IL-2 superantagonist constructs, methods, and uses thereof

Abstract

Interleukin-2 (IL-2) muteins or variants thereof are provided. Pharmaceutical compositions comprising such IL-2 muteins are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 375,675, filed September 14, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Interleukin-2 (IL-2) is a pluripotent cytokine produced primarily by activated CD4+ T cells and plays a key role in mediating normal immune responses. IL-2 promotes the proliferation and expansion of activated T lymphocytes, enhances B cell proliferation, and activates monocytes and natural killer cells. These activities have led to the testing of IL-2 and its use as an approved cancer treatment (aldesleukin, Proleukin®). In eukaryotic cells, human IL-2 is synthesized as a 153-amino acid precursor polypeptide, from which 20 amino acids are removed to generate the mature, secreted form (Taniguchi 1983). Recombinant human IL-2 has been produced in E. coli (Rosenberg 1984), insect cells (Smith 1985), and mammalian COS cells (Taniguchi 1983).

[0003] Interleukin-2 (IL-2) is a type I cytokine with a four alpha-helical bundle structure that was originally identified as a T-cell growth factor (Morgan et al., Science 193:1007 (1976)), but has since been shown to have a wide range of actions. IL-2 promotes T helper differentiation (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 natural killer activity and lymphokine-activated killer activity (Liao et al., Immunity 38:13 (2013)), and mediates activation-induced cell death (AICD) (Lenardo et al., Nature 353:858 (1991)).

[0004] IL-2 functions by interacting with three distinct 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 identified was IL-2Rα, a 55 kD polypeptide (p55) that appears upon T cell activation and was originally called Tac (T activation) antigen. IL-2Rα interacts with IL-2 approximately 10 -8 K of M dIL-2 binds to the IL-2 receptor at a specific site, also known as the "low affinity" IL-2 receptor. IL-2 binding to cells expressing only IL-2Rα does not result in any detectable biological response. In most cases, IL-2 acts through three distinct receptors: IL-2Rα, IL-2Rβ, and IL-2Rγ. Most cells, such as resting T cells, do not respond to IL-2 because they express only IL-2Rβ and IL-2Rγ, which have low affinity for IL-2. Upon stimulation, resting T cells express the relatively high affinity IL-2 receptor, IL-2Rα. When IL-2 binds to IL-2Rα, this receptor sequentially binds to IL-2Rβ and IL-2Rγ, resulting in T cell activation. IL-2 "superkines" have previously been developed with enhanced binding affinity for IL-2Rβ (Levin et al., Nature 2015). re 484:529(2012)).

[0005] Despite the wealth of knowledge related to IL-2, including IL-2 superantagonists, there remains a need in the art for more effective therapies for the treatment of cancer, including new therapies involving the IL-2 muteins provided herein. Summary of the Invention

[0006] IL-2 exerts a wide range of effects on the immune system, playing an important role in regulating both immune activation and homeostasis, and is used in the treatment of cancer.

[0007] The present invention includes the amino acid substitutions L18R, Q22E, and Q126T, numbered according to wild-type human IL-2 (hIL-2) (SEQ ID NO: 8), and further includes a group of amino acid substitutions selected from the group consisting of F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T, and S130R.

[0008] In some embodiments, the IL-2 mutein comprises the amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, and S130R, and optionally the IL-2 mutein comprises the amino acid sequence of SEQ ID NO:1.

[0009] In some embodiments, the IL-2 mutein comprises the amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A, and E62A, and optionally, the IL-2 mutein comprises the amino acid sequence of SEQ ID NO:2.

[0010] In some embodiments, the IL-2 mutein comprises the amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, and F42A, and optionally the IL-2 mutein comprises the amino acid sequence of SEQ ID NO:3.

[0011] In some embodiments, the IL-2 mutein comprises the amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A, and Y45A, and optionally the IL-2 mutein comprises the amino acid sequence of SEQ ID NO:4.

[0012] In some embodiments, the IL-2 mutein comprises the amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, and Q126T, and optionally the IL-2 mutein comprises the amino acid sequence of SEQ ID NO:5.

[0013] In some embodiments, the IL-2 mutein comprises the amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, F42A, and E62A, and optionally the IL-2 mutein comprises the amino acid sequence of SEQ ID NO:6.

[0014] In some embodiments, the IL-2 mutein comprises the amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, and F42A, and optionally the IL-2 mutein comprises the amino acid sequence of SEQ ID NO:7.

[0015] In some embodiments, the IL-2 mutein is fused to an albumin molecule, an Fc molecule, and / or another mutein, optionally the other mutein being an IL-13 mutein or an IL-4 mutein.

[0016] In some embodiments, the IL-2 mutein is fused to an albumin molecule.

[0017] In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:18.

[0018] In some embodiments, the IL-2 mutein is fused to an Fc region.

[0019] In some embodiments, the IL-2 mutein is fused to an Fc molecule and an IL-13 mutein, and optionally the IL-13 mutein contains the amino acid substitutions L10V, V18I, D87S, T88S, L101F, K104R, and K105T (A11), numbered according to wild-type human IL-13 (hIL-13).

[0020] In some embodiments, the IL-2 mutein is fused to an Fc molecule and an IL-4 mutein, optionally the IL-4 mutein containing the 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).

[0021] In some embodiments, the fusion protein comprises the amino acid sequence of one of SEQ ID NOs: 19-22.

[0022] In some embodiments, the IL-2 mutein has increased binding to CD122 compared to wild-type IL-2.

[0023] In some embodiments, the IL-2 mutein has reduced binding to CD25 compared to wild-type IL-2.

[0024] In some embodiments, the IL-2 mutein has inhibitory activity as determined using the HEKBlue IL-2 and / or CTLL2 assay.

[0025] In some embodiments, the IL-2 mutein inhibits IL-2-induced pSTAT5 signaling in human PBMCs.

[0026] In some embodiments, the IL-2 mutein exhibits no toxicity in mice as determined using a maximum tolerated dose (MTD) assay.

[0027] In some embodiments, the IL-2 mutein reduces disease scores as determined using an experimental autoimmune encephalomyelitis (EAE) assay.

[0028] The present invention also provides nucleic acids encoding the IL-2 muteins described herein.

[0029] The present invention also provides vectors comprising the nucleic acids described herein.

[0030] The present invention also provides a host cell comprising a nucleic acid or vector described herein. [Brief explanation of the drawings]

[0031] [Figure 1] 1 shows a schematic diagram of MDNA209, an IL-2 / IL-15 antagonist with a unique mechanism of action. [Figure 2A] 1 shows SPR sensorgrams of IL-2 antagonist constructs binding to the IL-2Rββc complex. [Figure 2B]1 shows SPR sensorgrams of IL-2 antagonist constructs binding to human CD25 and CD122. [Figure 2C] 1 shows SPR sensorgrams of IL-2 antagonist constructs binding to human CD25 and CD122. [Figure 2D] 1 shows SPR sensorgrams of IL-2 antagonist constructs binding to human CD25 and CD122. [Figure 3] Representative dose-response curves of different MDNA209 antagonist molecules in the HEK-Blue™ IL-2 reporter assay are shown. Each compound was tested in agonist (A, C) and antagonist (B) formats. In A, the EC values ​​for activated and background levels of IL-2 are indicated by the upper and lower dotted lines, respectively. [Figure 4] Representative graphs of dose response in a CTLL2 proliferation assay with IL-2-Fc alone or in the presence of 30 nM MDNA209-Fc are shown. The four-parameter logistic curve fit is shown as a solid line. Error bars represent the standard error of the mean from replicate wells. [Figure 5A] Representative dose-response curves of pSTAT5 (%) in response to antagonists in the presence of 67 pM rhIL-2 are shown. [Figure 5B] Representative dose-response curves of pSTAT5 (%) in response to antagonists in the presence of 670 pM rhIL-2 (FIG. 5B) are shown. [Figure 6] 1 shows the effects of MDNA209FEAA-Fc-A11 (30 mg / kg) and A11-Fc (21 and 11 mg / kg) on ​​the body weight of C57BL / 6J mice. [Figure 7] Figure 1 shows EAE scores (mean ± SEM) in 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). [Figure 8]Figure 1 shows EAE scores and body weights for mice treated with PBS, MDNA209FEAA-Fc-A11 (20 mg / kg), or the 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). For the combination of MDNA209-Fc and A11-Fc, n = 7 for days 6-10, n = 6 for day 12, and n = 1 for days 15 and beyond. For MDNA209FEAA-Fc-A11, n = 7 for days 6-14, and n = 4 for days 15 and beyond. Scores for dead mice were carried forward. [Figure 9] Figure 1 shows EAE scores in mice treated with PBS or MDNA209FEAA-Fc-A11 at a therapeutic dosing schedule. Mice were treated at 20 mg / kg and 5 mg / kg as indicated (dotted lines) on days 12, 16, and 19 after immunization. Data are presented as mean ± SEM, n = 7 for PBS treatment and n = 5 for MDNA209FEAA-Fc-A11 treatment. [Figure 10] IFNγ levels were quantified by ELISA in PBMCs treated with increasing concentrations of MDNA209-Fc for 48 hours in the presence of the indicated concentrations of rhIL-2. [Figure 11] MDNA209-Fc induced a dose-dependent inhibition (expressed as a percentage of maximum) of IFNγ production in human PBMCs stimulated with 0.3 μg / ml rhIL-2 from four different donors. [Figure 12] Body weights (A and B) and total lymphocyte counts (C and D) of animals administered PBS or MDNA209-Fc are shown. [Figure 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). [Figure 14]MDNA209-Fc inhibits proliferation of PBMCs in an MLR assay. Representative nonlinear fitted curves of the stimulation index (SI) of MDNA209-Fc, MDNA209-albumin, and MDNA209FEAA-Fc are plotted against the respective dose ranges tested. [Figure 15A] 1 shows sensorgrams of IL-2 antagonist binding to human IL2R (CD25) and IL2Rβ (CD122). [Figure 15B] 1 shows sensorgrams of IL-2 antagonist binding to human IL2R (CD25) and IL2Rβ (CD122). DETAILED DESCRIPTION OF THE INVENTION

[0032] In order that the present disclosure may be more readily understood, certain terms and phrases are defined below and throughout the specification.

[0033] definition All references cited herein are incorporated by reference in their entirety as if fully set forth. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons (New York, NY 2001), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5th ed., J. Wiley & Sons (New York, NY 2001), and Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001) can provide those skilled in the art with a general guide to many of the terms used in this disclosure. Where appropriate, procedures involving the use of commercially available kits and reagents are generally performed according to manufacturer-defined protocols and / or parameters unless otherwise noted.

[0034] As used herein, "IL-2" refers to wild-type IL-2, whether native or recombinant. Mature human IL-2 occurs as a 133 amino acid sequence (excluding a signal peptide consisting of an additional 20 N-terminal amino acids) as described in Fujita, et al., PNAS USA, 80, 7437-7441 (1983). The amino acid sequence of human IL-2 (SEQ ID NO: 11, full length) can be found in Genbank under accession number NP_000577.2. The amino acid sequence of mature human IL-2 is shown in SEQ ID NO: 8 (human wild-type mature; numbering of substitution positions is based on this sequence). The amino acid sequence of mouse (Mus musculus) IL-2 can be found in Genbank under accession number SEQ ID NO: 13. The amino acid sequence of mature mouse IL-2 is shown in SEQ ID NO: 12.

