. These nucleic acid molecules may be composed of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, such as DNA produced by phosphoramidite-based synthesis) or combinations or modifications of nucleotides within these types of nucleic acids. In addition, the nucleic acid molecules may be double-stranded or single-stranded (i.e., sense strand or antisense strand).

[0154] Nucleic acid molecules are not limited to sequences encoding polypeptides; they may also include some or all non-coding sequences located upstream or downstream of a coding sequence (e.g., a coding sequence of IL-2). Those of ordinary skill in the field of molecular biology are familiar with conventional procedures for isolating nucleic acid molecules. They can be produced, for example, by treating genomic DNA with restriction endonucleases, or by performing a 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.

[0155] Exemplary isolated nucleic acid molecules of the present disclosure may include fragments that are not found in nature as such. Thus, the present disclosure encompasses recombinant molecules such as those 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 a location in the genome of a homologous cell other than the natural chromosomal location).

[0156] As described above, the subject IL-2 mutant protein can exist as part of a chimeric polypeptide. In addition to or in place of the above-mentioned heterologous polypeptide, the subject nucleic acid molecule can contain a sequence encoding a "marker" or "reporter factor". Examples of markers or reporter genes include β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo r 、G418 r ), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacz (encoding β-galactosidase) and xanthine guanine phosphoribosyl transferase (XGPRT). Those skilled in the art will know other useful reagents, such as other sequences that can function as markers or reporters.

[0157] The subject nucleic acid molecule can be obtained by introducing a mutation into a DNA encoding IL-2 obtained from any biological cell such as a mammalian cell. Therefore, the subject nucleic acid (and its encoded polypeptide) can be those of a mouse, rat, guinea pig, cattle, sheep, horse, pig, rabbit, monkey, baboon, dog or cat. In one embodiment, the nucleic acid molecule will be a human nucleic acid molecule.

[0158] E. Expression of mutant IL-2 gene products

[0159] The above nucleic acid molecules can be contained in a vector that is capable of directing the expression of these nucleic acid molecules in, for example, cells that have been transduced with the vector. Therefore, in addition to the subject IL-2 mutant protein, expression vectors containing nucleic acid molecules encoding the subject IL-2 mutant protein and cells transfected with these vectors are also in preferred embodiments.

[0160] Of course, it should be understood that not all vectors and expression control sequences will work equally well to express the DNA sequences described herein. Not all hosts can work equally well with the same expression system. However, those skilled in the art can choose between these vectors, expression control sequences and hosts without having to perform too many experiments. For example, when selecting a vector, the host must be considered because the vector must be replicated in the host. The copy number of the vector, the ability to control the copy number, and the expression of any other protein (such as an antibiotic marker) encoded by the vector should also be considered. For example, the vectors that can be used include those vectors that allow the DNA encoding the IL-2 mutant protein to be amplified in copy number. Such amplifiable vector is well known in the art. These include, for example, vectors capable of amplification by DHFR amplification (see, e.g., Kaufman, U.S. Pat. No. 4,470,461, Kaufman and Sharp, "Construction of a Modular Dihydrafolate Reductase cDNA Gene: Analysis of Signals Utilized for Efficient Expression", Mol. Cell. Biol., 2, pp. 1304-1319 (1982)) or glutamine synthetase ("GS") amplification (see, e.g., U.S. Pat. No. 5,122,464 and European Published Application 338,841).

[0161] In some embodiments, human IL-2 mutant proteins disclosed herein will be expressed from a vector, preferably from an expression vector. The vector can be used for autonomous replication in a host cell, or can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating with the host genome (e.g., a non-free mammalian vector). The expression vector can guide the expression of the coding sequence operably connected thereto. In general, expression vectors useful in recombinant DNA technology are typically in the form of plasmids (vectors). However, other forms of expression vectors are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0162] Exemplary recombinant expression vectors can include one or more regulatory sequences, selected based on the host cell to be used for expression, operably linked to the nucleic acid sequence to be expressed.

[0163] Expression constructs or vectors can be designed to express the IL-2 mutein or variant thereof in prokaryotic or eukaryotic host cells.

[0164] Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd Edition, Cold Spring Harbor Laboratory Press, Plainview, NY) and other standard molecular biology laboratory manuals.

[0165] Protein expression in prokaryotes is most often carried out in Escherichia coli using vectors containing constitutive or inducible promoters. Strategies for maximizing expression of recombinant proteins in Escherichia coli can be found in, for example, Gottesman (1990), Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.), pages 119-128 and Wada et al. (1992) Nucleic Acids Res. 20: 2111-2118. Methods for growing, harvesting, disrupting or extracting IL-2 muteins or variants thereof from cells are disclosed, for example, in U.S. Pat. Nos. 4,604,377, 4,738,927, 4,656,132, 4,569,790, 4,748,234, 4,530,787, 4,572,798, 4,748,234, and 4,931,543, which are incorporated herein by reference in their entireties.

[0166] In some embodiments, recombinant IL-2 muteins or biologically active variants thereof can also be produced in eukaryotic organisms such as yeast or human cells. Suitable eukaryotic host cells include insect cells (examples of baculovirus vectors that can be used to express 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 Saccharomyces 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) Diego, Calif.) and pPicZ (Invitrogen Corporation, San Diego, Calif.); 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 generally provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma virus, adenovirus 2, cytomegalovirus, and simian virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells, see Chapters 16 and 17 of Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd Edition, Cold Spring Harbor Laboratory Press, Plainview, NY). See Goeddel (1990), Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.).

[0167] The sequence encoding the human IL-2 mutant protein of the present disclosure can be optimized for expression in the target host cell. The GC content of the sequence can be adjusted to the average level of a given cell host, as calculated with reference to known genes expressed in host cells. Methods for codon optimization are well known in the art. Codons within the IL-2 mutant protein coding sequence can be optimized to enhance expression in host cells, such that about 1%, about 5%, about 10%, about 25%, about 50%, about 75% or up to 100% of the codons within the coding sequence have been optimized for expression in a specific host cell.

[0168] Vectors suitable for use include T7-based vectors for bacteria (see, e.g., Rosenberg et al., Gene 56:125, 1987), the pMSXND expression vector for mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988), and baculovirus-derived vectors for insect cells (e.g., the expression vector pBacPAK9 from Clontech, Palo Alto, Calif.).

[0169] In some embodiments, a nucleic acid insert encoding a subject IL-2 mutein in such a vector may be operably linked to a promoter selected based on, for example, the cell type in which expression is sought.

[0170] In selecting an expression control sequence, a variety of factors should also be considered. These factors include, for example, the relative strength of the sequence, its controllability and its compatibility with the actual DNA sequence encoding the subject IL-2 mutant protein, particularly with respect to potential secondary structures. The choice of host should take into account their compatibility with the selected vector, the toxicity of the product encoded by the DNA sequence of the present invention, their secretion characteristics, their ability to correctly fold polypeptides, their fermentation or culture requirements, and the ease of purification of the product encoded by the DNA sequence.

[0171] Within these parameters, one skilled in the art can select various vector / expression control sequence / host combinations that will express the desired DNA sequence either in fermentation or in large-scale animal culture, for example using CHO cells or COS 7 cells.