[0035] SEQ ID NO: 11 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT SEQ ID NO:8 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT SEQ ID NO: 13 MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYL PKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ SEQ ID NO: 12 APTSSSTSSSTAEAQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ

[0036] As used herein, "IL-2 mutein" refers to an IL-2 polypeptide in which a specific substitution has been made in the interleukin-2 protein. The IL-2 mutein is characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites in the native IL-2 polypeptide chain or at other residues in the native IL-2 polypeptide chain. According to the present disclosure, any of these insertions, deletions, substitutions, and modifications results in an IL-2 mutein that retains IL-2Rβ binding activity. Exemplary muteins can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid substitutions.

[0037] Muteins also contain conservative modifications and substitutions at other positions of IL-2 (i.e., positions that have minimal effect on the secondary or tertiary structure of the mutein). Such conservative substitutions include those described by Dayhoff in *The Atlas of Protein Sequence and Structure* 5 (1978) and by Argos in *EMBO J., 8:779-785 (1989). For example, amino acids belonging to one of the following groups are conservatively altered: Group I (ala, pro, gly, gln, asn, ser, thr), Group II (cys, ser, tyr, thr), Group III (val, ile, leu, met, ala, phe), Group IV (lys, arg, his), Group V (phe, tyr, trp, his), and Group VI (asp, glu).

[0038] "Numbered according to IL-2" means that the selected amino acid is identified with reference to the position at which that amino acid is normally found in the mature sequence of wild-type IL-2; for example, R81 refers to arginine, the 81st amino acid found in SEQ ID NO:8. L80 refers to leucine, the 80th amino acid found in SEQ ID NO:8. L85 refers to leucine, the 85th amino acid found in SEQ ID NO:8. I86 refers to isoleucine, the 86th amino acid found in SEQ ID NO:8. I92 refers to isoleucine, the 92nd amino acid found in SEQ ID NO:8. F42 refers to phenylalanine, the 42nd amino acid found in SEQ ID NO:8. K43 refers to lysine, the 43rd amino acid found in SEQ ID NO:8. L18 refers to leucine, the 18th amino acid found in SEQ ID NO:8. Q22 refers to glutamine, the 22nd amino acid found in SEQ ID NO:8. Q126 refers to glutamine, the 126th amino acid found in SEQ ID NO:8. S130 refers to the 130th amino acid, serine, found in SEQ ID NO: 8. E62 refers to the 62nd amino acid, glutamic acid, found in SEQ ID NO: 8. Y45 refers to the 45th amino acid, tyrosine, found in SEQ ID NO: 8.

[0039] As used herein, the abbreviations for the genetically encoded L-enantiomeric amino acids used in the methods of the present disclosure are conventional and are shown in Table 1 below.

[0040] [Table 1]

[0041] "Hydrophilic amino acid" refers to an amino acid that has a hydrophobicity of less than 0 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).

[0042] The term "cell type having an IL-2Rαβγ receptor" refers to cells known to have this type of receptor, i.e., T cells, activated T cells, B cells, activated monocytes, and activated NK cells. The term "cell type having an IL-2Rβγ receptor" refers to cells known to have that type of receptor, i.e., B cells, resting monocytes, and resting NK cells.

[0043] The term "identity," as used herein in reference to polypeptide or DNA sequences, refers to the identity of subunit sequences between two molecules. When a subunit position in both molecules is occupied by the same monomeric subunit (i.e., the same amino acid residue or the same nucleotide), the molecules are identical at that position. Similarity between two amino acid sequences or two nucleotide sequences is measured on a first-order basis based on the number of identical positions. The function is to align sequences so that the best match is generally obtained. If necessary, identity can be calculated using published techniques and widely available computer programs such as the GCS program package (Devereux et al., Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, and FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990). Sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705) with its default parameters.

[0044] The terms "polypeptide," "protein," or "peptide" refer to any chain of amino acid residues, regardless of its length or post-translational modification (eg, glycosylation or phosphorylation).

[0045] When a variant IL-2 polypeptide of the disclosure is "substantially pure," the polypeptide can be at least about 60% by weight (dry weight) of the polypeptide of interest, e.g., a polypeptide comprising the amino acid sequence of the variant IL-2. For example, the polypeptide can be at least about 75%, 80%, 85%, 90%, 95%, or 99% by weight of the polypeptide of interest. Purity can be measured by any appropriate standard method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0046] An "agonist" is a compound that interacts with a target to increase or promote increased activation of that target.

[0047] A "partial agonist" is a compound that interacts with the same target as an agonist, but where increasing doses of the partial agonist do not exert as great a biochemical and / or physiological effect as an agonist.

[0048] "Superagonists" (also called "superkines") are a class of agonists that can produce a maximal response that is greater than that of the endogenous agonist for the target receptor, i.e., greater than 100% effective.

[0049] "Operably linked" is intended to mean that the nucleotide sequence of interest (i.e., the sequence encoding the IL-2 mutein) is linked to a regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence of interest (e.g., in an in vitro transcription / translation system or within 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, e.g., Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.). Regulatory sequences include sequences that direct constitutive expression of a nucleotide sequence in many types of host cell and sequences that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be apparent to those skilled in the art that the design of the expression vector can depend on factors such as the choice of host cell to be transformed, the level of expression of the desired protein, etc. The expression constructs of the present invention can be introduced into host cells to thereby produce the human IL-2 muteins disclosed herein, or to produce biologically active variants thereof. This can be done.

[0050] The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in successive generations, either 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 terms as used herein.

[0051] As used herein, the terms "transformation" and "transfection" refer to various art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into host cells, including calcium phosphate co-precipitation, calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, particle gun or electroporation.

[0052] As used herein, the term "pharmaceutically acceptable carrier" includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics) can also be added to the compositions.

[0053] As used herein, the terms "cancer" (or "cancerous"), "hyperproliferative," and "neoplastic" refer to cells capable of autonomous growth (i.e., an abnormal state or condition characterized by rapidly proliferating cell growth). Hyperproliferative and neoplastic disease states can be classified as pathological (i.e., characterizing or constituting a disease state) or non-pathological (i.e., deviating from normal but not associated with a disease state). These terms are intended to include any type of cancerous growth, oncogenic process, metastatic tissue, or malignantly transformed cell, tissue, or organ, regardless of invasive histopathological type or stage. "Pathological hyperproliferative" cells are found in disease states characterized by malignant tumor growth. An example of non-pathological hyperproliferative cells is cell proliferation associated with wound repair. The terms "cancer" or "neoplasm" are used to refer to malignancies of various organ systems, including those affecting the lung, breast, thyroid, lymph nodes and tissues, reproductive system, gastrointestinal tract, and genitourinary tract, and adenocarcinomas, which are generally considered to include malignancies such as most colon cancers, renal cell carcinoma, prostate and / or testicular cancer, non-small cell carcinoma of the lung, cancer of the small intestine, and cancer of the esophagus. Cancers generally 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.

[0054] The term "carcinoma" is art-recognized and refers to malignant tumors of epithelial or endocrine tissue, including respiratory, gastrointestinal, genitourinary, testicular, breast, prostate, endocrine, and melanoma. "Adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0055] As used herein, the term "hematopoietic neoplastic disorder" refers to a disease involving hyperplastic / neoplastic cells of hematopoietic origin, e.g., diseases arising from the myeloid, lymphoid, or erythroid lineages, or their precursor cells. Preferably, the disease results from a poorly differentiated acute leukemia (e.g., erythroblastic leukemia and acute megakaryoblastic leukemia). Additional exemplary bone marrow disorders include, but are not limited to, acute promyelocytic leukemia (APML), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML) (discussed in Vaickus, L. (1991) Crit Rev. in Oncol. / Hemotol. 11:267-97). Lymphoid malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL) (including B-cell lineage ALL and T-cell lineage ALL), chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL), and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphoma include, but are not limited to, non-Hodgkin's lymphoma and its subtypes, 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-Sternberg disease.

[0056] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic, in terms of partially or completely curing a disease and / or adverse effects caused by the disease. "Treatment," as used herein, encompasses any treatment of a disease in a mammal, particularly a human, and includes (a) preventing the disease from occurring in a subject predisposed to or at risk of 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., reversing the disease. A therapeutically effective amount can be an amount that reduces tumor number, tumor size, and / or increases survival rates.

[0057] 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 apes and humans), sport mammals (e.g., horses), livestock mammals (e.g., sheep, goats, etc.), pet mammals (dogs, cats, etc.), and rodents (e.g., mice, rats, etc.).

[0058] The terms "pharmaceutically acceptable" and "physiologically acceptable" refer to a biologically acceptable formulation, gas, liquid, or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery, or contact. A "pharmaceutically acceptable" or "physiologically acceptable" composition is a substance that is not biologically or otherwise undesirable; e.g., the substance can be administered to a subject without causing any significant undesirable biological effects. That is, such a pharmaceutical composition can be used, for example, when administering an IL-2 mutein to a subject. In some embodiments, the administered IL-2 mutein further comprises a substitution at position F42A. In some embodiments, the administered IL-2 mutein further comprises a substitution at position K43N.

[0059] The phrase "unit dosage form," as used herein, refers to physically discrete units suitable for unitary administration to a subject to be treated, each unit containing a predetermined amount, optionally with a pharmaceutical carrier (excipient, diluent, vehicle, or filler), that, 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 tumor number. In some embodiments, the therapeutic effect is a reduction in tumor size. In some embodiments, the therapeutic effect is an increase in survival rate.

[0060] In some embodiments, unit dosage forms include liquid compositions or freeze-dried or lyophilized compositions, which may be contained, for example, in ampoules and vials, to which, for example, a sterile liquid carrier can be added prior to administration or delivery in vivo. Individual unit dosage forms may be included in multi-dose kits or containers. The IL-2 muteins and pharmaceutical compositions thereof may be administered in a convenient manner. For size and uniformity of dosage, the compound may be packaged in single or multiple unit dosage forms.

[0061] A "therapeutically effective amount" will fall in a relatively broad range that can be determined through experimentation and / or clinical trials. For example, in vivo injection, e.g., direct injection into the subject's tissue or vascular system (e.g., liver tissue or vein). Other effective dosages can be readily determined by one of ordinary skill in the art through routine testing to determine dose-response curves.

[0062] An "effective amount" or "sufficient amount" refers to an amount, in one or more doses, that alone or in combination with one or more other compositions (therapeutic agents such as drugs), treatments, protocols or therapeutic regimens (including, for example, vaccine regimens), results in a detectable response of any duration (long or short term), an expected or desired outcome or benefit in a subject, to any measurable or detectable degree, or of any duration (e.g., minutes, hours, days, months or years, or a cure), of any time period (long or short term).

[0063] An "effective amount" or "sufficient amount" for treatment (e.g., treatment to ameliorate or produce a therapeutic effect or improvement) is typically effective to measurably produce a response to one, more than one, or all of the adverse symptoms, outcomes, or complications of the disease, e.g., one or more adverse symptoms, disorders, illnesses, pathologies, or complications caused by or associated with the disease. However, decreasing, reducing, inhibiting, suppressing, limiting, or controlling the progression or worsening of the disease is also a sufficient 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 tumors. In some embodiments, an effective amount is an amount sufficient to increase survival rates.

[0064] "Prevention" and grammatical variations thereof refer to methods in which contact, administration, or in vivo delivery to a subject occurs prior to the onset of disease. Administration to a subject or in vivo delivery can occur prior to the onset of adverse symptoms, conditions, complications, etc., caused by or associated with the disease. For example, a screen (e.g., a genetic screen) can be used to identify such subjects as candidates for the described methods and uses, even if the disease is not yet apparent in the subject. Thus, such subjects include those who screen positive for producing an insufficient or missing functional gene product (protein), or an abnormal, partially functional, or non-functional gene product (protein) that leads to disease, even if the subject is not yet exhibiting symptoms of the disease, and those who screen positive for an abnormal or defective (mutant) gene product (protein) that leads to disease.

[0065] I. MODE FOR CARRYING OUT THE INVENTION Provided herein are IL-2 muteins containing the amino acid substitutions L18R, Q22E, and Q126T, numbered according to wild-type human IL-2 (hIL-2) (SEQ ID NO: 8), and further containing an amino acid substitution selected from the group consisting of F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T, and S130R. Such IL-2 muteins are useful, for example, in the treatment of cancer. Nucleic acids encoding such IL-2 muteins, methods of making such IL-2 muteins, pharmaceutical compositions containing such IL-2 muteins, and therapeutic methods using such IL-2 muteins are also provided.

[0066] A. IL-2 muteins The substituted amino acid residue(s) generally include substitutions within the group: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. These mutations may be, but are not necessarily, conservative substitutions. These mutations may be at amino acid residues that contact IL-2Rβ and / or IL-2Rγ.

[0067] More specifically, mutations (either conservative or non-conservative substitutions by addition(s) or deletion(s)) can be made at one or more positions. For example, the mutations can be L18R, Q22E, and Q126T, and can further include mutations selected from the group consisting of F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T, and S130R.

[0068] For example, the mutations can be L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A, and E62A.

[0069] For example, the mutations may be L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, and F42A.

[0070] For example, the mutations can be L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A, and Y45A.

[0071] For example, the mutations can be L18R, Q22E, L80F, R81D, L85V, I86V, I92F, and Q126T.

[0072] For example, the mutations can be L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, F42A, and E62A.

[0073] For example, the mutations may be L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, and F42A.

[0074] [Table 2]

[0075] In some embodiments, the substitutions in the IL-2 mutein comprise L18R, Q22E, and Q126T, numbered according to wild-type human IL-2 in SEQ ID NO: 8, and further comprise 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, the substitutions in the IL-2 mutein comprise L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, and S130R, numbered according to wild-type human IL-2 in SEQ ID NO: 8. In some embodiments, the substitutions in the IL-2 mutein are L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S13 0R, F42A, and E62A. In some embodiments, the substitutions in the IL-2 mutein include L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, and F42A, numbered according to wild-type human IL-2 in SEQ ID NO: 8. In some embodiments, the substitutions in the IL-2 mutein include L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A, and Y45A, numbered according to wild-type human IL-2 in SEQ ID NO: 8. In some embodiments, the substitutions in the IL-2 mutein include L18R, Q22E, L80F, R81D, L85V, I86V, I92F, and Q126T, numbered according to wild-type human IL-2 in SEQ ID NO: 8. In some embodiments, the substitutions in the IL-2 mutein include L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, F42A, and E62A, numbered according to wild-type human IL-2 in SEQ ID NO: 8. In some embodiments, the substitutions in the IL-2 mutein include L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, and F42A, numbered according to wild-type human IL-2 in SEQ ID NO: 8.

[0076] In some embodiments, substitutions in an IL-2 mutein that increase and / or enhance binding to IL-2Rβ include L18R, Q22E, L80F, R81D, L85V, I86V, I92F, and Q126T, numbered according to wild-type human IL-2 in SEQ ID NO: 8. In some embodiments, an IL-2 mutein used herein includes L18R, Q22E, L80F, R81D, L85V, I86V, I92F, and Q126T and exhibits increased binding to IL-2Rβ. In some embodiments, an IL-2 mutein used in the invention further includes a substitution at position S130R. In some embodiments, an IL-2 mutein used in the invention further includes a substitution at position S130R. In some embodiments, an IL-2 mutein used in the invention further includes a substitution at position F42A. In some embodiments, an IL-2 mutein used in the invention further includes a substitution at position E62A. In some embodiments, an IL-2 mutein for use in the invention further comprises a substitution at position Y45A. In some embodiments, the substitutions in the IL-2 mutein comprise L18R, Q22E, and Q126T, numbered according to wild-type human IL-2 of SEQ ID NO: 8, and further comprise 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, the IL-2 mutein comprises the substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, and S130R, numbered according to wild-type human IL-2 of SEQ ID NO: 8. In some embodiments, the IL-2 mutein comprises the substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A, and E62A, numbered according to wild-type human IL-2 in SEQ ID NO: 8. In some embodiments, the IL-2 mutein comprises the substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, and F42A, numbered according to wild-type human IL-2 in SEQ ID NO: 8.In some embodiments, the IL-2 mutein comprises the substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A, and Y45A, numbered according to wild-type human IL-2 in SEQ ID NO: 8. In some embodiments, the IL-2 mutein comprises the substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, and Q126T, numbered according to wild-type human IL-2 in SEQ ID NO: 8. In some embodiments, the IL-2 mutein comprises the substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, F42A, and E62A, numbered according to wild-type human IL-2 in SEQ ID NO: 8. In some embodiments, the IL-2 mutein comprises the substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, and F42A, numbered according to wild-type human IL-2 in SEQ ID NO:8.

[0077] In some embodiments, the mutein comprises one or more substitutions selected from the group consisting of the substitutions L18R, Q22E, and Q126T, and F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T, and S130R, all relative to wild-type human IL-2 (SEQ ID NO: 8).

[0078] In some embodiments, amino acid substitutions that increase binding affinity to IL-2Rβ include L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A, E62A, and / or Y45A. In some embodiments, amino acid substitutions that increase binding affinity to IL-2Rβ include L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A, E62A, and / or Y45A.

[0079] In some embodiments, an IL-2 mutein of the invention having a higher binding affinity to IL-2Rβ compared to wild-type human IL-2 comprises the amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, and S130R. In some embodiments, the IL-2 mutein has the amino acid sequence: I have TIFF2025532554000004.tif22170.

[0080] In some embodiments, an IL-2 mutein of the invention having a higher binding affinity to IL-2Rβ compared to wild-type human IL-2 comprises the amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A, and E62A. In some embodiments, the IL-2 mutein has the amino acid sequence: I have TIFF2025532554000005.tif21170.

[0081] In some embodiments, an IL-2 mutein of the invention having a higher binding affinity to IL-2Rβ compared to wild-type human IL-2 comprises the amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, and F42A. In some embodiments, the IL-2 mutein has the amino acid sequence: I have TIFF2025532554000006.tif23170.

[0082] In some embodiments, an IL-2 mutein of the invention having a higher binding affinity to IL-2Rβ compared to wild-type human IL-2 comprises the amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A, and Y45A. In some embodiments, the IL-2 mutein has the amino acid sequence: I have TIFF2025532554000007.tif21170.

[0083] In some embodiments, an IL-2 mutein of the invention having a higher binding affinity to IL-2Rβ compared to wild-type human IL-2 comprises the amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, and Q126T. In some embodiments, the IL-2 mutein has the amino acid sequence: I have TIFF2025532554000008.tif21170.

[0084] In some embodiments, an IL-2 mutein of the invention having a higher binding affinity to IL-2Rβ compared to wild-type human IL-2 comprises the amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, F42A, and E62A. In some embodiments, the IL-2 mutein has the amino acid sequence: I have TIFF2025532554000009.tif21170.

[0085] In some embodiments, an IL-2 mutein of the invention having a higher binding affinity to IL-2Rβ compared to wild-type human IL-2 comprises the amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, and F42A. In some embodiments, the IL-2 mutein has the amino acid sequence: I have TIFF2025532554000010.tif21170.

[0086] In some embodiments, the IL-2 mutein sequence is at least about 90% identical to any one of SEQ ID NOs:1-7. In some embodiments, the IL-2 mutein sequence is at least about 95% identical to any one of SEQ ID NOs:1-7. In some embodiments, the IL-2 mutein sequence is at least about 98% identical to any one of SEQ ID NOs:1-7. In some embodiments, the IL-2 mutein sequence is at least about 99% identical to any one of SEQ ID NOs:1-7.

[0087] B. IL-2 Mutein Fusion Protein IL-2 muteins can be prepared as fusion or chimeric polypeptides comprising an IL-2 mutein of the invention and a heterologous polypeptide (i.e., a polypeptide that is not IL-2 or a variant thereof) (see, e.g., U.S. Pat. No. 6,451,308). Exemplary heterologous polypeptides can extend the circulating half-life of the chimeric polypeptide in vivo, thereby further enhancing the properties of the mutant IL-2 polypeptides of the invention. In various embodiments, the polypeptide that extends circulating half-life is a serum antibody. The Fc region may be albumin (such as human serum albumin), PEG, a PEG derivative, or the Fc region of an IgG subclass antibody (lacking the heavy chain variable region of IgG). Exemplary Fc regions may contain mutations that inhibit complement fixation and Fc receptor binding, or may be lytic, i.e., capable of lysing cells by binding complement or another mechanism such as antibody-dependent complement lysis (ADCC, U.S. Patent No. 08 / 355,502, filed December 12, 1994).

[0088] The "Fc region" can be a naturally occurring polypeptide or a synthetic polypeptide homologous to the IgG C-terminal domain produced by digesting IgG with papain. IgG Fc has a molecular weight of approximately 50 kDa. A mutant IL-2 polypeptide can comprise the entire Fc region, or a smaller Fc region 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 Fc region or a fragmented Fc region can be a variant of the wild-type molecule. In some embodiments, an IL-2 mutein fusion protein (e.g., an IL-2 mutein as described herein) comprises an IgG1, IgG2, IgG3, or IgG4 Fc region (see, e.g., the sequences in Figures 2A-2B). In some embodiments, the Fc region comprises the substitution N297A.

[0089] In some embodiments, the IL-2 mutein is linked directly or indirectly to a heterologous fusion polypeptide.

[0090] In some embodiments, the IL-2 mutein is linked directly to the Fc region. In some embodiments, the IL-2 mutein is linked to the Fc region via a linker peptide, such as GGGGS. In some embodiments, the linker is (GGGGS)n, where n is an integer from 1 to 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 GGGGSGGGGGSGGGGGS (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).

[0091] Fc regions can be "lytic" or "nonlytic," but are typically nonlytic. Nonlytic Fc regions typically lack the high-affinity Fc receptor binding site and the C'1q binding site. The high-affinity Fc receptor binding site of mouse IgG Fc contains a Leu residue at position 235 of IgG Fc. That is, the Fc receptor binding site can be disrupted by mutating or deleting Leu235. For example, substituting Leu235 with Glu inhibits the ability of the Fc region to bind to the high-affinity Fc receptor. The function of the mouse C'1q binding site can be disrupted by mutating or deleting residues Glu318, Lys320, and Lys322 of IgG. For example, substituting Glu318, Lys320, and Lys322 with Ala residues disrupts the binding of IgG1 The Fc region of a soluble IgG lacks the ability to induce antibody-dependent complement lysis. In contrast, the 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 contains a Leu residue at position 235 of IgG Fc, and the C'1q binding site contains residues Glu318, Lys320, and Lys322 of IgG1. Soluble IgG Fc contains wild-type residues or conservative amino acid substitutions at these sites. Soluble IgG Fc can target cells for antibody-dependent cellular cytotoxicity or complement-induced cytolysis (CDC). Mutations suitable for human IgG are also known (see, e.g., Morrison et al., The Immunologist 2:119-124, 1994 and Brekke et al., The Immunologist 2:125, 1994).

[0092] In another embodiment, a chimeric polypeptide can comprise an IL-2 mutein of the present invention and a polypeptide that functions as an antigenic tag, such as a FLAG sequence. The FLAG sequence is recognized by a highly specific biotinylated anti-FLAG antibody, as described herein (see also Blanar et al., Science 256:1014, 1992; LeClair et al., Proc. Natl. Acad. Sci. USA 89:8145, 1992). In some embodiments, the chimeric polypeptide further comprises a C-terminal c-myc epitope tag.