[0172] In some embodiments, the choice of expression control sequences and expression vectors will depend on the choice of the host. A variety of expression host / vector combinations can be used. Expression vectors useful for eukaryotic hosts include, for example, vectors with expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids, such as plasmids from Escherichia coli, including col El, pCRI, pER32z, pMB9, and their derivatives; plasmids with a wider host range such as RP4; phage DNA, such as many derivatives of lambda phage such as NM989, and other DNA phages such as M13 and filamentous single-stranded DNA phages. Expression vectors useful for yeast cells include 2μ plasmids and their derivatives. Vectors useful for insect cells include pVL 941 and pFastBac TM 1 (Gibco BRL, Gaithersburg, Md.). Cate et al., "Isolation Of The Bovine And Human Genes For Mullerian Inhibiting Substance And Expression Of The Human Gene In Animal Cells", Cell, 45, pp. 685-98 (1986).

[0173] In addition, any of a variety of expression control sequences can be used in these vectors. Such useful expression control sequences include expression control sequences associated with the structural genes of the aforementioned expression vectors. Examples of useful expression control sequences include, for example, early and late promoters of SV40 or adenovirus, lac systems, trp systems, TAC or TRC systems, major operators and promoter regions of lambda phages such as PL, control regions of fd coat proteins, promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, promoters of acid phosphatases such as PhoA, promoters of yeast a mating systems, polyhedral promoters of baculoviruses, and other sequences known to control gene expression of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.

[0174] The T7 promoter can be used for bacteria, the polyhedrin promoter can be used for insect cells, and the cytomegalovirus or metallothionein promoters can be used for mammalian cells. In addition, with respect to higher eukaryotic organisms, tissue-specific and cell-type-specific promoters are widely available. These promoters are named for their ability to direct the expression of nucleic acid molecules in a given tissue or cell type in vivo. The technician is familiar with many promoters and other regulatory elements that can be used to direct nucleic acid expression.

[0175] In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, a vector may also contain an origin of replication and other genes encoding selectable markers. For example, neomycin resistance (neo r ) gene confers G418 resistance to cells expressing it and thus allows phenotypic selection of transfected cells. One skilled in the art can readily determine whether a given regulatory element or selectable marker is appropriate for use in a particular experimental setting.

[0176] Viral vectors useful in the present invention include, for example, retroviral, adenoviral and adeno-associated viral 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).

[0177] Prokaryotic or eukaryotic cells containing and expressing nucleic acid molecules encoding the subject IL-2 mutant proteins disclosed herein are also features of the present invention. The cells of the present invention are transfected cells, i.e., cells into which nucleic acid molecules (e.g., nucleic acid molecules encoding mutant IL-2 polypeptides) have been introduced by recombinant DNA technology. The progeny of such cells are also considered to be within the scope of the present invention.

[0178] The exact components of the expression system are not important. For example, IL-2 mutant proteins can be produced in prokaryotic hosts such as bacterial Escherichia coli or in eukaryotic hosts such as insect cells (e.g., Sf21 cells) or mammalian cells (e.g., CHO, HEK293, COS cells, NIH 3T3 cells or HeLa cells). These cells can be obtained from many sources, including the American Type Culture Collection (Manassas, Va.). When selecting an expression system, it is important whether the components are compatible with each other. A technician or a skilled person can make such a decision. In addition, if it is necessary to obtain guidance in selecting an expression system, a skilled person can consult Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, New York, NY, 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Supplement, 1987).

[0179] The expressed polypeptides can be purified from the expression system using conventional biochemical procedures and can be used, for example, as therapeutic agents as described herein.

[0180] In some embodiments, depending on the host organism used to produce the mutant protein, the IL-2 mutant protein obtained will be glycosylated or unglycosylated. If bacteria are selected as the host, the IL-2 mutant protein produced will be unglycosylated. On the other hand, eukaryotic cells will glycosylate the IL-2 mutant protein, but may be different from the way the natural IL-2 is glycosylated. The IL-2 mutant protein produced by the transformed host can be purified according to any suitable method. A variety of methods for purifying IL-2 are known. See, for example, Current Protocols in Protein Science, Vol. 2. Editors: 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 mutant proteins can be isolated from inclusion bodies produced in Escherichia coli, or isolated from conditioned medium of mammalian or yeast cultures producing a given mutant protein using cation exchange, gel filtration and / or reversed phase liquid chromatography.

[0181] Another exemplary method for constructing a DNA sequence encoding an IL-2 mutant protein is by chemical synthesis. This includes direct peptide synthesis by chemical means from a protein sequence encoding an IL-2 mutant protein exhibiting the properties. The method can incorporate natural and non-natural amino acids at positions that affect the interaction of IL-2 with IL-2Rα, IL-2Rβ and / or IL-2Rγ. Alternatively, an oligonucleotide synthesizer can be used to synthesize genes encoding the desired IL-2 mutant protein by chemical means. Such oligonucleotides are designed based on the amino acid sequence of the desired IL-2 mutant protein, and preferably those codons that are favorable in the host cell where the recombinant mutant protein will be produced are selected. In this regard, it is well known that the genetic code is degenerate, that is, an amino acid can be encoded by more than one codon. For example, Phe (F) is encoded by two codons TIC or TTT, Tyr (Y) is encoded by TAC or TAT, and his (H) is encoded by CAC or CAT. Trp (W) is encoded by a single codon TGG. Therefore, it will be appreciated that for a given DNA sequence encoding a specific IL-2 mutant protein, there will be many degenerate DNA sequences encoding the IL-2 mutant protein. For example, it will be appreciated that in addition to the preferred DNA sequence of the mutant protein H9, there will be many degenerate DNA sequences encoding the IL-2 mutant protein shown. These degenerate DNA sequences are considered to be within the scope of the present disclosure. Therefore, in the context of the present invention, "degenerate variants thereof" refers to all DNA sequences that encode a specific mutant protein and thus enable the expression of the specific mutant protein.

[0182] The biological activity of the IL-2 mutein can be determined by any suitable method known in the art. Such assays include PHA-blast proliferation and NK cell proliferation.

[0183] F. Treatment Methods

[0184] In some embodiments, the subject IL-2 mutant proteins and / or nucleic acids expressing them can be administered to a subject to treat disorders associated with abnormal apoptosis or differentiation processes (e.g., cell proliferative disorders or cell differentiation disorders, such as cancer, for example, by producing active or passive immunotherapy). In the treatment of such diseases, the disclosed IL-2 mutant proteins may have advantageous properties, such as reducing vascular leakage syndrome. In some embodiments, the IL-2 mutant protein is any IL-2 mutant protein or variant disclosed herein. In some embodiments, the IL-2 mutant protein sequence is at least about 90% identical to any one of SEQ ID NO:1 to SEQ ID NO:7. In some embodiments, the substitution in the IL-2 mutant protein is according to the wild-type human IL-2 numbering of SEQ ID NO:8. In some embodiments, the IL-2 mutant protein is a fusion protein. In some embodiments, the IL-2 mutant protein is associated with a CAR-T construct and / or expressed by a CAR-T construct. In some embodiments, the IL-2 mutant protein is expressed by an oncolytic virus and / or is associated with an oncolytic virus.

[0185] Examples of cell proliferative and / or differentiative disorders include cancer (e.g., carcinoma, sarcoma, metastatic disorder, or hematopoietic neoplastic disorder, such as leukemia). Metastatic tumors may arise from a variety of primary tumor types, including but not limited to prostate cancer, ovarian cancer, breast cancer, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer including small cell lung cancer, kidney cancer, liver cancer, colon cancer, colorectal cancer, pancreatic cancer, gastric cancer, and brain cancer.

[0186] The mutant IL-2 polypeptides can be used to treat patients who have, are suspected of having, or may be at high risk of developing any type of cancer, including renal cancer or melanoma, or any viral disease. Exemplary carcinomas include those formed from tissues of the cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also includes carcinosarcoma, which includes malignant tumors composed of carcinoma tissue and sarcoma tissue.