[0093] In another embodiment, the chimeric polypeptide comprises a mutant IL-2 polypeptide and a heterologous polypeptide (such as an agglutinin subunit called Aga2p) that functions to enhance expression of the mutant IL-2 polypeptide or to induce cellular localization of the mutant IL-2 polypeptide (see, e.g., Boder and Wittrup, Nature Biotechnol. 15:553-7, 1997).

[0094] In another embodiment, a chimeric polypeptide can be produced comprising a mutant IL-2 and an antibody or its antigen-binding portion. The antibody or antigen-binding portion of the chimeric protein can function as a targeting moiety. For example, the antibody or antigen-binding portion 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.

[0095] In other embodiments, chimeric polypeptides can be made that include mutant IL-2 and IL-4 proteins. Any IL-4 sequence or variant thereof, including those described herein, can be used in fusions with IL-2 mutants or variants. In some embodiments, mutant IL-2 is fused to an IL-4 mutant having the sequence of SEQ ID NO: 23, as shown below: SEQ ID NO: 23 (KFR) KCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMKEKFRKCSS In some embodiments, the mutant IL-2 is fused to an IL-4 mutant having the sequence of SEQ ID NO: 24, shown below: SEQ ID NO: 24 (cpRGA) MDTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLRVIMQSKWFKCGAGGNGGHKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAAS In some embodiments, the mutant IL-2 is fused to an IL-4 mutant having the sequence of SEQ ID NO: 25, shown below: SEQ ID NO: 25 (RGA) HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLRVIMQSKWFKCGA

[0096] In some embodiments, the mutant IL-2 is fused to an IL-4 variant having a sequence at least about 90% identical to SEQ ID NO: 23. In some embodiments, the mutant IL-2 is fused to an IL-4 variant 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 variant 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 variant having a sequence at least about 95% identical to SEQ ID NO:23. In some embodiments, mutant IL-2 is fused to an IL-4 variant 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 variant 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 variant 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 variant 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 variant 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 variant having a sequence at least about 99% identical to SEQ ID NO:23. In some embodiments, the mutant IL-2 is fused to an IL-4 variant having a sequence at least about 99% identical to SEQ ID NO: 24. In some embodiments, the mutant IL-2 is fused to an IL-4 variant having a sequence at least about 99% identical to SEQ ID NO: 25.

[0097] In some embodiments, SEQ ID NO: 23 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 24 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 25 is linked to an IL-2 or IL-2 mutein described herein.

[0098] In some embodiments, the IL-2 mutein sequence is at least about 90% identical to any one of SEQ ID NOs: 1-7 (e.g., any of the IL-2 sequences presented herein). In some embodiments, the IL-2 mutein sequence is at least about 95% identical to any one of SEQ ID NOs: 1-7 (e.g., any of the IL-2 sequences presented herein). In some embodiments, the IL-2 mutein sequence is at least about 98% identical to any one of SEQ ID NOs: 1-7 (e.g., any of the IL-2 sequences presented herein). In some embodiments, the IL-2 mutein sequence is at least about 99% identical to any one of SEQ ID NOs: 1-7 (e.g., any of the IL-2 sequences presented herein). In some embodiments, the IL-2 mutein further comprises and / or is combined with an A11 mutein 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).

[0099] In some embodiments, the sequence of the IL-2 variant fusion protein is at least about 90% identical to any one of SEQ ID NOs: 18-22. In some embodiments, the sequence of the IL-2 variant fusion protein is at least about 95% identical to any one of SEQ ID NOs: 18-22. In some embodiments, the sequence of the IL-2 variant fusion protein is at least about 98% identical to any one of SEQ ID NOs: 18-22. In some embodiments, the sequence of the IL-2 variant fusion protein is at least about 99% identical to any one of SEQ ID NOs: 18-22. In some embodiments, the sequence of the IL-2 variant fusion protein comprises any one of SEQ ID NOs: 18-22.

[0100] [Table 3-1] [Table 3-2]

[0101] C. Recombinant Expression of IL-2 Muteins, Expression Vectors and Host Cells In various embodiments, the polypeptides used in practicing the invention are synthesized or produced by expressing a recombinant nucleic acid molecule. When the polypeptide is chimeric (e.g., a fusion protein comprising at least a mutant IL-2 polypeptide and a heterologous polypeptide), the polypeptide encodes all or part of an IL-2 mutein. The IL-2 muteins of the invention can be encoded by a hybrid nucleic acid molecule comprising one sequence encoding a heterologous polypeptide and a second sequence encoding all or part of the heterologous polypeptide. For example, the IL-2 muteins of the invention described herein can be fused to a hexahistidine tag to facilitate purification of proteins expressed in bacteria, or a hemagglutinin tag to facilitate purification of proteins expressed in eukaryotic cells.

[0102] Methods for constructing DNA sequences encoding IL-2 muteins and expressing these sequences in an appropriately transformed host include, but are not limited to, the use of PCR-mediated mutagenesis. Mutations consisting of deletions or additions of amino acid residues to an IL-2 polypeptide can also be made using standard recombinant techniques. For deletions or additions, the nucleic acid molecule encoding IL-2 is optionally digested with an appropriate restriction enzyme. The resulting fragments can be expressed directly or further manipulated, for example, by ligating the fragment to a second fragment. Ligation can be facilitated if the two ends of the nucleic acid molecule contain overlapping complementary nucleotides, although blunt-ended fragments can also be ligated. Nucleic acids generated by PCR can also be used to generate a variety of mutant sequences.

[0103] The complete amino acid sequence can be used to construct a reverse-translated gene. DNA oligomers containing nucleotide sequences encoding IL-2 muteins can be synthesized. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0104] In addition to producing mutant polypeptides by expressing nucleic acid molecules altered by molecular biological recombinant techniques, the IL-2 muteins of the present invention can be chemically synthesized, which can be produced by conventional methods by those skilled in the art.

[0105] Once assembled (by synthesis, site-directed mutagenesis, or otherwise), the DNA sequence encoding the IL-2 mutein is inserted into an expression vector and operably linked to expression control sequences suitable for expressing the IL-2 mutein in the desired host to be transformed. Proper assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, to obtain high levels of expression of a transfected gene in a host, the gene must be operably linked to transcriptional and translational expression control sequences that are functional in the selected expression host.

[0106] The DNA sequence encoding an IL-2 mutein, whether 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 one that is recognized by the cell selected for expression of the IL-2 mutein. The signal sequence can be a prokaryotic sequence, a eukaryotic sequence, or a combination of the two. It can also be the signal sequence of native IL-2. The inclusion of a signal sequence depends on whether it is desired to secrete the IL-2 mutein from the recombinant cell in which it is produced. If the selected cell is a prokaryotic cell, it is generally preferred that the DNA sequence does not encode a signal sequence. If the selected cell is a eukaryotic cell, it is generally preferred that a signal sequence be encoded, with the wild-type IL-2 signal sequence being most preferred.

[0107] D. Nucleic Acid Molecules Encoding Variant IL-2 In some embodiments, the IL-2 muteins of the present invention are administered alone or in combination with any of the above-described The IL-2 muteins can be obtained by expression of a nucleic acid molecule as part of a chimeric polypeptide, such as that described herein. Just as IL-2 muteins can be described in terms of their identity to a wild-type IL-2 polypeptide, a nucleic acid molecule encoding an IL-2 mutein will necessarily have a certain identity to a nucleic acid molecule encoding wild-type IL-2. For example, a nucleic acid molecule encoding an IL-2 mutein of the invention 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 a nucleic acid encoding wild-type IL-2 (e.g., SEQ ID NO: 8).

[0108] The provided nucleic acid molecules can include naturally occurring sequences or sequences that differ from sequences found in nature but, due to the degeneracy of the genetic code, encode the same polypeptide. These nucleic acid molecules can be composed of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA (such as DNA produced by phosphoramidite-based synthesis)), or combinations or modifications of the nucleotides within these types of nucleic acids. In addition, the nucleic acid molecules can be double-stranded or single-stranded (i.e., either the sense or antisense strand).

[0109] The nucleic acid molecule is not limited to a sequence encoding a polypeptide, but can also include some or all of the non-coding sequences upstream or downstream from the coding sequence (e.g., the coding sequence for IL-2). Those skilled in the art of molecular biology are familiar with conventional procedures for isolating nucleic acid molecules. These molecules can be prepared, for example, by treating genomic DNA with restriction enzymes or by performing the polymerase chain reaction (PCR). If the nucleic acid molecule is ribonucleic acid (RNA), the molecule can be prepared, for example, by in vitro transcription.

[0110] Exemplary isolated nucleic acid molecules of the present disclosure can include fragments not found in nature, etc. That is, the present disclosure includes recombinant molecules in which a nucleic acid sequence (e.g., a sequence encoding a mutant IL-2) is incorporated into a vector (e.g., a plasmid or viral vector) or into the genome of a heterologous cell (or into the genome of a homologous cell at a location other than its natural chromosomal location).

[0111] As described above, the IL-2 muteins of the present invention may be present as part of a chimeric polypeptide. In addition to, or instead of, the heterologous polypeptides described above, the nucleic acid molecules of the present invention may contain sequences encoding a "marker" or "reporter." Examples of marker or reporter genes include β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo), and the like. r , G418 r ), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacz (encoding β-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT). Those skilled in the art will recognize additional useful reagents, for example, additional sequences that can serve the function of a marker or reporter.

[0112] The nucleic acid molecules of the present invention can be obtained by introducing mutations into DNA encoding IL-2 obtained from any living cell (e.g., a mammalian cell). That is, the nucleic acids of the present invention (and the polypeptides they encode) can be from a mouse, rat, guinea pig, cow, sheep, horse, pig, rabbit, monkey, baboon, dog, or cat. In one embodiment, the nucleic acid molecule is a human molecule.

[0113] E. Expression of Mutant IL-2 Gene Products The nucleic acid molecules described above can be included, for example, in a vector capable of directing expression in cells transduced with the vector. Thus, in addition to the IL-2 muteins of the present invention, Expression vectors containing nucleic acid molecules encoding the IL-2 muteins of the present invention, and cells transfected with these vectors, are included in preferred embodiments.

[0114] It should be understood, of course, that not all vectors and expression control sequences function equally well to express the DNA sequences described herein. Also, not all hosts function equally well in the same expression system. However, one of skill in the art can make a selection from among these vectors, expression control sequences, and hosts without undue experimentation. For example, when selecting a vector, the host must be considered, since the vector must replicate within the host. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector (such as antibiotic markers) must also be considered. For example, vectors that can be used include those that amplify the copy number of DNA encoding an IL-2 mutein. Such amplifiable vectors are well known in the art. Examples of such vectors include those that can be amplified by DHFR amplification (see, e.g., U.S. Pat. No. 4,470,461 to Kaufman; Kaufman and Sharp, "Construction of a Modular Dihydrafolate Reductase cDNA Gene: Analysis of Signals Utilized for Efficient Expression," Mol. Cell. Biol., 2, pp. 1304-19 (1982)) or glutamine synthetase ("GS") amplification (see, e.g., U.S. Pat. No. 5,122,464 and European Patent Application Publication No. 338,841).

[0115] In some embodiments, the human IL-2 muteins of the present disclosure will be expressed from a vector, preferably an expression vector. The vector may be useful for autonomous replication in a host cell or may be integrated into the genome of the host cell upon introduction into the host cell, thereby replicating along with the host genome (e.g., non-episomal mammalian vectors). Expression vectors are capable of directing the expression of coding sequences that are operably linked to them. Generally, expression vectors useful in recombinant DNA techniques are often in the form of plasmids (vectors). However, other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses and adeno-associated viruses), are also included.

[0116] 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.

[0117] The expression construct or vector can be designed to express the IL-2 mutein or variant thereof in a prokaryotic or eukaryotic host cell.

[0118] Vector DNA can be introduced into prokaryotic or eukaryotic cells via 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 (2d ed., Cold Spring Harbor Laboratory Press, Plainview, NY) and other standard molecular biology laboratory manuals.

[0119] Protein expression in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters. Strategies for maximizing recombinant protein expression in E. coli are described, for example, by Gotte et al. and Wada et al. (1992) Nucleic Acids Res. 20:2111-2118. Processes for growing, recovering, disrupting, or extracting IL-2 muteins or variants thereof from cells are substantially as described in, for example, U.S. Patent 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, each of which is incorporated by reference in its entirety.

[0120] 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 available for expression of proteins in cultured insect cells, such as Sf9 cells, include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39)), yeast cells (examples of vectors for expression in the yeast S. cerenvisiae include pYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1989) Cell 30:943-943), pYpSec1 (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:94 ...YpSec1 (Schultz et al. (1989) Cell 30:943-943), pYpSec1 (Schultz et al. (1989) Cell 30:943), pYpSec1 (Schultz et al. (1989) Cell 3 Expression vectors include vectors derived from vectors such as pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187:195) or mammalian cells (mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187:195)). Suitable mammalian cells include Chinese hamster ovary cells (CHO) or COS cells. In mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other expression systems suitable for both prokaryotic and eukaryotic cells, see Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2 nd See Chapters 16 and 17 of Gene Expression Technology: Methods in Enzymology, ed., Cold Spring Harbor Laboratory Press, Plainview, NY. 185 (Academic Press, San Diego, Calif.).

[0121] The sequences encoding the human IL-2 muteins of the present disclosure can be optimized for expression in the target host cell. The GC content of the sequences can be adjusted to the average level for that cellular host, as calculated by reference to known genes expressed in a given host cell. Methods for optimizing codons are well known in the art. The codons in the IL-2 mutein coding sequence can be optimized to enhance expression in the host cell, such that about 1%, about 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons in the coding sequence are optimized for expression in a particular host cell.

[0122] Suitable vectors for use include T7-based vectors used in bacteria (see, e.g., Rosenberg et al., Gene 56:125, 1987). Examples of vectors that can be used include the pMSXND expression vector (Lee and Nathans, J. Biol. Chem. 263:3521, 1988) used in mammalian cells, and baculovirus-derived vectors used in insect cells (e.g., the expression vector pBacPAK9 manufactured by Clontech, Palo Alto, Calif.).

[0123] In some embodiments, in such vectors, the nucleic acid insert encoding the IL-2 mutein of the present invention can be operably linked to a promoter, which is selected, for example, based on the cell type in which expression is desired.

[0124] A variety of factors must also be considered when selecting an expression control sequence. These factors include, for example, the relative strength and control of the sequence, and compatibility with the actual DNA sequence encoding the IL-2 mutein of the present invention, particularly with respect to potential secondary structure. A host should be selected taking into consideration compatibility with the selected vector, the toxicity of the product encoded by the DNA sequence of the present invention, the secretion characteristics of the host, the ability to correctly fold the polypeptide, fermentation or cultivation requirements, and the ease of purification of the product encoded by the DNA sequence.

[0125] One skilled in the art can select various vector / expression control sequence / host combinations within these parameters that will express the desired DNA sequence during fermentation or large scale animal culture using, for example, CHO or COS7 cells.

[0126] The choice of expression control sequences and expression vectors will, in some embodiments, depend on the choice of host. A wide variety of expression host / expression vector combinations can be used. Useful expression vectors for eukaryotic hosts include, for example, vectors having expression control sequences derived from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids such as E. coli-derived plasmids, including colEl, pCRI, pER32z, pMB9, and their derivatives; broad-host-range plasmids such as RP4; phage DNA, e.g., numerous derivatives of lambda phage, e.g., NM989; and DNA phages such as M13 and filamentous single-stranded DNA phages. Useful expression vectors for yeast cells include the 2μ plasmid and its derivatives. Useful vectors for insect cells include pVL941 and pFastBac™1 (GibcoBRL, Gaithersburg, Md.). See 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).

[0127] In addition, any of a wide variety of expression control sequences can be used in these vectors. Such useful expression control sequences include those associated with the structural genes of the expression vectors described above. Examples of useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac, trp, TAC, or TRC systems, the major operator and promoter regions of lambda phage, e.g., PL, the coat protein control region of fd, promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, promoters for acid phosphatases such as PhoA, promoters of the yeast a mating system, the polyhedrin promoter of baculovirus, and various combinations thereof, along with other sequences known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.

[0128] The T7 promoter can be used in bacteria, and the polyhedrin promoter can be used in insect cells. For example, cytomegalovirus promoters or metallothionein promoters can be used in mammalian cells. Additionally, in higher eukaryotes, tissue- and cell-type-specific promoters are widely available. These promoters are named for their ability to direct the expression of nucleic acid molecules in specific tissue or cell types within the body. Those skilled in the art are well aware of the numerous promoters and other regulatory elements that can be used to direct the expression of nucleic acids.

[0129] In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, vectors can include an origin of replication and other genes that encode selectable markers, e.g., neomycin resistance (neomycin resistance). r The ) gene confers G418 resistance to expressing cells, thereby allowing for phenotypic selection of transfected cells. One of skill in the art can readily determine whether a given regulatory element or selectable marker is suitable for use in a particular experimental context.

[0130] Viral vectors that can be used in the present invention include, for example, retroviral vectors, adenoviral vectors, adeno-associated vectors, herpes virus vectors, simian virus 40 (SV40) vectors, and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, NY).

[0131] Prokaryotic or eukaryotic cells that contain and express a nucleic acid molecule encoding an IL-2 mutein of the invention disclosed herein are also a feature of the invention. The cells of the invention are transfected cells, i.e., cells into which a nucleic acid molecule, e.g., a nucleic acid molecule encoding a mutant IL-2 polypeptide, has been introduced by recombinant DNA techniques. The progeny of such cells are also considered within the scope of the invention.

[0132] The exact components of the expression system are not critical. For example, IL-2 muteins can be produced in prokaryotic hosts such as the bacterium E. coli, or eukaryotic hosts such as insect cells (e.g., Sf21 cells) or mammalian cells (e.g., CHO cells, HEK293 cells, COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, Va.). In selecting an expression system, it is important only that the components are compatible with each other. One of skill in the art can make such a judgment. Furthermore, if guidance is needed in selecting an expression system, one of skill in the art can refer to Ausubel et al. (Current Protocols in Molecular Biology, Vol. 1, No. 1, pp. 111-114, 1997). Biology, John Wiley and Sons, New York, NY, 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987) can be used as references.

[0133] The expressed polypeptide can be purified from the expression system using conventional biochemical procedures and used, for example, as a therapeutic agent as described herein.

[0134] In some embodiments, the resulting IL-2 muteins will be glycosylated or deglycosylated depending on the host organism used to produce the mutein. If a bacterial host is selected, the IL-2 muteins produced will be deglycosylated. In eukaryotic cells, on the other hand, the IL-2 muteins will be glycosylated, but probably not in the same way that native IL-2 is glycosylated. The IL-2 muteins produced by the transformed host can be purified according to any suitable method. Various methods for purifying IL-2 are known, for example, Curre et al. See, "Protocols in Protein Science," Vol. 2, Eds. John E. Coligan, Ben M. Dunn, Hidde L. Ploehg, David W. Speicher, Paul T. Wingfield, Unit 6.5 (Copyright 1997, John Wiley and Sons, Inc.). IL-2 muteins can be isolated from inclusion bodies produced in E. coli or from conditioned medium derived from either mammalian or yeast cultures producing the given mutein using cation exchange chromatography, gel filtration chromatography, and / or reversed-phase liquid chromatography.

[0135] Another exemplary method for constructing a DNA sequence encoding an IL-2 mutein is by chemical synthesis. This method involves directly synthesizing a peptide by chemical means to encode an IL-2 mutein exhibiting the described properties. This method allows for the incorporation of both natural and unnatural amino acids at positions that affect the interaction of IL-2 with IL-2Rα, IL-2Rβ, and / or IL-2Rγ. Alternatively, a gene encoding the desired IL-2 mutein can be synthesized by chemical means using an oligonucleotide synthesizer. Such oligonucleotides are designed based on the amino acid sequence of the desired IL-2 mutein, preferably based on the selection of those codons that are preferred in the host cell in which the recombinant mutein will be produced. In this regard, it is well recognized that the genetic code is degenerate, i.e., an amino acid can be coded for by more than one codon. For example, Phe (F) is coded for by two codons, TIC or TTT, Tyr (Y) is coded for by TAC or TAT, and his (H) is coded for by CAC or CAT. Trp(W) is encoded by a single codon, TGG. Thus, for a given DNA sequence encoding a particular IL-2 mutein, it will be apparent that there are many degenerate DNA sequences that will encode that IL-2 mutein. For example, in addition to the preferred DNA sequence for mutein H9, it will be apparent that there are many degenerate DNA sequences that will encode the indicated IL-2 mutein. These degenerate DNA sequences are considered to be within the scope of this disclosure. Thus, in the context of this invention, "degenerate variants thereof" refers to all DNA sequences that encode a particular mutein and thereby provide for the expression of that mutein.

[0136] The biological activity of the IL-2 muteins can be assayed by any suitable method known in the art, including PHA blastocyte proliferation and NK cell proliferation.

[0137] F. Treatment method In some embodiments, the IL-2 muteins of the present invention and / or nucleic acids expressing them can be administered to a subject to treat diseases associated with abnormal apoptotic or differentiation processes (e.g., to provide active or passive immunity, thereby treating, for example, cell proliferative or cell differentiation disorders (such as cancer)). In treating such diseases, the disclosed IL-2 muteins may have beneficial properties, such as reducing vascular leak syndrome. In some embodiments, the IL-2 mutein is any IL-2 mutein or variant disclosed herein. In some embodiments, the IL-2 mutein sequence is at least about 90% identical to any one of SEQ ID NOs: 1-7. In some embodiments, substitutions in the IL-2 mutein are numbered according to wild-type human IL-2 in SEQ ID NO: 8. In some embodiments, the IL-2 mutein is a fusion protein. In some embodiments, the IL-2 mutein is coupled to and / or expressed by a CAR-T construct. In some embodiments, the IL-2 mutein is expressed by and / or associated with an oncolytic virus.

[0138] Examples of cell proliferative and / or cell differentiative disorders include cancer (e.g., carcinoma, sarcoma, metastatic disorders, or hematopoietic neoplastic disorders such as leukemia). Metastatic tumors can arise from many types of primary tumors, including, but not limited to, primary tumors of the prostate, ovarian, breast, endometrial, multiple myeloma, melanoma, lymphoma, lung (including small cell lung cancer), kidney, liver, colon, colorectal, pancreatic, gastric, and brain.

[0139] The mutant IL-2 polypeptides of the present invention can be used to treat patients suspected of having or who may be at high risk of developing any type of cancer, including kidney cancer or melanoma, or any viral disease. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also includes carcinomas, including malignant tumors composed of carcinomatous and sarcomatous tissue.

[0140] Further examples of proliferative disorders include hematopoietic neoplastic disorders.

[0141] Instead of, or in addition to, administering the mutant IL-2 polypeptide to a patient, in some embodiments, the mutant IL-2 polypeptide can be used in an ex vivo manner. 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 involve adding an IL-2 mutant to the culture medium. The culturing step can further include stimulating or treating the cells with other substances, for example, to stimulate proliferation or expand a cell population that is responsive to an antigen of interest (e.g., a cancer antigen or a viral antigen). After the cells have been treated, they are administered to the patient.