[0187] Additional examples of proliferative disorders include neoplastic disorders of the hematopoietic system.

[0188] Alternatively, or in addition to methods of direct administration to a patient, in some embodiments, mutant IL-2 polypeptides can also be used in ex vivo methods. For example, cells (e.g., peripheral blood lymphocytes or purified lymphocyte populations isolated from a patient and placed or maintained in culture) can be cultured in vitro in a culture medium, and the contacting step can be achieved by adding the IL-2 mutant to the culture medium. The culturing step can include additional steps in which the cells are stimulated or treated with other agents, such as to stimulate proliferation or expand a cell population reactive to a target antigen (e.g., a cancer antigen or a viral antigen). Then, after the cells have been treated, they are administered to a patient.

[0189] In some embodiments, the IL-2 mutant protein is used to treat cancer, according to the numbering of human wild-type IL-2 (SEQ ID NO: 8), the mutant protein comprises substitutions L18R, Q22E and Q126T, and one or more substitutions selected from the group consisting of: F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T and S130R. In some embodiments, the IL-2 mutant protein is used in combination with nivolumab to treat cancer, according to the numbering of human wild-type IL-2 (SEQ ID NO: 8), the mutant protein comprises substitutions L18R, Q22E and Q126T, and one or more substitutions selected from the group consisting of: F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T and S130R. In some embodiments, the IL-2 mutant protein is used in combination with BMS-936558 to treat cancer, and the mutant protein comprises substitutions L18R, Q22E and Q126T according to human wild-type IL-2 (SEQ ID NO: 8), and one or more substitutions selected from the group consisting of: F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T and S130R. In some embodiments, the IL-2 mutant protein is used in combination with MDX-1106 to treat cancer, and the mutant protein comprises substitutions L18R, Q22E and Q126T according to human wild-type IL-2 (SEQ ID NO: 8), and one or more substitutions selected from the group consisting of: F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T and S130R. In some embodiments, the IL-2 mutant protein is used in combination with ONO-4538 to treat cancer, and the mutant protein comprises substitutions L18R, Q22E and Q126T according to human wild-type IL-2 (SEQ ID NO: 8), and one or more substitutions selected from the group consisting of: F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T and S130R. In some embodiments, the IL-2 mutant protein is used in combination with AMP224 to treat cancer, and the mutant protein comprises substitutions L18R, Q22E and Q126T according to human wild-type IL-2 (SEQ ID NO: 8), and one or more substitutions selected from the group consisting of: F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T and S130R.In some embodiments, the IL-2 mutant protein is used in combination with CT-011 to treat cancer, and the mutant protein comprises substitutions L18R, Q22E and Q126T according to human wild-type IL-2 (SEQ ID NO: 8), and one or more substitutions selected from the group consisting of: F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T and S130R. In some embodiments, the IL-2 mutant protein is used in combination with MK-3475 to treat cancer, and the mutant protein comprises substitutions L18R, Q22E and Q126T according to human wild-type IL-2 (SEQ ID NO: 8), and one or more substitutions selected from the group consisting of: F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T and S130R. In some embodiments, the IL-2 mutant protein comprises substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F and Q126T. In some embodiments, the IL-2 mutant protein further comprises a substitution at position S130R. In some embodiments, the IL-2 mutant protein further comprises a substitution at position S130R. In some embodiments, the IL-2 mutant protein further comprises a substitution at position F42A. In some embodiments, the IL-2 mutant protein further comprises a substitution at position E62A. In some embodiments, the IL-2 mutant protein further comprises a substitution at position Y45A. In some embodiments, according to the wild-type human IL-2 numbering of SEQ ID NO: 8, the substitutions in the IL-2 mutant protein include L18R, Q22E and Q126T, and further include a set of amino acid substitutions selected from the group consisting of: F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T and S130R. In some embodiments, according to the wild-type human IL-2 numbering of SEQ ID NO: 8, the IL-2 mutant protein includes the substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T and S130R. In some embodiments, the IL-2 mutant protein comprises substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A and E62A according to the wild-type human IL-2 numbering of SEQ ID NO: 8. In some embodiments, the IL-2 mutant protein comprises substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A and E62A according to the wild-type human IL-2 numbering of SEQ ID NO: 8.In some embodiments, the IL-2 mutant protein comprises substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A and Y45A according to the wild-type human IL-2 numbering of SEQ ID NO: 8. In some embodiments, the IL-2 mutant protein comprises substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F and Q126T according to the wild-type human IL-2 numbering of SEQ ID NO: 8. In some embodiments, the IL-2 mutant protein comprises substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F and Q126T according to the wild-type human IL-2 numbering of SEQ ID NO: 8. In some embodiments, the IL-2 mutant protein comprises substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, and F42A, according to the wild-type human IL-2 numbering of SEQ ID NO:8.

[0190] In some embodiments, an IL-2 mutant protein is used in combination with an antibody and / or immunotherapy to treat cancer, wherein the mutant protein comprises substitutions L18R, Q22E and Q126T and one or more substitutions selected from the group consisting of: F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T and S130R according to the numbering of human wild-type IL-2 (SEQ ID NO: 8); the antibody and / or immunotherapy includes but is not limited to anti-CT LA4 mAb, such as ipilimumab, tremelimumab; anti-PD-L1 antagonist antibodies, such as BMS-936559 / MDX-1105, MEDI4736, RG-7446 / MPDL3280A; anti-LAG-3, such as IMP-321; agonistic antibodies targeting immunostimulatory proteins, including anti-CD40 mAbs, such as CP-870,893, lucatumumab, dacetuzumab; anti-CD137 mAbs (anti-4-1-BB antibodies), such as BMS-663513 urelumab (anti-4-1BB antibody; see, e.g., U.S. Pat. Nos. 7,288,638 and 8,962,804, which are incorporated herein by reference in their entireties), lirilumab (anti-KIR mAB; IPH2102 / BMS-986015; blocks NK cell inhibitory receptors), and PF-05082566 (utomilumab; see, e.g., U.S. Pat. Nos. 8,821,867, 8,337,850, and 9,468,678, and International Patent Application Publication No. WO 2012 / 032433, which is incorporated herein by reference in its entirety); anti-OX40 mAb (see, e.g., WO 2006 / 029879 or WO 2010 / 096418, which is incorporated herein by reference in its entirety); anti-GITR mAb, such as TRX518 (see, e.g., U.S. Pat. No. 7,812,135, which is incorporated herein by reference in its entirety); anti-CD27 mAb, such as varlilumab CDX-1127 (see, e.g., WO 2016 / 145085 and U.S. Patent Publication Nos. US2011 / 0274685 and US2012 / 0213771, which are incorporated herein by reference in their entirety); anti-ICOS mAb (e.g., MEDI-570, JTX-2011;and anti-TIM-3 antibodies (see, e.g., WO 2013 / 006490 or U.S. Patent Publication No. US2016 / 0257758, which are incorporated herein by reference in their entirety). ;

[0191] In some embodiments, an IL-2 mutant protein is used in combination with another antibody to treat cancer, wherein the mutant protein comprises substitutions L18R, Q22E and Q126T, and one or more substitutions selected from the group consisting of: F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T and S130R, based on the numbering of human wild-type IL-2 (SEQ ID NO: 8), and the antibody may include monoclonal antibodies against prostate cancer, ovarian cancer, breast cancer, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer including small cell lung cancer, renal cancer, colorectal cancer, pancreatic cancer, gastric cancer, brain cancer (see generally www.clinicaltrials.gov).

[0192] In some embodiments, an IL-2 mutant protein is used in combination with an antibody for antibody-dependent cell-mediated cytotoxicity (ADCC) to treat cancer, and the mutant protein comprises substitutions L18R, Q22E and Q126T, and one or more substitutions selected from the group consisting of: F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T and S130R, numbered according to human wild-type IL-2 (SEQ ID NO: 8).

[0193] G. Pharmaceutical Compositions and Methods of Administration

[0194] In some embodiments, the subject IL-2 mutant proteins and nucleic acids can be incorporated into compositions, including pharmaceutical compositions. Such compositions generally include a polypeptide or nucleic acid molecule and a pharmaceutically acceptable carrier.

[0195] The pharmaceutical composition is formulated to be compatible with its intended route of administration. The mutant IL-2 polypeptides of the present invention can be administered orally, but they are more likely to be administered by a parenteral route, including, for example, intravenous administration. Examples of parenteral routes of administration include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral application may contain the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methyl paraben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate or phosphate; and an agent for adjusting tension, such as sodium chloride or glucose. The pH can be adjusted with an acid or base, such as sodium dihydrogen phosphate and / or disodium hydrogen phosphate, hydrochloric acid or sodium hydroxide (e.g., to a pH of about 7.2-7.8, e.g., 7.5). The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0196] Pharmaceutical compositions suitable for injectable use 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 TM . (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should flow to a degree that is easy to inject. Under manufacturing and storage conditions, the composition should be stable and must prevent the contamination of microorganisms such as bacteria and fungi during storage. The carrier can be a solvent or a dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and a suitable mixture thereof. Suitable fluidity can be maintained, for example, by using a coating (such as lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant (e.g., sodium lauryl sulfate). The effect of microorganisms can be prevented by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferred to include an isotonic agent in the composition, such as sugar, a polyol (such as mannitol, sorbitol), sodium chloride. The absorption of the injectable composition can be extended by including an agent (e.g., aluminum monostearate and gelatin) that delays absorption in the composition.

[0197] Sterile injectable solutions can be prepared by incorporating the required amount of active compound (as required) with one or a combination of the ingredients listed above into an appropriate solvent followed by filtration sterilization. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying, which obtain powders of the active ingredient and any other desired ingredients from their previously sterile filtered solutions.

[0198] If an oral composition is used, it generally includes an inert diluent or edible carrier. For the purpose of oral therapeutic administration, the active compound can be combined with an excipient and used in the form of tablets, lozenges, or capsules, such as 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 any of the following ingredients, or compounds of a similar nature: binders, such as microcrystalline cellulose, tragacanth or gelatin; excipients, such as starch or lactose; disintegrants, such as alginic acid, Primogel TM or corn starch; lubricants such as magnesium stearate or Sterotes TM ; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring such as mint, methyl salicylate, or orange flavoring.

[0199] If administered by inhalation, the IL-2 mutant protein or its encoding nucleic acid is delivered in the form of an aerosol spray from a pressure container or dispenser containing a suitable propellant (e.g., a gas such as carbon dioxide), or from a nebulizer. Such methods include those described in U.S. Pat. No. 6,468,798.

[0200] Systemic administration of IL-2 mutant proteins or nucleic acids can also be carried out by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are generally known in the art, and include, for example, detergents, bile salts and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into an ointment, salves, gel or cream generally known in the art.

[0201] In some embodiments, the compounds (mutant 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.

[0202] In some embodiments, the compound (subject IL-2 mutant protein or nucleic acid) can also be administered by transfection or infection using methods known in the art, including but not limited to the methods described in McCaffrey et al. (Nature 418:6893, 2002), Xia et al. (Nature Biotechnol. 20:1006-1010, 2002), or Putnam (Am. J. Health Syst. Pharm. 53:151-160, 1996, erratum see Am. J. Health Syst. Pharm. 53:325, 1996).

[0203] In one embodiment, the IL-2 mutant protein or nucleic acid is prepared with a carrier that protects the mutant IL-2 polypeptide from rapid clearance 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 preparations can be prepared using standard techniques. Materials can also be commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as the method described in U.S. Patent No. 4,522,811.

[0204] Dosage, toxicity, and therapeutic efficacy of IL-2 muteins or nucleic acid compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as for determining LD 50 (the dose that is lethal to 50% of the population) and ED 50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as LD 50 / ED 50 Compounds that exhibit high therapeutic indices are preferred. Although compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets the compound to the site of the affected tissue in order to minimize potential damage to uninfected cells and thereby reduce side effects.

[0205] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds preferably lies within the range that includes the ED with little or no toxicity. 50The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve an IC as determined in cell culture. 50 The circulating plasma concentration range of the test compound (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms) can be used to more accurately determine useful doses for humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0206] As defined herein, the therapeutically effective amount (i.e., effective dose) of the subject IL-2 mutant protein depends on the selected polypeptide or antibody. In some embodiments, the amount of a single dose of the IL-2 mutant protein that can be administered can be in the range of about 0.001 mg to 0.1 mg per kilogram of patient body weight. In some embodiments, the dose of the IL-2 mutant protein that can be administered is about 0.005 mg / kg, 0.01 mg / kg, 0.025 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 5.0 mg / kg, 10.0 mg / kg. In some embodiments, 600,000 IU / kg is administered (IU can be determined by lymphocyte proliferation bioassay and expressed in international units (IU), as established by the World Health Organization Interleukin 2 (Human) First International Standard). The dose may be different from The dosage of the prescription dose is similar to that of the IL-2 mutant protein, but it is expected to be lower than this dosage. The composition can be applied once or more per day to once or more per week; including once every other day. The technician will recognize that certain factors may affect the dosage and time required for effective treatment of the subject, including but not limited to the severity of the disease or condition, previous treatment, the overall health and / or age of the subject, and other diseases present. In addition, the treatment of the subject with a therapeutically effective amount of the subject IL-2 mutant protein may include a single treatment, or may include a series of treatments. In one embodiment, the composition is applied every 8 hours for five days, then stopped for 2 to 14 days (e.g., 9 days), and then applied every 8 hours for another five days. In some embodiments, the application is 3 doses every 4 days.

[0207] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0208] The following examples are provided to illustrate certain embodiments of the invention provided herein and should not be construed as limiting.

[0209] Example

[0210] Example 1: IL-2 antagonist analysis

[0211] Combination - Surface Plasmon Resonance (SPR) method

[0212] Surface plasmon resonance (SPR) technology enables label-free, real-time analysis of biomolecular interactions. Binding interactions between proteins in their native state can be quantitatively examined using purified preparations or complex mixtures, and can assess binding specificity, stoichiometry, concentration, thermodynamics, kinetics (association rate constant k a and the dissociation rate constant k d ) and overall affinity (equilibrium dissociation constant, K D =k d / k a ). The sensor chip (CM5) was activated (420 seconds, flow rate was 10 μL / min) and 50 μg / mL of anti-histidine antibody (in fixed buffer 10 mM sodium acetate (pH 4.5)) was injected (420 seconds, flow rate was 10 μL / min). For ligand capture, the sample was diluted to 5 μg / mL (HEPES, containing 0.005% Tween-20, ph7.4) and injected (flow rate was 10 μL / min) to achieve a capture level of approximately 200 RU. The receptor was diluted and up to 8 specified concentrations were obtained and injected (flow rate was 30 μL / min), maintaining an association period of 120 seconds, followed by a dissociation period of 300 seconds. The constructs examined by SPR are shown in Tables 4 and 5.