[0142] In some embodiments, an IL-2 mutein comprising one or more substitutions selected from the group consisting of substitutions L18R, Q22E, and Q126T, numbered according to wild-type human IL-2 (SEQ ID NO: 8), and F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T, and S130R, is used to treat cancer. In some embodiments, an IL-2 mutein comprising one or more substitutions selected from the group consisting of substitutions L18R, Q22E, and Q126T, numbered according to wild-type human IL-2 (SEQ ID NO: 8), and F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T, and S130R, is used to treat cancer in combination with nivolumab. In some embodiments, IL-2 muteins containing one or more substitutions selected from the group consisting of substitutions L18R, Q22E, and Q126T, numbered according to wild-type human IL-2 (SEQ ID NO: 8), and F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T, and S130R, are used in combination with BMS-936558 to treat cancer. In some embodiments, IL-2 muteins containing one or more substitutions selected from the group consisting of substitutions L18R, Q22E, and Q126T, numbered according to wild-type human IL-2 (SEQ ID NO: 8), and F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T, and S130R, are used in combination with MDX-1106 to treat cancer. In some embodiments, IL-2 muteins comprising one or more substitutions selected from the group consisting of substitutions L18R, Q22E, and Q126T, numbered according to wild-type human IL-2 (SEQ ID NO: 8), and F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T, and S130R, are used in combination with ONO-4538 to treat cancer. In some embodiments, IL-2 muteins comprising one or more substitutions selected from the group consisting of substitutions L18R, Q22E, and Q126T, numbered according to wild-type human IL-2 (SEQ ID NO: 8), and F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T, and S130R, are used in combination with ONO-4538 to treat cancer. and S130R are used in combination with AMP224 to treat cancer. In some embodiments, IL-2 muteins containing one or more substitutions selected from the group consisting of substitutions L18R, Q22E, and Q126T, numbered according to wild-type human 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 are used in combination with CT-011 to treat cancer. In some embodiments, an IL-2 mutein comprising the substitutions L18R, Q22E, and Q126T, numbered according to wild-type human 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, is used in combination with MK-3475 to treat cancer. In some embodiments, the IL-2 mutein comprises the substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, and Q126T. In some embodiments, the IL-2 mutein further comprises a substitution at position S130R. In some embodiments, the IL-2 mutein further comprises a substitution at position S130R. In some embodiments, the IL-2 mutein further comprises a substitution at position F42A. In some embodiments, the IL-2 mutein further comprises a substitution at position E62A. In some embodiments, the IL-2 mutein further comprises a substitution at position Y45A. In some embodiments, the substitutions in the IL-2 mutein comprise L18R, Q22E, and Q126T, numbered according to wild-type human IL-2 of SEQ ID NO: 8, and further comprise 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, the IL-2 mutein comprises the substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, and S130R, numbered according to wild-type human IL-2 of SEQ ID NO: 8.In some embodiments, the IL-2 mutein comprises the substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A, and E62A, numbered according to wild-type human IL-2 in SEQ ID NO: 8. In some embodiments, the IL-2 mutein comprises the substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, and F42A, numbered according to wild-type human IL-2 in SEQ ID NO: 8. In some embodiments, the IL-2 mutein comprises the substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, S130R, F42A, and Y45A, numbered according to wild-type human IL-2 in SEQ ID NO: 8. In some embodiments, the IL-2 mutein comprises the substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, and Q126T, numbered according to wild-type human IL-2 in SEQ ID NO: 8. In some embodiments, the IL-2 mutein comprises the substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, F42A, and E62A, numbered according to wild-type human IL-2 in SEQ ID NO: 8. In some embodiments, the IL-2 mutein comprises the substitutions L18R, Q22E, L80F, R81D, L85V, I86V, I92F, Q126T, and F42A, numbered according to wild-type human IL-2 in SEQ ID NO: 8.

[0143] In some embodiments, IL-2 muteins comprising one or more substitutions selected from the group consisting of substitutions L18R, Q22E, and Q126T, numbered according to wild-type human IL-2 (SEQ ID NO: 8), and F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T, and S130R, are used in combination with anti-CTLA4 mAbs (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), agonist antibodies targeting immune stimulatory proteins (including anti-CD40 mAbs such as CP-870,893, lucatumumab, dacetuzumab), anti-CD137 ... anti-CD137 mAbs (such as i mAb (anti-4-1BB antibody) (BMS-663513 urelumab (anti-4-1BB antibody, e.g., U.S. Pat. Nos. 7,288,638 and 8, 962,804, which is incorporated by reference in its entirety), 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 Publication No. WO2012 / 032433, which is incorporated by reference in its entirety), anti-OX40 mAb (see, e.g., WO2006 / 029879 or WO2010 / 096418, which is incorporated by reference in its entirety), anti-GITR and for use in the treatment of cancer in combination with antibodies and / or immunotherapies, including, but not limited to, mAbs (such as TRX518 (see, e.g., U.S. Pat. No. 7,812,135, incorporated herein by reference in its entirety)), anti-CD27 mAbs (such as varlilumab CDX-1127 (see, e.g., WO2016 / 145085, and U.S. Patent Application Publication Nos. 2011 / 0274685 and 2012 / 0213771, incorporated herein by reference in their entireties)), anti-ICOS mAbs (such as MEDI-570, JTX-2011, and anti-TIM-3 antibodies (see, e.g., WO2013 / 006490 or U.S. Patent Application Publication No. 2016 / 0257758, incorporated herein by reference in their entireties)).

[0144] In some embodiments, an IL-2 mutein comprising one or more substitutions selected from the group consisting of substitutions L18R, Q22E, and Q126T, numbered according to wild-type human IL-2 (SEQ ID NO: 8), and F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T, and S130R, is used to treat cancer in combination with another antibody, which may include monoclonal antibodies directed 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).

[0145] In some embodiments, IL-2 muteins containing one or more substitutions selected from the group consisting of substitutions L18R, Q22E, and Q126T, numbered according to wild-type human IL-2 (SEQ ID NO: 8), and F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T, and S130R, are used to treat cancer in combination with antibody-dependent cellular cytotoxicity (ADCC).

[0146] G. Pharmaceutical Compositions and Methods of Administration In some embodiments, the IL-2 muteins and nucleic acids of the present invention can be added to compositions, including pharmaceutical compositions, which typically include a polypeptide or nucleic acid molecule and a pharmaceutically acceptable carrier.

[0147] Pharmaceutical compositions are formulated to be compatible with the intended route of administration. While the mutant IL-2 polypeptides of the present invention may be administered orally, they are often administered parenterally, including intravenously. Examples of parenteral routes include intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral application may include the following components: a sterile diluent (water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents), an antibacterial agent (e.g., benzyl alcohol or methylparaben), an antioxidant (e.g., ascorbic acid or sodium bisulfite), a chelating agent (e.g., ethylenediaminetetraacetic acid), a buffer (e.g., acetate, citrate, or phosphate), and an agent for adjusting tonicity (e.g., sodium chloride or dextrose). The pH can be adjusted (e.g., to about pH 7.2-7.8, e.g., 7.5) with an acid or base, such as mono- and / or dibasic sodium phosphate, hydrochloric acid, or sodium hydroxide. Parenteral preparations are It may be packaged in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

[0148] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and must be preserved so as to be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants such as sodium dodecyl sulfate. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols (for example, mannitol, sorbitol), sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0149] Sterile injectable solution can be prepared by incorporating active compound in the required amount into suitable solvent with one or a combination of the above-listed components if necessary, and then sterilize by filtration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains basic dispersion medium and the other components listed above that are required.For the sterile powder used to prepare sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying, by which powder of active ingredient and any desired additional ingredients can be obtained from the solution that has been previously sterilized and filtered.

[0150] When an oral composition is used, the oral composition generally contains an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of a tablet, a troche, or a capsule, such as a gelatin capsule. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binders and / or adjuvants can be included as part of the composition. Tablets, pills, capsules, troches and the like may contain any of the following ingredients: binders (such as microcrystalline cellulose, gum tragacanth or gelatin), fillers (such as starch or lactose), disintegrants (such as alginic acid, Primogel™ or cornstarch), lubricants (such as magnesium stearate or Sterotes™), flow agents (such as colloidal silicon dioxide), sweeteners (such as sucrose or saccharin), or flavoring agents (such as peppermint, methyl salicylate or orange flavor), or compounds of a similar nature.

[0151] For administration by inhalation, the IL-2 muteins or nucleic acids encoding them are delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer, such as those described in U.S. Patent No. 6,468,798.

[0152] Systemic administration of IL-2 muteins or nucleic acids may be by transmucosal or transdermal means. It can also be. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated into an ointment, salve, gel, or cream, as is generally known in the art.

[0153] In some embodiments, the compounds (variant IL-2 polypeptides or nucleic acids) can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0154] In some embodiments, the compound (IL-2 mutein or nucleic acid of the invention) can be administered by transfection or infection using methods known in the art, such as those described by 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 at Am. J. Health Syst. Pharm. 53:325, 1996), but are not limited to these.

[0155] In one embodiment, the IL-2 mutein or nucleic acid is prepared with a carrier that protects the mutant IL-2 polypeptide from rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Such formulations can be prepared using standard techniques. Materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells by monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0156] The dosage, toxicity and therapeutic effect of the IL-2 mutein or nucleic acid compound can be determined, for example, by the LD 50 (median lethal dose in the population) and ED 50 The LD (the dose therapeutically effective in 50% of a population) can be determined by standard pharmaceutical procedures in cell culture or experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD 50 / ED 50 The therapeutic index can be expressed as a ratio of . Compounds with a high therapeutic index are preferred. Although compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to reduce side effects by minimizing the potential for damage to uninfected cells.

[0157] Data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for human use. The dosage of such compounds can be determined to be ED5 with little or no toxicity. 50The dosage may vary within this range depending on 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. The dose should be within the range of circulating concentrations that are within the IC50 / IC ... 50 Animal models may be developed to achieve a circulating plasma concentration range that includes the concentration of the test compound that achieves a 50% maximal inhibition of symptoms. Such information can be used to more accurately determine useful doses in humans. Plasma levels may be measured, for example, by high performance liquid chromatography.

[0158] As defined herein, a therapeutically effective amount (i.e., an effective dosage) of an IL-2 mutein of the present invention will depend on the polypeptide or antibody selected. In some embodiments, a single dose of an IL-2 mutein can range from approximately 0.001 mg / kg to 0.1 mg / kg of patient body weight. In some embodiments, an IL-2 mutein can be administered at a dose of about 0.005 mg / kg, about 0.01 mg / kg, about 0.025 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 0.25 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 5.0 mg / kg, or about 10.0 mg / kg. In some embodiments, 600,000 IU / kg is administered (IU can be determined by lymphocyte proliferation bioassay and is used in accordance with the World Health Organization 1 stThe dosage is expressed in international units (IU) as defined by the International Standard for Interleukin-2 (human). It is expected that the dosage may be similar to, but less than, the amount prescribed for PROLEUKIN®. The composition can be administered from once or more times daily to once or more times weekly, including once every two days. It will be apparent to one of skill in the art that certain factors, including, but not limited to, the severity of the disease or disorder, previous treatments, the subject's general health and / or age, and other diseases present, can affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of an IL-2 mutein of the invention can include a single treatment or a series of treatments. In one embodiment, the composition is administered every 8 hours for 5 days, followed by a rest period of 2 to 14 days, e.g., 9 days, followed by administration every 8 hours for another 5 days. In some embodiments, administration is three times every four days.