[0213] Table 4. Constructs examined by SPR

[0214]

[0215] Table 5: List of MDNA209 variants analyzed

[0216]

[0217]

[0218] SPR binding results

[0219] IL-2 binds to IL2Rβ (CD122) and γ c (CD132), or a combination of IL2Rα (CD25), IL2Rβ and IL2Rγ c MDNA209 is a high-affinity heterotrimeric IL-2 receptor. MDNA209 is an IL-2 super antagonist mutant protein that has enhanced affinity for IL2Rβ and has the ability to interfere with γ c Combined mutations (Mitra et al., 2015). See also Figure 2A .

[0220] Sensorgrams evaluating the binding affinity of the MDNA209-Fc, MDN209FEAA-Fc, MDNA209FEAA-Fc-A11, MDNA209(3)FEAA-Fc, MDNA209(3)FEAA-Fc-MDNA209(3)FEAA, MDNA209FA-Fc, and MDNA209FEY-Fc variants are shown in Figure 2B -D and their binding is summarized in Table 6. D Compared with Fc-IL-2 as a positive control. K of MDNA209-Fc, MDNA209FA-Fc, MDNA209FEY-Fc, MDNA209FEAA-Fc, MDNA209FEAA-Fc-A11, MDNA209(3)FEAA-Fc, MDNA209(3)FEAA-Fc-MDNA209(3)FEAA against IL2Rβ (CD122) D The value was significantly lower than the K for Fc-IL-2 D Other mutations in MDNA209FEAA-Fc, MDNA209FEY-Fc, MDNA209FA-Fc, MDNA209(3)FEAA-Fc, MDNA209(3)FEAA-Fc-MDNA209(3)FEAA resulted in no binding to IL2Rα (CD25).

[0221] Table 6. Binding affinity K of IL-2 antagonists to human CD25 and CD122 D value

[0222]

[0223] *K D Based on steady-state affinity calculated from observed fit, rather than typical 1:1 binding

[0224] SPR binding conclusion

[0225] According to SPR analysis, all tested IL-2 antagonists showed enhanced binding to CD122 compared to Fc-IL-2 (Table 6). Addition of FEAA, FA or FEY mutations resulted in a lack of binding to CD25. In the bispecific format, MDNA209FEAA-Fc-A11 showed comparable binding affinity to MDNA209FEAA-Fc.

[0226] Signal transduction assay

[0227] HEK-Blue TM IL-2 reporter gene antagonist assay

[0228] HEK-Blue TM IL-2 reporter cells express the high affinity IL-2 receptor and are designed to monitor activation of the JAK-STAT pathway. HEK-Blue is produced by stably transfecting HEK293 cells with human IL-2Rα, IL-2Rβ, IL-2Rγ, JAK3 and STAT5 genes and a STAT5-inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene. TM IL-2 reporter cells. IL-2 stimulation will lead to activation of STAT5 and subsequent expression and secretion of SEAP, which can be detected using QUANTI-Blue TM Quantitative.

[0229] HEK-Blue TM IL-2 reporter cells (InvivoGen, 50,000 cells / well) were manipulated in two formats: 1) fixed agonist format using increasing concentrations of construct and EC 80 Concentration of rhIL-2 (determined experimentally to be 0.1 nM); or 2) antagonist form, in which 30 nM test compound is exposed to a range of IL-2-Fc (1 nM to 0.001 pM) (Table 7). The cells were incubated for 24 hours, and after incubation, the cell supernatant (20 μL) was moved to a new plate, 180 μL of QUANTI-Blue solution was added and incubated at 37°C for 2 hours. The plate was scanned on a conventional plate reader to determine the absorbance at 650 nm.

[0230] Table 7: HEK Blue TM Constructs and reagents tested in the IL-2 reporter gene assay

[0231]

[0232] Results of HEK-Blue IL-2 reporter assay

[0233] Preliminary experiments were performed to determine the dose of IL-2 to be used in the antagonist assay (EC 50 =0.025 nM). Using this assay, HEK-Blue TM Antagonists were screened in IL-2 reporter cell lines, and MDNA209-Fc showed significant antagonistic activity, with EC 50 The value is 8.396nM( Figure 3A and Table 7). In addition, MDNA209-Fc was tested in the form of an antagonist, where increasing amounts of IL-2 and a fixed concentration of compound were added. MDNA209-Fc clearly exhibited antagonist activity, as shown by the rightward shift of the curve, where IC 50is 59.31( Figure 3B ).

[0234] In a previous publication, a series of IL-2 antagonists with various levels of antagonist efficacy, including weak partial agonists, were designed (Mitra et al., 2015). The MDNA209 (3) molecule has three mutations (RET) that have been reported to strongly but not completely inhibit γ c Unlike MDNA209 (RETR), which completely eliminates γ c Therefore, the MDNA209(3) compound was tested for its weak partial agonist activity.

[0235] In this assay, both MDNA209(3)FEAA-Fc and MDNA209(3)FEAA-Fc-MDNA209(3)FEAA exhibited weak partial agonist activity (approximately 60-fold less potent than free IL-2) ( Figure 3C and Table 8). MDNA209(3)-Fc-MDNA209(3)FEAA contains four MDNA209(3) moieties and exhibits enhanced agonist activity compared to MDNA209(3)FEAA-Fc ( Figure 3C and Table 8).

[0236] Table 8. HEK Blue TM Agonist EC in IL-2 reporter gene assay 50 IC 50 value

[0237]

[0238]

[0239] * The experimental results are shown in Figure 3A middle

[0240] ** The experimental results are shown in Figure 3C middle

[0241] # The experimental results are shown in Figure 3B middle

[0242] Conclusions of the HEK Blue IL-2 reporter assay

[0243] MDNA209-Fc exhibited antagonist activity against IL-2, while MDNA209(3)FEAA-Fc and MDNA209(3)FEAA-Fc-MDNA209(3)FEAA exhibited weak agonist activity.

[0244] CTLL-2 cell proliferation assay

[0245] CTLL-2 cells are cytotoxic T cell lines that depend on IL-2 for survival and proliferation. Compared with the HEK-Blue signal transduction assay, this assay provides functional readout (proliferation) and the assay duration is longer. In duplicate, the samples shown in Table 9 are performed CTLL2 proliferation assays. CTLL2 cells are plated in a culture medium lacking T-STIM proliferation supplements with 30,000 cells per well. Cells are treated with increasing concentrations of test samples or control samples for 48 hours. Cell Titer Blue cell viability reagent (Promega G8080) is added to each well, and after generating a fluorescent viability signal (about 6 hours), the plate is scanned at 560Ex / 590Em.

[0246] Table 9. Compounds tested in the CTLL-2 assay

[0247] Test object batch number Test range IL-2-Fc 11977-828988 3nM was diluted approximately 4 times to 0.011pM MDNA209-Fc Construct #8 IL-2-Fc titration in the presence of 30 nM

[0248] Results of CTLL-2 proliferation assay

[0249] The data of RFU versus IL-2-Fc concentration were fitted to a 4-parameter logistic curve and the EC 50 The values ​​were calculated as well as the average of 2 or 4 plates (Table 10). The antagonism fold is calculated as the EC of IL-2-Fc in the presence of the test sample. 50 EC with IL-2-Fc alone 50 The ratio calculation ( Figure 4 and Table 11). MDNA209-Fc antagonizes IL-2-Fc, and its EC 50 The value is about 70 times higher.

[0250] Table 10: EC of CTLL2 50 Value (in pM).

[0251] Test compound / plate 1 2 3 4 average value IL-2-Fc 3.2 2.6 3.6 4.7 4.4 MDNA209-Fc 193.9 221.4 207.7

[0252] Table 11: Fold antagonism relative to IL-2-Fc.

[0253] Tested compounds 1* 2 3 4 average value IL-2-Fc 1.00 1.00 1.00 1.00 1.00 MDNA209-Fc 60.35 85.52 72.935

[0254] * Antagonism fold is based on the EC of the test sample 50 Divide by the EC of IL2-Fc 50 calculate.

[0255] Conclusions of CTLL2 Assay

[0256] MDNA209-Fc is a potent inhibitor of IL-2-induced CTLL2 proliferation.

[0257] pSTAT5 signaling assay using human peripheral blood mononuclear cells (PBMC) method.

[0258] To examine the activity and potential cell population selectivity of various IL-2 superantagonist variants, immune cell populations (T cells) present in human peripheral blood mononuclear cells (PBMCs) were examined. reg , CD4+T cells, CD8+T cells (resting and activated cells), and NK cells). PBMCs purified from whole blood were left to rest overnight. The cells were counted and plated in serum-free medium (60 minutes). Medium alone or medium containing compounds (10 point 5-fold dilutions starting from 25nM) was added to the cells and incubated at 37°C for 15 minutes. rhIL-2 (10 point 5-fold dilutions starting from 25nM) was used as a positive control for pSTAT5. After activation with IL-2 or other compounds (listed in Table 13), the cells were fixed with PFA for 15 minutes at room temperature, permeabilized (BD transcription factor phosphorylation buffer set), and stored in methanol (-20°C). After storage in methanol, the samples were stained with antibodies to detect pSTAT5, CD4, CD8, CD25, CD56, and FOXP3, and analyzed by flow cytometry. The compounds tested in the pSTAT5 assay are shown in Table 12.

[0259] Table 12. Constructs tested in the pSTAT5 assay

[0260]

[0261] Results of pSTAT5 signaling assay

[0262] Cells were pretreated with increasing concentrations of antagonist or neutralizing antibody control and subsequently exposed to 67 pM or 670 pM of rhIL-2 (equivalent to approximately 1 ng / mL and 10 ng / mL used in the 2015 publication by Mitra et al.).

[0263] Representative dose-response curves are shown in Figure 5 , and the IC 50 The results are summarized in Tables 14 and 15. Analysis of pSTAT5 signaling in human PBMCs showed that MDNA209-Fc exhibited higher potency in different cell types compared to all other antagonists and antibody controls. reg The inhibitory ratio was also better than that of other immune cells, including CD8+CD25+T cells, which also express high-affinity receptors. When comparing these two cell types, anti-CD25 (daclizumab biosimilar) appeared to have higher inhibitory activity against CD8+CD25+T cells than against T cells. regof the inhibitory activity.

[0264] Due to T reg Cells limit T cell activity in autoimmune diseases, thereby preferentially targeting effector T cells without affecting T reg Adding FEAA mutations that do not bind to the high-affinity CD25 receptor appears to better discriminate between T cells. reg The addition of the FEAA mutation also resulted in a significant reduction in potency against all immune cell types tested and was stronger than anti-CD122. This was also observed for the MDNA209FEAA-Fc and MDNA209(3)FEAA-Fc compounds, which showed similar levels of activity.

[0265] The bispecific molecule MDNA209FEAA-Fc-A11 was further analyzed to confirm that the activity of the MDNA209FEAA portion was not affected by the addition of the A11 portion. A11 was shown to be inactive in this assay (data not shown) and the addition of a second portion to MDNA209FEAA reduced its activity in inhibiting pSTAT5 signaling.

[0266] Table 13. IC 50 Summary of ratiometric analysis of values ​​and percentage of pSTAT5-positive cells in human PBMCs (67 pM rhIL-2).

[0267]

[0268] Table 14: IC 50 Summary of ratiometric analysis of values ​​and percentage of pSTAT5-positive cells in human PBMCs (670 pM rhIL-2).

[0269]

[0270] Conclusions of pSTAT5 signaling assay

[0271] MDNA209-Fc is a potent inhibitor of pSTAT5 signaling in human PBMCs and targets T reg Compared with MDNA209-Fc, MDNA209FEAA-Fc showed a higher proportional efficacy against T cells than against other immune cells. reg The inhibitory activity against other immune cells was better distinguished, but also had reduced overall potency. MDNA209(3)FEAA-Fc and MDNA209FEAA-Fc showed similar potency in this assay. It was observed that MDNA209FEAA-Fc-A11 showed the lowest inhibitory activity.

[0272] In vivo maximum tolerated dose (MTD) approach

[0273] The purpose of this study was to determine the tolerability of MDNA209FEAA-Fc-A11 in naive C57BL / 6 mice. A preliminary maximum tolerated dose (MTD) study was conducted to evaluate the acute toxicity of the MDNA209FEAA-Fc-A11 compound in naive C56Bl / 6 mice. MDNA209FEAA-Fc-A11 (30 mg / kg) and A11-Fc (21 mg / kg or 11 mg / kg) were administered to three mice per group. Animals were dosed intraperitoneally on days 1, 5, and 8.

[0274] MTD Results

[0275] Animals receiving doses of MDNA209FEAA-Fc-A11 (30 mg / kg) or A11-Fc (21 mg / kg, molar equivalent) showed no significant weight loss or overt signs of toxicity and were tolerant to the drugs over a 12-day period ( Figure 6 ).

[0276] MTD Conclusion

[0277] The MDNA209FEAA-Fc-A11 compound was well tolerated and did not induce any signs of acute toxicity.

[0278] Experimental Autoimmune Encephalomyelitis (EAE) Model Methods

[0279] Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis. C57BL / 6 mice were grouped on day 0 and injected with MOG in complete Freund's adjuvant at two different sites on the posterior flank on day 2. 35-55 Peptide is immunized to mice to induce EAE. Pertussis toxin is administered 2 hours and 48 hours after immunization. Mice are treated 8 days, 12 days and 15 days after immunization (Table 16). Animal body weight and clinical EAE score are assessed every day, and mice are scored on a 0-5.0 scale (as defined in Table 15).

[0280] Table 15: Clinical EAE Scores

[0281]

[0282]

[0283] Table 16. Dosing Groups in EAE Study

[0284]

[0285] a:ip: intraperitoneal injection

[0286] b: BIW: Administered twice a week. Administered on the 8th, 12th, and 15th days after immunization

[0287] c: Treatment group n=5, initial dose was 20 mg / kg and subsequent dose was 5 mg / kg

[0288] On day 18 after immunization, it was observed that the scores of mice treated with MDNA209-Fc were significantly lower than those of mice treated with PBS (p<0.05, t-test, n=7) ( Figure 7 ). Compared with the PBS control group, the MDNA209FA-Fc group showed a decreased score.

[0289] Mice were treated with MDNA209FEAA-Fc-A11 or a combination of MDNA209-Fc and A11-Fc. Both treatments resulted in a decrease in scores compared to the PBS control group ( Figure 8 ). Compared with the PBS control group, mice treated with the combination of A11-Fc and MDNA209-Fc showed a significant increase in body weight on study days 12 and 14 (p<0.05, t-test, n=6) ( Figure 8 ).

[0290] Mice that received a therapeutic dose of MDNA209FEAA-Fc-A11 showed a decrease in scores after administration ( Fig. 9 ).