[0159] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0160] The following examples are presented for the purpose of illustrating certain embodiments of the invention depicted herein and should not be construed as limiting.

[0161] Example Example 1. Analysis of IL-2 antagonists Binding - Surface Plasmon Resonance (SPR) Method Surface plasmon resonance (SPR) technology allows for the analysis of biomolecular interactions in real time without labeling. Binding interactions between proteins using purified preparations or complex mixtures can be quantitatively investigated in their native state, revealing binding specificity, stoichiometry, concentration, thermodynamics, and kinetics (binding rate constant k). a and the dissociation rate constant k d ), and overall affinity (equilibrium dissociation constant K D =k d / ka ) can be evaluated. A sensor chip (CM5) was activated (10 μL / min flow rate for 420 seconds), and 50 μg / mL of anti-histidine antibody (in 10 mM sodium acetate (pH 4.5) as immobilization buffer) was injected (10 μL / min flow rate for 420 seconds). For ligand capture, samples were diluted to 5 μg / mL (in HEPES, pH 7.4, containing 0.005% Tween-20) and injected (10 μL / min flow rate) to achieve a capture level of approximately 200 RU. Receptor was diluted to the eight concentrations shown and injected (30 μL / min flow rate) with a 120-second binding phase followed by a 300-second dissociation phase. The constructs examined by SPR are listed in Tables 4 and 5.

[0162] [Table 4] [Table 5-1] [Table 5-2]

[0163] SPR binding results IL-2 binds IL2Rβ (CD122) and γ c The intermediate-affinity heterodimeric IL-2 receptor, consisting of IL2Rα (CD25), IL2Rβ, and IL2Rγ (CD132). c MDNA209 binds to the high-affinity heterotrimeric IL-2 receptor, which is composed of IL2Rβ and IL2Rγ. c It is an IL-2 superantagonist mutated to prevent binding (Mitra et al., 2015). See also Figure 2A.

[0164] Evaluation of binding affinity in the sensorgrams of MDNA209-Fc, MDN209FEAA-Fc, MDNA209FEAA-Fc-A11, MDNA209(3)FEAA-Fc, MDNA209(3)FEAA-Fc-MDNA209(3)FEAA, MDNA209FA-Fc, and MDNA209FEY-Fc variants is shown in Figure 2B-D, and the binding is summarized in Table 6. D The results were compared with those of Fc-IL-2 as a positive control. The K of MDNA209-Fc, MDNA209FA-Fc, MDNA209FEY-Fc, MDN209FEAA-Fc, MDNA209FEAA-Fc-A11, MDNA209(3)FEAA-Fc, MDNA209(3)FEAA-Fc-MDNA209(3FEAA) against IL2Rβ (CD122) was D The values ​​are dramatically lower than those of Fc-IL-2. Further mutations in MDNA209FEAA-Fc, MDNA209FEY-Fc, MDNA209FA-Fc, MDNA209(3)FEAA-Fc, and MDNA209(3)FEAA-Fc-MDNA209(3)FEAA did not result in binding to IL2Rα (CD25).

[0165] [Table 6]

[0166] SPR binding conclusions Based on SPR analysis, all IL-2 antagonists tested showed higher binding to CD122 compared to Fc-IL-2 (Table 6). Addition of FEAA, FA, or FEY mutations abolished binding to CD25. In the bispecific format, MDNA209FEAA-Fc-A11 showed binding affinity comparable to MDNA209FEAA-Fc.

[0167] Signal transduction assays HEK-Blue™ IL-2 Reporter Antagonist Assay HEK-Blue™ IL-2 reporter cells express the high-affinity IL-2 receptor and are designed to monitor activation of the JAK-STAT pathway. HEK-Blue™ IL-2 reporter cells express the human IL-2Rα, IL-2Rβ, IL-2Rγ, JAK3, and STAT5 genes, as well as the STAT5-inducible SEAP (SEAP) pathway. The IL-2 expression vector was generated by stable transfection of HEK293 cells with a secretory embryonic alkaline phosphatase (ESP) reporter gene. IL-2 stimulation induces STAT5 activation and subsequent SEAP expression and secretion, which is quantified using Quanti-Blue™.

[0168] HEK-Blue™ IL-2 reporter cells (InvivoGen, 50,000 cells / well) were incubated with 1) increasing concentrations of constructs and EC 80 The assay was performed in two formats: 1) a fixed agonist format using rhIL-2 at a concentration (experimentally determined to be 0.1 nM) and 2) an antagonist format in which 30 nM of test compound was exposed to a range of IL-2-Fc (1 nM to 0.001 pM) (Table 7). Cells were incubated for 24 hours, and after incubation, cell supernatants (20 μL) were removed to a new plate, followed by the addition of 180 μL of QUANTI-Blue solution and incubation at 37°C for 2 hours. Each plate was read for absorbance at 650 nm on a conventional plate reader.

[0169] [Table 7]

[0170] HEK-Blue IL-2 reporter results Dose of IL-2 for antagonist assay (EC 50 Initial experiments were performed to establish an EC50 / EC60 / EC60 (EC50 / EC6 ...50 MDNA209-Fc showed clear antagonist activity with an IC value of 8.396 nM (Figure 3A and Table 7). MDNA209-Fc was also tested in an antagonist format with a fixed concentration of compound and increasing amounts of IL-2. MDNA209-Fc showed an IC 50 This clearly demonstrated antagonist activity as seen by a right shift of .DELTA.=59.31 (FIG. 3B).

[0171] In previous publications, a wide range of IL-2 antagonists with different levels of antagonist activity, including weak partial agonists, have been designed (Mitra et al., 2015). The MDNA209(3) molecule is a γ c Unlike MDNA209(RETR), which completely inhibits binding to γ c It has three mutations (RET) that have been reported to potently but incompletely inhibit binding to β-glucan. Therefore, the MDNA209 (3) compound was tested for weak partial agonist activity.

[0172] 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) in this assay (Figure 3C and Table 8). MDNA209(3)-Fc-MDNA209(3)FEAA, which contains four MDNA209(3) moieties, exhibited higher agonist activity than MDNA209(3)FEAA-Fc (Figure 3C and Table 8).

[0173] [Table 8]

[0174] HEK-Blue IL-2 reporter conclusions MDNA209-Fc exhibited antagonistic activity against IL-2, while MDNA209(3)FEAA-Fc and MDNA209(3)FEAA-Fc-MDNA209(3)FEAA exhibited weak agonistic activity.

[0175] CTLL-2 proliferation assay CTLL-2 cells are a cytotoxic T cell line that is dependent on IL-2 for survival and proliferation. This assay provided a functional readout (proliferation) over a longer assay period than the HEK-Blue signaling assay. CTLL2 proliferation assays were performed in duplicate with the samples shown in Table 9. CTLL2 cells were seeded at 30,000 cells / well in medium lacking T-SIM proliferation supplements. Cells were treated with increasing concentrations of test or control samples for 48 hours. After treatment, Cell Titer Blue viability reagent (Promega G8080) was added to each well, and after 3 hours, each plate was incubated for 3 hours after the development of a fluorescent viability signal (approximately 6 h). The reading was taken at 560Ex / 590Em.

[0176] [Table 9]

[0177] Consequences of CTLL-2 expansion The RFU versus IL-2-Fc concentration data were fitted to a four-parameter logistic curve to determine the EC 50 Values ​​were calculated and averaged from duplicate or quadruple plates (Table 10). Fold antagonism was calculated as the EC50 of IL-2-Fc alone. 50 EC50 of IL-2-Fc in the presence of test samples against 50 The EC ratio was calculated as the ratio of EC (Figure 4 and Table 11). MDNA209-Fc showed approximately 70-fold higher EC 50 antagonized IL-2-Fc.

[0178] [Table 10] [Table 11]

[0179] CTLL2 assay results MDNA209-Fc was a potent inhibitor of IL-2-induced CTLL2 proliferation.

[0180] pSTAT5 signaling assay using human peripheral blood mononuclear cells (PBMCs) To investigate the activity and potential cell population selectivity of different IL-2 superantagonist variants, inhibition of STAT5 phosphorylation was assessed in a subset of immune cell populations (T cells) present in human peripheral blood mononuclear cells (PBMCs). reg PBMCs purified from whole blood were rested overnight. Cell counts were performed. Cells were seeded in serum-free medium (60 min). Medium alone or medium containing each compound (10-point 5-fold dilutions starting from 25 nM) was added to the cells and incubated at 37°C for 15 min. rhIL-2 (10-point 5-fold dilutions starting from 25 nM) was used as a positive control for pSTAT5. After activation with IL-2 or other compounds (listed in Table 13), cells were fixed in PFA at room temperature for 15 min, permeabilized (BD Transcription Factor Phosphorylation Buffer Set), and stored in methanol (-20°C). After storage in methanol, each sample was 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 listed in Table 12.

[0181] [Table 12]

[0182] Results of pSTAT5 signaling assay Cells were pretreated with increasing concentrations of antagonist or neutralizing antibody control, then exposed to 67 pM or 670 pM rhIL-2 (equivalent to approximately 1 ng / mL and 10 ng / mL, respectively, as used in the Mitra et al., 2015 publication). Click or tap here to enter text. Representative dose-response curves are shown in Figure 5, and IC 50A summary of the results is shown in Tables 14 and 15. Analysis of pSTAT5 signaling in human PBMCs revealed that MDNA209-Fc exhibited high potency across different cell types compared to all other antagonists and antibody controls. MDNA209-Fc also inhibited T cells more effectively than other immune cells, including CD8+CD25+ T cells, which also express the high affinity receptor. reg Comparing these two cell types, anti-CD25 (a biosimilar to daclizumab) inhibited T cells more effectively than CD8+CD25+ T cells. reg It was thought to have a higher inhibitory activity than

[0183] T reg T cells limit T cell activity in autoimmune diseases. reg Molecules that selectively target effector T cells while leaving the population intact would be advantageous. Adding the FEAA mutation, which abolishes binding to the high-affinity CD25 receptor, reduces T reg This appears to allow for better separation of the inhibitory effects on CD122 and other immune cells. The addition of the FEAA mutation also significantly reduced potency against all immune cell types tested, although potency was higher than that of anti-CD122. This was observed for both the MDNA209FEAA-Fc and MDNA209(3)FEAA-Fc compounds, which showed similar levels of activity.

[0184] Further analysis of the bispecific molecule MDNA209FEAA-Fc-A11 confirmed that the activity of the MDNA209FEAA moiety was unaffected by the addition of the A11 moiety, which was shown to be inactive in this assay (data not shown), and the addition of a second moiety to MDNA209FEAA reduced its pSTAT5 inhibitory signaling activity.

[0185] [Table 13] [Table 14]

[0186] Conclusions of the pSTAT5 signaling assay MDNA209-Fc is a potent inhibitor of pSTAT5 signaling in human PBMCs and T cells more than other immune cells reg MDNA209FEAA-Fc had a higher proportional potency against T cells compared to MDNA209-Fc. reg MDNA209(3)FEAA-Fc and MDNA209FEAA-Fc showed similar potency in this assay. MDNA209FEAA-Fc-A11 showed the lowest inhibitory activity observed.

[0187] In vivo maximum tolerated dose (MTD) method The purpose of this study was to evaluate the efficacy of MDNA209FEAA-Fc-A in naive C57BL / 6 mice. The objective of this study was to determine the tolerability of MDNA209FEAA-Fc-A11. An initial maximum tolerated dose (MTD) study was conducted to evaluate the acute toxicity of the MDNA209FEAA-Fc-A11 compound in naive C56B1 / 6 mice. A total of three mice per group were administered MDNA209FEAA-Fc-A11 (30 mg / kg) and A11-Fc (21 mg / kg or 11 mg / kg). Animals were dosed intraperitoneally on days 1, 5, and 8.