[0291] EAE Conclusion

[0292] MDNA209-Fc, MDNA209FA-Fc, and MDNA209FEAA-Fc-A11 reduced disease scores in the EAE model in both prophylactic and therapeutic dosing regimens compared to PBS control animals.

[0293] MDNA209-Fc blocks IL-2-induced IFNγ secretion

[0294] Interleukin 2 (IL-2) stimulates human peripheral mononuclear cells (PBMCs) and induces the secretion of cytokines including proinflammatory interferon (IFN)-γ. PBMCs from three different healthy donors were incubated in vitro with increasing concentrations of MDNA209-Fc in the presence of 0.3 or 0.1 μg / mL human recombinant IL-2 (rhIL-2) for 48 hours. The culture supernatants were collected and analyzed for IFNγ levels using an enzyme-linked immunosorbent assay (ELISA). Fig.10As shown in , MDNA209-Fc inhibited rhIL-2-induced IFNγ secretion in three different PBMCs in a dose-dependent manner. Values ​​below the lower limit of quantification (LLOQ) were plotted as 0.5×LLOQ (4 pg / mL).

[0295] in conclusion

[0296] The described MDNA209 variants were tested for binding, signaling (HEKBlue IL-2 reporter, pSTAT5 in human PBMCs), CTLL2 proliferation, MTD in mice, and efficacy in EAE in vivo studies. All compounds showed enhanced binding to CD122 (compared to IL-2), while compounds containing FEAA, FA, or FEY mutations showed reduced binding to CD25. MDNA209-Fc showed inhibitory activity in HEKBlue IL-2 and CTLL2 assays. All compounds inhibited IL-2-induced pSTAT5 signaling in human PBMCs. MDNA209FEAA-Fc-A11 was non-toxic to mice (MTD assay); MDNA209-Fc reduced disease scores in EAE.

[0297] MDNA209's differentiated mechanism of action provides an opportunity, namely, MDNA209 is an antagonist of effector cells, directly targeting effector immune cells that drive disease and blocking CD4+ and CD8+ T cells and NK cells. Therefore, MDNA209 has the opportunity to be widely used in autoimmune diseases with minimal T-reg involvement.

[0298] Example 2: Human PBMC cytokine release assay

[0299] The aim of this study was to test the activity of MDNA209-Fc in blocking IL-2 signaling in normal healthy human PBMCs using IFN-γ secretion as a readout.

[0300] method:

[0301] PBMCs from four healthy human donors were purchased from STEMCELL Technologies ( Table 1) and screened for their response to IL-2 stimulation. Briefly, cells were pre-incubated with 0-1000nM MDNA209-Fc (2:1) for 15 minutes and then stimulated with rhIL-2 (3μg / ml, 1μg / ml or 0.3μg / ml) for 48 hours. The supernatant was subjected to IFN-γ ELISA. Since IFN-γ was above the quantitative level when PBMCs were treated with 1μg / ml and 3μg / ml rhIL-2, the data obtained by 0.3μg / ml rhIL-2 stimulation were used in the analysis and presented in this report. The data for each donor were normalized by calculating the percentage inhibition of the average maximum response in the absence of MDNA209-Fc treatment.

[0302] Table 17: Overview of PBMC donors used in the study

[0303] Donor# gender age nationality blood type Smokers 1 female 50 years old Caucasian O+ no 2 female 43 years old Caucasian B+ no 3 male 20 years old Caucasian O+ no 4 female 21 years old Caucasian A+ no

[0304] Results and Conclusions:

[0305] Four donors who had a clear response to 0.3 μg / mL rhIL-2 stimulation were selected for MDNA209-Fc experiments. In all four unique PMBC donor samples, MDNA209-Fc showed dose-dependent inhibition of rhIL-2-induced IFN-γ release (Table 17 and Fig.11 ). Thus, MDNA209-Fc exhibited IL-2 antagonist activity by inhibiting IL-2-induced activation and IFN-γ cytokine release in human PBMCs.

[0306] Example 3: MDNA209 exposure and in vivo pharmacokinetics

[0307] The purpose of this study was to determine the tolerability and pharmacokinetics of MDNA209-Fc in preclinical in vivo studies to develop an appropriate dosing regimen.

[0308] method:

[0309] BALB / c mice (10-11 weeks old) were randomly divided into 7 groups (3 mice per group) according to body weight (Table 18), and different dosing regimens were used to treat different doses of MDNA209-Fc by intraperitoneal (IP) injection. Daily clinical cage observations and twice weekly body weight measurements were recorded. Food and water consumption was also monitored. At the specified non-end point time point, about 100 μL of whole blood was collected and processed into plasma and stored at -80°C for analysis. About 200 μl of whole blood was collected for CBC analysis. A gross autopsy was performed at termination. MDNA209-Fc was detected using MDS ELISA. R&D Systems MAB202-100 was used as the capture antibody, and the absorbed anti-human Fc cross species (Sigma#SAB3701284) was used as the detection antibody, followed by detection with HRP-conjugated anti-goat IgG (Millipore#401515).

[0310] Table 18: Animal study design for MDNA209-Fc administration and blood collection

[0311]

[0312] Results and Conclusions:

[0313] In acute (4 doses over 5 days) or long-term (8 doses over 21 days) studies, MDNA209-Fc was well tolerated when repeated doses up to 20 mg / kg were administered by IP injection. There was no change in body weight and no abnormal findings at autopsy (Figure 12). Despite multiple administrations of MDNA209-Fc (Figure 12), there was no effect on lymphocyte counts, which may be because lymphocytes have a long lifespan (15-20 days) and long-term studies are required to detect changes. MDNA209-Fc was detectable in the blood 24 hours after dosing, but was rapidly cleared from the mice, and was undetectable in the blood 72 hours after dosing ( Fig.13 ).

[0314] Example 4: Mixed Lymphocyte Reaction (MLR) Assay

[0315] The aim of this study was to determine the efficacy of MDNA209 constructs (MDNA209-Fc, MDNA209FEAA-Fc, and MDNA209-albumin) in inhibiting the proliferation of allogeneic human peripheral blood mononuclear cells (PBMCs) in a mixed lymphocyte reaction (MLR) assay.

[0316] MDNA209-Albumin Protein Sequence:

[0317]

[0318] ● Adapter sequence In italics and underlined.

[0319] The MDNA209 sequence is shown in bold. Mutations in wild-type human IL-2 are highlighted in grey and are as follows: L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R

[0320] Albumin sequences are in normal font.

[0321] method:

[0322] Four healthy donors provided by Xeno Diagnostics were used in this study (Table 19). Blood samples were collected by venipuncture, and PBMCs were isolated by Ficoll density gradient centrifugation and cryopreserved. Cryopreserved PBMCs were thawed and left overnight before use. PBMCs from two unrelated donors (200K cells / donor) were co-cultured with MDNA209-Fc, MDNA209-albumin, and MDNA209FEAAC-Fc at different dilutions (0.0016nM–100nM; 5-fold dilution). The mitogenic stimulator phytohemagglutinin (PHA) and the proliferation inhibitor dexamethasone were used as positive and negative controls, respectively. Samples were cultured in triplicate for 4 days. On day 3, proliferation was observed under a microscope and BrdU was added. On day 4, a colorimetric ELISA for BrdU incorporation was performed. The stimulation index (SI) was calculated by dividing the test absorbance by the absorbance of the baseline background control. The half maximal inhibitory concentration (IC50) of SI was calculated using a four-parameter curve fit.