[0188] MTD results Animals receiving a dose of 30 mg / kg MDNA209FEAA-Fc-A11) or A11-Fc (21 mg / kg, molar equivalent) tolerated the drug for 12 days without significant weight loss or overt signs of toxicity (Figure 6).

[0189] MTD Conclusion The MDNA209FEAA-Fc-A11 compound was well tolerated and showed no signs of acute toxicity.

[0190] Experimental autoimmune encephalomyelitis (EAE) model Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis. C57BL / 6 mice were divided into groups on day 0 and injected with MOG in complete Freund's adjuvant at two different sites in the hind flank on day 2. 35-55 EAE was induced by immunization with peptides. Pertussis toxin was administered 2 hours and 48 hours after immunization. Mice were treated 8, 12, and 15 days after immunization (Table 16). Animal weights and clinical EAE scores were assessed daily, and mice were scored on a scale of 0 to 5.0 (defined in Table 15).

[0191] [Table 15]

[0192] [Table 16]

[0193] Mice treated with MDNA209-Fc were observed to have significantly lower scores than PBS-treated mice on day 18 after immunization (p<0.05, t-test, n=7) (Figure 7). MDNA209FA-Fc also showed a reduced score compared to the PBS control.

[0194] Mice were treated with MDNA209FEAA-Fc-A11 or a combination of MDNA209-Fc and A11-Fc. Both treatments resulted in a decrease in the score compared to the PBS control (Figure 8). Mice treated with the combination of A11-Fc and MDNA209-Fc showed a significant increase in body weight on days 12 and 14 compared to the PBS control (p<0.05, t-test, n=6) (Figure 8).

[0195] Mice administered a therapeutic dose of MDNA209FEAA-Fc-A11 showed a decrease in scores after administration (FIG. 9).

[0196] EAE Conclusion MDNA209-Fc, MDNA209FA-Fc, and MDNA209FEAA-Fc-A11 reduced disease scores in the EAE model compared to PBS control animals in both prophylactic and therapeutic administration schedules.

[0197] Blockade of IL-2-induced IFNγ secretion by MDNA209-Fc Interleukin-2 (IL-2) stimulates human peripheral mononuclear cells (PBMCs) to secrete cytokines, including pro-inflammatory interferon (IFN)-γ. PBMCs from three different healthy donors were incubated in vitro for 48 hours with increasing concentrations of MDNA209-Fc in the presence of 0.3 or 0.1 μg / mL of human recombinant IL-2 (rhIL-2). Culture supernatants were collected and analyzed for IFNγ levels using an enzyme-linked immunosorbent assay (ELISA). As shown in Figure 10, MDNA209-Fc dose-dependently inhibited rhIL-2-induced IFNγ secretion in three different PBMCs. Values ​​below the lower limit of quantitation (LLOQ) are plotted as 0.5 × LLOQ (4 pg / mL).

[0198] conclusion Each of the listed MDNA209 variants was tested for binding, signaling (pSTAT5, a HEKBlue IL-2 reporter in human PBMCs), CTLL2 proliferation, MTD in mice, and efficacy in an EAE in vivo assay. All compounds showed enhanced binding to CD122 (compared to IL-2), and compounds with FEAA, FA, or FEY mutations showed reduced binding to CD25. MDNA209-Fc demonstrated inhibitory activity in HEKBlue IL-2 and CTLL2 assays. All compounds inhibited IL-2-induced pSTAT5 signaling in human PBMCs. MDNA209FEAA-Fc-A11 showed no toxicity in mice (MTD assay), and MDNA209-Fc reduced disease scores in EAE.

[0199] The distinct mechanism of action of MDNA209 offers advantages: it is an effector cell antagonist, directly targeting disease-causing effector immune cells and blocking CD4+ and CD8+ T cells and NK cells. Thus, MDNA209 has broad application in autoimmune indications, with minimal involvement of Tregs.

[0200] Example 2. Cytokine release assay in human PBMCs This study was conducted to test the activity of MDNA209-Fc in blocking IL-2 signaling in normal healthy human PBMCs, using IFN-γ secretion as a readout.

[0201] method: PBMCs from four healthy human donors (Table 1) were purchased from STEMCELL Technologies and screened for their response to IL-2 stimulation. Briefly, cells were preincubated with 0–1000 nM MDNA209-Fc (2:1) for 15 min and then stimulated with rhIL-2 (3, 1, or 0.3 μg / ml) for 48 h. IFN-γ ELISA was performed on the supernatants. Because IFN-γ levels were above the quantifiable level when PBMCs were treated with 1 μg / ml and 3 μg / ml rhIL-2, data from stimulation with 0.3 μg / ml rhIL-2 were used for analysis and are presented in this report. Data were normalized for each donor by calculating the percent inhibition of the mean maximal response without MDNA209-Fc treatment.

[0202] [Table 17]

[0203] Results and conclusions: Four donors who showed a clear response to 0.3 μg / mL rhIL-2 stimulation were selected for experiments with MDNA209-Fc. In all four unique PBMC donor samples, MDNA209-Fc demonstrated dose-dependent inhibition of rhIL-2-induced IFN-γ release (Table 17 and Figure 11). Thus, MDNA209-Fc demonstrated IL-2 antagonist activity by inhibiting IL-2-induced activation and IFN-γ cytokine release in human PBMCs.

[0204] Example 3. MDNA209 Exposure and Pharmacokinetics In Vivo This study was conducted to determine the tolerability and pharmacokinetics of MDNA209-Fc for an appropriate dosing regimen in a preclinical in vivo study.

[0205] method: BALB / c mice (10-11 weeks old) were randomized into seven groups (3 animals per group) based on body weight (Table 18) and treated with different doses of MDNA209-Fc via intraperitoneal (IP) injection using different dosing schedules. Daily cageside clinical observations and body weights were recorded twice weekly. Food and water consumption were also monitored. At the indicated non-terminal time points, approximately 100 μL of whole blood was collected, processed to plasma, and stored at -80°C until analysis. Approximately 200 μL of whole blood was collected for CBC analysis. Necropsy was performed at termination. MDNA209-Fc detection was performed using an MDS ELISA. R&D Systems MAB202-100 was used as the capture antibody, and anti-human Fc Cross Species Absorbed (Sigma part number SAB3701284) was used as the detection antibody, followed by probing with HRP-conjugated anti-goat IgG (Millipore part number 401515).

[0206] [Table 18]

[0207] Results and conclusions: MDNA209-Fc was well tolerated when administered at repeated doses up to 20 mg / kg via IP injection in acute (four doses over five days) or chronic (eight doses over 21 days) studies. There were no changes in body weight, and no abnormal findings were noted at necropsy (Figure 12). Multiple doses of MDNA209-Fc did not affect lymphocyte counts (Figure 12), likely due to the long life span of lymphocytes (15–20 days), necessitating long-term studies to detect changes. In mice, MDNA209-Fc was detectable in the blood 24 hours after administration but was rapidly eliminated, becoming undetectable in the blood 72 hours after administration (Figure 13).

[0208] Example 4. Mixed Lymphocyte Reaction (MLR) Assay The purpose of this study was to evaluate 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. The aim was to determine the effectiveness.

[0209] TIFF2025532554000029.tif134170

[0210] method: Four healthy donors were obtained from Xeno Diagnostics for use 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 allowed to settle overnight prior to use. PBMCs (200,000 cells / donor) from two unrelated donors were co-cultured with MDNA209-Fc, MDNA209-albumin, and MDNA209FEAAC-Fc at different dilutions (0.0016 nM to 100 nM, 5-fold dilutions). Phytohemagglutinin (PHA) as a mitogen and dexamethasone as a growth inhibitor were used as positive and negative controls, respectively. Each sample was run in triplicate and cultured for four days. Growth was observed microscopically on day three, and BrdU was added. Colorimetric ELISA for BrdU incorporation was performed on day four. The stimulation index (SI) was calculated by dividing the test absorbance by the absorbance of the baseline background control. The 50% inhibitory concentration (IC50) was calculated for the SI by a four-parameter curve fit.

[0211] [Table 19]

[0212] Results and conclusions: MDNA209-Fc inhibited PBMC proliferation in all five donor pairs (IC 50 Within the dose range tested, MDNA209FEAA-Fc was the only pair (IC 50 = 28.54 nM), whereas 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 under MLR conditions. MDNA209FEAA-Fc had limited or no effect on PBMC proliferation. See Figure 14 and Table 20.

[0213] [Table 20]

[0214] Example 5. Further Binding (SPR) Data Results and conclusions: Based on SPR analysis, all IL-2 antagonists tested (free and fused with Fc) showed increased binding to CD122 compared to their respective IL-2 or Fc-IL-2 controls (Table 21). Addition of FEAA abolished binding to CD25. MDNA209(3)-Fc retained binding to CD25 and showed high binding to CD122. Addition of FA to MDNA209(3) reduced its affinity for CD25.

[0215] Fusion of MDNA209 with the IL-4 agonist KFR or RGA moieties maintained binding affinity to CD25 and CD122 (Table 21, Figures 15A-15B).

[0216] [Table 21]

[0217] The above examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use embodiments of the compositions, systems, and methods of this 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 invention that are obvious to those of ordinary skill in the art are intended to be within the scope of the following claims. Any patents and publications mentioned herein are indicative of the level of knowledge of those skilled in the art to which this invention pertains. All references cited in this disclosure are incorporated by reference in their entirety to the same extent as if each reference were individually incorporated by reference.

[0218] Any heading and section designations are used for clarity and reference purposes only and should not be construed as limiting in any way. For example, those skilled in the art will understand the utility of combining various aspects from the different headings and sections as appropriate in accordance with the spirit and scope of the invention described herein.

[0219] Any references cited herein are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0220] Many modifications and variations of this application can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are offered by way of example only, and this application is limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Claims

1. 1. An IL-2 mutein comprising the 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 group of amino acid substitutions selected from the group consisting of F42A, Y45A, E62A, L80F, R81D, L85V, I86V, I92F, Q126T, and S130R.

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

1.

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

1.

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

3.

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

4.

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

5.

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

6.

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

7.

9. 9. An IL-2 mutein according to any one of claims 1 to 8, which is fused to an albumin molecule, an Fc molecule and / or another mutein, optionally the other mutein being an IL-13 mutein or an IL-4 mutein.

10. 10. The IL-2 mutein of claim 9, fused to an albumin molecule.

11. 11. The IL-2 mutein of claim 10, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:

18.

12. 10. The IL-2 mutein of claim 9, which is fused to an Fc molecule.

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

14. 10. The IL-2 mutein of claim 9 fused to an Fc molecule and an IL-4 mutein, optionally wherein the IL-4 mutein comprises the 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. 15. The IL-2 mutein of claim 14, wherein the fusion protein comprises the amino acid sequence of one of SEQ ID NOs: 19-22.

16. 16. The IL-2 mutein of any one of claims 1 to 15, which has increased binding to CD122 compared to wild-type IL-2.

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

18. 18. An IL-2 mutein according to any one of claims 1 to 17, having inhibitory activity as determined using a HEKBlue IL-2 and / or CTLL2 assay.

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

20. 20. The IL-2 mutein of any one of claims 1 to 19, which exhibits no toxicity in mice as determined using a maximum tolerated dose (MTD) assay.

21. 21. An IL-2 mutein according to any one of claims 1 to 20, which reduces disease scores as determined using an experimental autoimmune encephalomyelitis (EAE) assay.

22. A nucleic acid encoding an IL-2 mutein according to any one of claims 1 to 21.

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

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

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