[0323] Table 19: PBMC donor samples used in the study

[0324] Donor# gender age Race / Ethnicity 50 years old male 30 years old African Americans 59 years old female 37 years old Caucasian 90 years old male 31 years old Caucasian 80 years old male 26 years old Hispanic

[0325] Results and Conclusions:

[0326] MDNA209-Fc inhibited the proliferation of PBMCs from all five donor pairs (IC 50 =0.0294±0.01 nM). Although MDNA209FEAA-Fc showed inhibitory effect in only one pair (IC 50 =28.54 nM), but within the tested dose range, MDNA209-albumin was able to inhibit PBMC proliferation in two of the three tested pairs (IC 50=0.18±0.02 nM). Overall, MDNA209-Fc was 15-fold more potent than MDNA209-albumin in blocking proliferation in the context of an MLR. MDNA209FEAA-Fc had limited or no effect on PBMC proliferation. Fig.14 and Table 20.

[0327] Table 20: IC50 values ​​and fold potency of the test constructs in the MLR assay

[0328]

[0329] *Maximum tested concentration

[0330] #IC 50 Value # Fold changes are calculated relative to the same donor pair Example 5: Additional binding (SPR) data

[0331] Results and Conclusions:

[0332] According to SPR analysis, all IL-2 antagonists (free and Fc fusion) examined showed enhanced binding to CD122 compared to the corresponding IL-2 or Fc-IL-2 controls (Table 21). Addition of FEAA resulted in a lack of binding to CD25. MDNA209(3)-Fc retained binding to CD25 and enhanced binding to CD122. Addition of FA to MDNA209(3) reduced its affinity for CD25.

[0333] Fusion of MDNA209 with the IL-4 agonist KFR or RGA moieties maintained binding affinity to CD25 and CD122 (Table 21, Figures 15A-15B ).

[0334] Table 21: Binding affinity KD values ​​of IL-2 antagonists to human CD25 (IL-2Rα) and CD122 (IL-2Rβ)

[0335]

[0336]

[0337] *K D Based on steady-state affinity calculated from observed fit, rather than typical 1:1 binding

[0338] The embodiments described above are provided to give those of ordinary skill in the art a complete disclosure and description of embodiments of the compositions, systems and methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the present invention that are obvious to those skilled in the art are intended to fall within the scope of the appended claims. All patents and publications mentioned in the specification are indicative of the skill level of those skilled in the art to which the present invention belongs. All references cited in this disclosure are incorporated by reference to the same extent as if each reference were individually incorporated by reference in its entirety.

[0339] All headings and section names are used for clarity and reference purposes only and should not be considered limiting in any way. For example, one skilled in the art will appreciate that it is useful to combine various aspects from different headings and sections as appropriate in accordance with the spirit and scope of the invention described herein.

[0340] All references cited herein are hereby incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0341] Without departing from the spirit and scope of the present application, many modifications and changes may be made to the present application, which will be apparent to those skilled in the art. The specific embodiments and examples described herein are provided by way of example only, and the present application is limited only by the terms of the appended claims and the full scope of equivalents to which the claims are entitled.

Claims

1. An IL-2 mutant protein, comprising amino acid substitutions L18R, Q22E and Q126T numbered according to wild-type human IL-2 (hIL-2) (SEQ ID NO: 8), and further comprising a set of amino acid substitutions selected from the group consisting of: F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T and S130R.

2. The IL-2 mutant protein of claim 1, wherein the IL-2 mutant protein comprises amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T and S130R, optionally wherein the IL-2 mutant protein comprises the amino acid sequence of SEQ ID NO:

1.

3. The IL-2 mutant protein of claim 1, wherein the IL-2 mutant protein comprises amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A and E62A, optionally wherein the IL-2 mutant protein comprises the amino acid sequence of SEQ ID NO:

2.

4. The IL-2 mutant protein of claim 1, wherein the IL-2 mutant protein comprises amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R and F42A, optionally wherein the IL-2 mutant protein comprises the amino acid sequence of SEQ ID NO:

3.

5. The IL-2 mutant protein of claim 1, wherein the IL-2 mutant protein comprises amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A and Y45A, optionally wherein the IL-2 mutant protein comprises the amino acid sequence of SEQ ID NO:

4.

6. The IL-2 mutant protein of claim 1, wherein the IL-2 mutant protein comprises amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F and Q126T, optionally wherein the IL-2 mutant protein comprises the amino acid sequence of SEQ ID NO:

5.

7. The IL-2 mutant protein of claim 1, wherein the IL-2 mutant protein comprises amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, F42A and E62A, optionally wherein the IL-2 mutant protein comprises the amino acid sequence of SEQ ID NO:

6.

8. The IL-2 mutant protein of claim 1, wherein the IL-2 mutant protein comprises amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T and F42A, optionally wherein the IL-2 mutant protein comprises the amino acid sequence of SEQ ID NO:

7.

9. The IL-2 mutant protein according to any one of claims 1 to 8, wherein the IL-2 mutant protein is fused to an albumin molecule, an Fc molecule and / or another mutant protein, optionally wherein the other mutant protein is an IL-13 mutant protein or an IL-4 mutant protein.

10. The IL-2 mutein of claim 9, wherein the IL-2 mutein is fused to an albumin molecule. The IL-2 mutant protein of claim 10 , wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:

18.

12. The IL-2 mutant protein of claim 9, wherein the IL-2 mutant protein is fused to an Fc molecule.

13. The IL-2 mutant protein of claim 9, wherein the IL-2 mutant protein is fused to an Fc molecule and an IL-13 mutant protein, optionally wherein the IL-13 mutant protein comprises amino acid substitutions L10V, V18I, D87S, T88S, L101F, K104R and K105T (A11) numbered according to wild-type human IL-13 (hIL-13).

14. The IL-2 mutant protein of claim 9, wherein the IL-2 mutant protein is fused to an Fc molecule and an IL-4 mutant protein, optionally wherein the IL-4 mutant protein comprises amino acid substitutions R121K, Y124F and S125R (KFR) or K117R, T118V, R121Q, E122S, Y124W, S125F, S128G and S129A (RGA) numbered according to wild-type human IL-4 (hIL-4).

15. The IL-2 mutant protein of claim 14, wherein the fusion protein comprises the amino acid sequence of one of SEQ ID NOs: 19-22.

16. The IL-2 mutant protein of any one of claims 1-15, wherein the IL-2 mutant protein has increased binding to CD122 compared to wild-type IL-2.

17. The IL-2 mutein of any one of claims 1-16, wherein the IL-2 mutein has reduced binding to CD25 compared to wild-type IL-2.

18. The IL-2 mutein of any one of claims 1-17, wherein the IL-2 mutein has inhibitory activity as determined using a HEKBlue IL-2 and / or CTLL2 assay.

19. The IL-2 mutein of any one of claims 1-18, wherein the IL-2 mutein inhibits IL-2-induced pSTAT5 signaling in human PBMCs.

20. The IL-2 mutein of any one of claims 1-19, wherein the IL-2 mutein is non-toxic to mice as determined using a maximum tolerated dose (MTD) assay.

21. The IL-2 mutein of any one of claims 1-20, wherein the IL-2 mutein reduces disease score as determined using an Experimental Autoimmune Encephalomyelitis (EAE) assay.

22. A nucleic acid encoding the IL-2 mutant protein according to any one of claims 1 to 21.

23. A vector comprising the nucleic acid of claim 22.

24. A host cell comprising the nucleic acid of claim 22 or the vector of claim 23.

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